JP7015232B2 - Method for Producing High-Purity Fluorescein Compounds - Google Patents

Description

The present invention relates to a method for producing a high-purity fluorescein compound which is efficient and highly safe on an industrial scale.

Fluoresceins compounds are known to exhibit strong fluorescent emission in solution and have biological uses as fluorescent labeling agents for proteins and fluorescent reagents for protein tracking.

Furthermore, fluorescein compounds are useful compounds in a wide range of industrial applications such as therapeutic agents for cancer and inflammatory diseases, cosmetic materials, organic EL panels, electronic information materials such as color filter substrate materials, and optical materials. ..

A general method for producing a fluorescein compound is obtained by reacting a compound having a phenolic hydroxyl group with an acid anhydride compound in the presence of an acidic catalyst.
Fluorescein was first synthesized from resorcinol and phthalic anhydride in 1871, and many researchers have since conducted fluorescence characteristic analysis and synthesis studies of analogs. Naftfluorescein, a type of fluorescein compound, was synthesized in 1989 from 1,6-dihydroxynaphthalene and phthalic anhydride.

Patent Document 1 proposes a method using zinc chloride as an acid catalyst used in a method for synthesizing a fluorescein compound. However, since zinc chloride is deliquescent, it is easily hydrolyzed and has a problem that it is difficult to handle industrially. It is also known that zinc chloride decomposes when heated to produce toxic fumes. Zinc chloride fume is irritating and has problems such as irritation to the eyes, respiratory tract or skin. Further, in recent years, there has been a demand for a method for efficiently producing a higher-purity fluorescein compound on an industrial scale.

Therefore, Patent Document 2, Non-Patent Documents 1, 2 and 3 propose a production method using methanesulfonic acid as an acidic catalyst and a reaction solvent.
However, since the production methods described in Patent Document 2 and Non-Patent Documents 1, 2 and 3 use methanesulfonic acid as an acid catalyst and a reaction solvent, the amount of methanesulfonic acid used becomes excessive and synthesis is performed. There is a problem that acid treatment after the reaction is difficult.

Further, in the above-mentioned production method, the reaction solution is added to water to precipitate fluorescein compounds, but the addition of methanesulfonic acid, which is a strong acid, causes intense heat generation, so it is a safe production method. I can't say. Further, if it is added little by little in order to suppress heat generation, the productivity is lowered, so that it is not an efficient production method. In addition, since impurities and related substances are also deposited at the same time in the precipitation operation with water, there is a problem that the chemical purity of the precipitated fluorescein compounds is low.

Further, in Patent Document 2 and Non-Patent Documents 1, 2 and 3, column chromatography is used for purification of fluorescein compounds, but purification by column chromatography is inefficient, and the purification cost is high and a stable industrial production method is used. I can't say.

That is, in the production of high-purity fluorescein compounds, an industrial production method having high production efficiency and high safety has not yet been established, and high-purity fluorescein compounds can be efficiently produced on an industrial scale. There was a demand for a manufacturing method for safe production.

European Patent No. 0357350 International Publication No. 2016/148215

Chemical Communications, 5974-5976, 2005. Bioorganic & Medicinal Chemistry Letters, 18 (22), 5948-5950, 2008. Journal of Organic Chemistry, 77 (7), 3492-3500, 2012.

An object of the present invention is to provide a method for efficiently and safely producing a high-purity fluorescein compound on an industrial scale.

The method for producing a high-purity fluorescein compound of the present invention is a method for producing a fluorescein compound by reacting a compound having a phenolic hydroxyl group with an acid anhydride compound in the presence of an acidic catalyst, wherein the phenolic hydroxyl group is produced. 1,6-Dihydroxynaphthalene as a compound having, the step of carrying out the reaction using a solvent containing acetic acid as a reaction solvent, and the above-mentioned A step of isolating the fluorescein compound, a step of dissolving the isolated fluorescein compound in an aprotonic solvent and crystallization in a poor solvent, or a step of purifying the isolated fluorescein compound by washing the slurry with a protonic polar solvent. It is characterized in that a high-purity fluorescein compound represented by the following formula (5) having a chemical purity of 95% or more is obtained.

According to the fluorescein compound of the present invention, the compound has a high chemical purity of 95% or more, preferably 98% or more, and thus has a wide range of industrial uses such as biological applications, electronic information materials and optical materials. Is useful in.

According to the method for producing a fluorescein compound of the present invention, the amount of an acidic catalyst can be reduced by using a solvent containing a carboxylic acid as a reaction solvent, so that the acidic waste liquid treatment after the reaction becomes easy. Further, since the fluorescein compound obtained by the present invention has low solubility in a solvent containing a carboxylic acid, it can be isolated and purified by simple operations such as crystallization, filtration and washing.

The method for producing a fluorescein compound of the present invention can safely produce a high-purity fluorescein compound on an industrial scale. The method for producing a fluorescein compound of the present invention preferably enables efficient and safe production of a high-purity fluorescein compound by purifying by crystallization.

The high-purity fluorescein compounds of the present invention will be described in detail below.
As used herein, fluorescein compounds refer to fluorescein and its derivatives, as well as naphthofluorescein and its derivatives.

上記式（１）において、ｘ、ｙ、ｚは、０～４の整数を表し、Ｒ^１、Ｒ^２、Ｒ^３は、それぞれ独立して、置換基を有する縮合したベンゼン環又は無置換の縮合したベンゼン環である。ｘ、ｙ、ｚは、好ましくは、１～４の整数である。Ｒ^１、Ｒ^２は、好ましくは、水酸基を有する縮合したベンゼン環であり、より好ましくはｘ，ｙが１、ｚが０であるとよい。 The fluorescein compound of the present invention is a compound represented by the following formula (1).

In the above formula (1), x, y, and z represent integers of 0 to 4, and R ¹ , R ² , and R ³ are independently condensed benzene rings having substituents or unsubstituted condensation. It is a benzene ring. x, y, and z are preferably integers of 1 to 4. R ¹ and R ² are preferably condensed benzene rings having a hydroxyl group, and more preferably x and y are 1 and z is 0.

The chemical purity of the fluorescein compound is 95% or more, preferably 98% or more. The upper limit of the chemical purity of fluorescein compounds is 100%. In the present specification, the chemical purity of a fluorescein compound is a fraction of the peak area of the fluorescein compound when measured by a method described later using a high performance liquid chromatography method (hereinafter, abbreviated as “HPLC”). HPLC area%).

上記式（２）～（４）において、ａ、ｂ、ｃ、ｄ、ｅ、ｆは、１～４の整数を表し、Ｒ^６、Ｒ^７、Ｒ^８、Ｒ^９、Ｒ^１０、Ｒ^１１は、それぞれ独立して、水素、水酸基、アルコキシ基、カルボキシル基、スルホン酸基、アミノ基、ハロゲン、ニトロ基及びアルキル基からなる群から選択される。Ｒ^６、Ｒ^７、Ｒ^８、Ｒ^９、Ｒ^１０、Ｒ^１１は、水酸基であることが好ましい。 The fluorescein compound of the present invention is a compound selected from the group of compounds represented by the following formulas (2) to (4). The fluorescein compound may be one kind of compound or a mixture of a plurality of kinds of compounds among the compounds represented by the following formulas (2) to (4).

In the above equations (2) to (4), a, b, c, d, e, and f represent integers of 1 to 4, and R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ are. , Each independently selected from the group consisting of hydrogen, hydroxyl group, alkoxy group, carboxyl group, sulfonic acid group, amino group, halogen, nitro group and alkyl group. It is preferable that R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ are hydroxyl groups.

The chemical purity of the fluorescein compound selected from the group of the compounds represented by the above formulas (2) to (4) is 95% or more, preferably 98% or more. The upper limit of chemical purity is 100%. The chemical purity of the fluorescein compound is a fraction of the peak area of the fluorescein compound measured by HPLC (HPLC area%).

The fluorescein compound of the present invention is preferably a compound selected from the group of compounds represented by the following formulas (5) to (7). The fluorescein compound may be one kind of compound or a mixture of a plurality of kinds of compounds among the compounds represented by the following formulas (5) to (7).

Next, a method for producing a high-purity fluorescein compound of the present invention will be described in detail.
The method for producing a fluorescein compound of the present invention is a method for producing a fluorescein compound by reacting a compound having a phenolic hydroxyl group with an acid anhydride compound in the presence of an acidic catalyst, and the reaction is used as a reaction solvent for carboxylic acid. This is done using a solvent containing an acid.

As the compound having a phenolic hydroxyl group, phenols and naphthols can be used. Further, one kind of compound having a phenolic hydroxyl group can be used, or two or more kinds can be mixed and used.

When phenols are used in the reaction, fluorescein compounds consisting of fluorescein and its derivatives are obtained. Examples of phenols include phenol, catechol, resorcinol, 2-aminoresorcinol, 4-aminoresorcinol, 2-chlororesorcinol, 4-chlororesorcinol, 2-bromoresorcinol, 4-bromoresorcinol, hydroquinone, pyrogallol, and fluoroglucolcinol. , Salicylic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2-aminophenol, 3-aminophenol, 4-aminophenol, o-cresol, m-cresol, p-cresol, o-chlorophenol, m-chlorophenol, p-chlorophenol, o-bromophenol, m-bromophenol, p-bromophenol, o-iodophenol, m-iodophenol, p- Examples thereof include iodophenol, 2-nitrophenol, 3-nitrophenol, and 4-nitrophenol. As the phenols, phenol, catechol, resorcinol, hydroquinone, pyrogallol, hydroxybenzoic acid and aminophenol are more preferable, and catechol, resorcinol and hydroquinone are particularly preferable.

On the other hand, when naphthols are used as the compound having a phenolic hydroxyl group, a naphthofluorescein compound composed of naphthofluorescein and a derivative thereof can be obtained.
Examples of naphthols include 1-naphthol, 2-naphthol, 1,2-dihydroxynaphthalene, 1,3-dihydroxynaphthalene, 1,4-dihydroxynaphthalene, 1,5-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, and the like. 1,7-Dihydroxynaphthalene, 1,8-dihydroxynaphthalene, 2,3-dihydroxynaphthalene, 2,6-dihydroxynaphthalene, 2,7-dihydroxynaphthalene, 1-hydroxy-3-naphthoic acid, 1-hydroxy-4- Naphtholic acid, 1-hydroxy-5-naphthoic acid, 1-hydroxy-6-naphthoic acid, 1-hydroxy-7-naphthoic acid, 1-hydroxy-8-naphthoic acid, 3-hydroxy-1-naphthoic acid, 3- Amino-1-naphthol, 4-amino-1-naphthol, 5-amino-1-naphthol, 6-amino-1-naphthol, 7-amino-1-naphthol, 8-amino-1-naphthol, 1-amino- 2-naphthol, 3-amino-2-naphthol, 4-amino-2-naphthol, 5-amino-2-naphthol, 6-amino-2-naphthol, 7-amino-2-naphthol, 8-amino-2- Naphthol, 3-chloro-1-naphthol, 4-chloro-1-naphthol, 5-chloro-1-naphthol, 6-chloro-1-naphthol, 7-chloro-1-naphthol, 8-chloro-1-naphthol, 1-Chloro-2-naphthol, 3-chloro-2-naphthol, 4-chloro-2-naphthol, 5-chloro-2-naphthol, 6-chloro-2-naphthol, 7-chloro-2-naphthol, 8- Chloro-2-naphthol, 3-bromo-1-naphthol, 4-bromo-1-naphthol, 5-bromo-1-naphthol, 6-bromo-1-naphthol, 7-bromo-1-naphthol, 8-bromo- 1-naphthol, 1-bromo-2-naphthol, 3-bromo-2-naphthol, 4-bromo-2-naphthol, 5-bromo-2-naphthol, 6-bromo-2-naphthol, 7-bromo-2- Examples thereof include naphthol, 8-bromo-2-naphthol, and the like. As the naphthols, naphthol, dihydroxynaphthalene, aminonaphthol and hydroxynaphthoic acid are preferable, and 1,5-dihydroxynaphthalene, 1,6-dihydroxynaphthalene, 1,7-dihydroxynaphthalene and 2,7-dihydroxynaphthalene are particularly preferable. preferable.

In the production method of the present invention, phthalic anhydride and its derivatives can be preferably used as the acid anhydride. The benzene ring of phthalic anhydride may optionally have a substituent. The type, number and position of the functional group of the substituent are not particularly limited. Further, the substituent may be a condensed benzene ring having a substituent or an unsubstituted condensed benzene ring.

Examples of phthalic acid anhydrides and derivatives thereof include phthalic acid anhydride, 2,3-naphthalenedicarboxylic acid anhydride, 1,2-naphthalenedicarboxylic acid anhydride, trimellitic acid anhydride, 3-methylphthalic acid anhydride, and the like. 4-Methylphthalic anhydride, 3-chlorophthalic anhydride, 4-chlorophthalic anhydride, 3-bromophthalic anhydride, 4-bromophthalic anhydride, 3-hydroxyphthalic anhydride, 4-hydroxyphthalic anhydride , 3-Aminophthalic acid anhydride, 4-aminophthalic acid anhydride, 3-nitrophthalic acid anhydride, 4-nitrophthalic acid anhydride, 3-methylphthalic acid anhydride, 4-methylphthalic acid anhydride and the like are exemplified.

In the method for producing a fluorescein compound, the amount of the compound having a phenolic hydroxyl group and the acid anhydride is not particularly limited, and the larger the amount of the compound having a phenolic hydroxyl group with respect to the acid anhydride, the easier the reaction may proceed. If it is too much, the raw material cost will be high, which is not preferable in terms of economic efficiency. Specifically, the amount of the compound having a phenolic hydroxyl group for synthesizing the fluorescein compound is preferably 1 to 10 mol times, more preferably 2 to 4 mol times, the molar amount of the acid anhydride used. Yes, more preferably 2-3 mol times. As an inexpensive industrial production method, it is preferable to react at a stoichiometric amount of 2 mol times.

In the method for producing a fluorescein compound, an acidic catalyst is used as a reaction accelerator. The acidic catalyst is preferably a protonic acid. Examples of the protonic acid include hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, fluorosulfonic acid, hexafluoroantimonic acid, tetrafluoroboric acid, hexafluorophosphate, chromium acid and boric acid. , Methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, trifluoromethanesulfonic acid and the like are exemplified. As the protonic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid and p-toluenesulfonic acid are preferable.

The amount of the acidic catalyst is 0.1 to 20 mol times, preferably 1 to 10 mol times, and more preferably 2 to 5 mol times the amount of the compound having a phenolic hydroxyl group. If the amount of the acidic catalyst is less than 0.1 mol times, the reaction rate may be significantly lowered, and the amount of the obtained fluorescein compound may be lowered. On the other hand, if it is used 20 mol times or more, a large amount of acidic waste is discharged, which makes acid treatment difficult and may not be a safe industrial production method.

A solvent that does not inhibit the reaction is selected as the reaction solvent used in the method for producing a fluorescein compound of the present invention, but a solvent containing a carboxylic acid having low solubility of the fluorescein compound is used for purification by crystallization. Use. The carbon number of the carboxylic acid is preferably 1 to 24, more preferably 1 to 9, and even more preferably 1 to 4. Specific examples of the carboxylic acid include for example, formic acid, acetic acid, propionic acid, butyric acid, isobutyric acid, pivalic acid, valeric acid, isovaleric acid, caproic acid, 2-ethylbutyric acid, 2-ethylhexanoic acid, capric acid and olein. Acids, lactic acids, citric acid, oxalic acid, octyl acid, naphthenic acid, neodecanoic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, ligrinoseric acid, cellotic acid, montanic acid, melicin Acids, toucic acid, lindelic acid, tsuzuic acid, maccoic acid, myristoleic acid, zomarinic acid, petroseric acid, baxenoic acid, gadrainic acid, whale oil acid, ercinic acid, shark oil acid, linoleic acid, hirago acid, eleo Examples thereof include stearate acid, bunica acid, tricosanoic acid, linolenic acid, moroctic acid, parinalic acid, arachidonic acid, sardine acid, hiragashiric acid, and heric acid. Of these, formic acid, acetic acid, propionic acid, butyric acid, and isobutyric acid are preferable, and acetic acid and propionic acid are more preferable. Further, one type of carboxylic acid may be used, or two or more types may be mixed and used. In this specification, the carboxylic acid does not contain an anhydrous carboxylic acid.

The reaction solvent containing a carboxylic acid may contain a solvent other than the carboxylic acid. The component of the other solvent to be mixed with the carboxylic acid is selected from commonly used solvents, but a solvent having an electron-withdrawing functional group that does not inhibit the synthetic reaction of the fluorescein compound is more preferable. Specifically, halogen-based solvents such as dichloromethane, dichloroethane and chloroform, nitro-based solvents such as nitromethane, nitroethane and nitrobenzene, nitrile-based solvents such as acetonitrile and benzonitrile, formaldehyde, acetaldehyde, propionaldehyde, butanal, pentanal and benzaldehyde and the like. Examples thereof include aldehyde-based solvents, ester-based solvents such as methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate and isobutyl acetate.

The content of the carboxylic acid is appropriately selected within a range that does not reduce the solubility of the fluorescein compound with respect to 100% by weight of the reaction solvent containing the carboxylic acid, but is preferably 1% by weight to 100% by weight, more preferably 30. It is from% by weight to 100% by weight, more preferably 50% by weight to 100% by weight. In the present invention, the reaction solvent containing the carboxylic acid may be composed only of the carboxylic acid.

The amount of the reaction solvent used in the method for producing a fluorescein compound is preferably 0.5 to 100 times by weight, more preferably 1 to 50 times by weight, still more preferably, as compared with the compound having a phenolic hydroxyl group. Is 1 to 20 times by weight.

The reaction temperature for producing the fluorescein compound is usually 40 to 150 ° C, preferably 50 to 130 ° C, and more preferably 70 to 120 ° C. 40 ° C. or higher is preferable from the viewpoint of reaction rate, and 150 ° C. or lower is preferable from the viewpoint of safe industrial production conditions.

The chemical purity of the fluorescein compound in the reaction solution obtained by the production method of the present invention is preferably 80% or more, more preferably 85% or more. The chemical purity of the fluorescein compound in the reaction solution is a fraction of the peak area of the fluorescein compound (HPLC area) as measured by HPLC.

In the present invention, the fluorescein compound can be (1) removed from unreacted raw materials, (2) removed from the reaction solvent, and (3) removed from the acidic catalyst by crystallization. In the present invention, fluorescein compounds are preferably isolated by crystallization. By isolating the fluorescein compound by crystallization, the fluorescein compound is purified, and the chemical purity of the obtained fluorescein compound is further improved. The crystallization includes cold crystallization, poor solvent crystallization, evaporation crystallization, non-solvent addition crystallization, etc., but in the present invention, cold crystallization and poor solvent crystallization, which are simple crystallization methods, are more preferable. ..

The cooling crystallization described in the present specification means a method of precipitating crystals of a fluorescein compound by cooling the reaction solution below the reaction temperature and utilizing the difference in solubility of the fluorescein compound depending on the solution temperature. do. Poor solvent crystallization is a method in which a solvent having a low solubility in a fluorescein compound is added to the reaction solution to reduce the solubility of the reaction solution to precipitate crystals of the fluorescein compound, or the reaction solution is insoluble in the fluorescein compound. It means a method of precipitating a fluorescein compound in addition to the solvent of the above, or a method of mixing a solvent having a high solubility of the fluorescein compound and a solvent having a low solubility and distilling off the solvent having a high solubility to precipitate the fluorescein compound.

The chemical purity of the fluorescein compound obtained by crystallization is preferably 95% or more, more preferably 98% or more.

Crystals of fluorescein compounds precipitated by crystallization can be separated from unreacted raw materials, acidic catalysts and reaction solvents by filtration. Further, it is preferable to completely remove the remaining unreacted raw materials, the acidic catalyst, and the reaction solvent by washing the obtained crystals.

Crystals of fluorescein compounds can be increased in chemical purity by filtering and then washing the slurry with a solvent. In the present specification, the slurry washing means a method in which a wet cake containing crystals obtained by filtration is transferred to a container such as a flask again, a solvent having a low solubility is added, and the mixture is stirred and washed. Therefore, it is a cleaning method different from the cleaning in which the wet cake on the funnel or the like is washed with a solvent.

As the solvent used for slurry cleaning, a protonic polar solvent can be exemplified. Moreover, examples of the protictic polar solvent used here include water, methanol, ethanol, 1-propanol, 2-propanol, acetic acid, propionic acid, ethylene glycol, glycerin and the like. Of these, water, methanol, ethanol, 1-propanol and 2-propanol are preferable, and water, methanol and 2-propanol are more preferable. Further, one type of protic and aprotic solvent may be used, or two or more types may be mixed and used.

Further, crystals of fluorescein compounds can be purified by filtering, dissolving in a solvent, and then crystallizing. Crystals of fluorescein compounds can be dissolved at room temperature or with a heated solvent. Examples of the solvent include an aprotic polar solvent and a protonic polar solvent.

The aprotonic polar solvent is, for example, N, N-dimethylacetamide, N, N-dimethylacetacetamide, N, N-diethylacetamide, N, N-dimethylformamide (DMF), 2-pyrrolidone, N-methyl-pyrrolidone. Amido compounds such as (NMP), hexamethylphosphoric triamide, 1,3-dimethyl-2-imidazolinozin; sulfoxide compounds such as dimethyl sulfoxide (DMSO) and diethyl sulfoxide; sulfolan compounds such as sulfolane and methyl sulfolane; acetonitrile , Nitrile compounds such as propionitrile; ether compounds such as monoglime, diglime, triglime, tetraglime, tetrahydrofuran (THF), 1,4-dioxane, 1,3-dioxolane; ketone compounds such as acetone, and the like. Of these, N-methylpyrrolidone, dimethylformamide, and dimethyl sulfoxide are preferable, and N-methylpyrrolidone is more preferably used.

Examples of the protonic polar solvent include water, methanol, ethanol, 1-propanol, 2-propanol, acetic acid, propionic acid, ethylene glycol, glycerin and the like. Of these, water, methanol, ethanol, 1-propanol and 2-propanol are preferable, and water, methanol and 2-propanol are more preferable. Further, one type of protic and aprotic solvent may be used, or two or more types may be mixed and used.

The temperature at which the crystals of the fluorescein compound are dissolved in the aprotic polar solvent and the protic polar solvent is preferably 20 to 100 ° C, more preferably 40 to 80 ° C, still more preferably 50 to 70 ° C. The amount of the aprotic polar solvent and the protic and aprotic polar solvent used is preferably 1 to 50 times by weight, more preferably 5 to 40 times by weight, still more preferably 10 to 30 times the weight of the fluorescein compound. It should be double the weight.

Next, the fluorescein compound dissolved in the aprotic polar solvent is preferably purified by antisolvent crystallization. Examples of the poor solvent include water, toluene and the like. The temperature of the poor solvent crystallization is preferably 5 to 80 ° C, more preferably 10 to 60 ° C, and even more preferably 10 to 35 ° C. The amount of the poor solvent used is preferably 0.1 to 20 times by weight, more preferably 0.1 to 10 times by weight, and even more preferably 0.1 to 5 times by weight with respect to the fluorescein compounds. It should be.

The chemical purity of the fluorescein compound obtained by purification with a solvent is preferably 95% or more, more preferably 98% or more. The upper limit of chemical purity is 100%. The chemical purity of the fluorescein compound is a fraction of the peak area of the fluorescein compound measured by HPLC (HPLC area%).

上記式において、ｘ、ｙ、ｚは、０～４の整数を表し、Ｒ^１、Ｒ^２、Ｒ^３は、それぞれ独立して、水素、水酸基、アルコキシ基、カルボキシル基、スルホン酸基、アミノ基、ハロゲン、ニトロ基及びアルキル基からなる群から選択される。ｘ、ｙ、ｚは、好ましくは、１～４の整数である。 The fluorescein compound obtained by the production method of the present invention is preferably a compound represented by the following formula.

In the above formula, x, y, and z represent integers from 0 to 4, and R ¹ , R ² , and R ³ are independently hydrogen, hydroxyl group, alkoxy group, carboxyl group, sulfonic acid group, and amino group, respectively. , Halogen, nitro group and alkyl group. x, y, and z are preferably integers of 1 to 4.

上記式（１）において、ｘ、ｙ、ｚは、０～４の整数を表し、Ｒ^１、Ｒ^２、Ｒ^３は、それぞれ独立して、置換基を有する縮合したベンゼン環又は無置換の縮合したベンゼン環である。ｘ、ｙ、ｚは、好ましくは、１～４の整数である。Ｒ^１、Ｒ^２は、好ましくは、水酸基を有する縮合したベンゼン環であり、より好ましくはｘ，ｙが１、ｚが０であるとよい。 The fluorescein compound obtained by the production method of the present invention is preferably a compound represented by the following formula (1).

In the above formula (1), x, y, and z represent integers of 0 to 4, and R ¹ , R ² , and R ³ are independently condensed benzene rings having substituents or unsubstituted condensation. It is a benzene ring. x, y, and z are preferably integers of 1 to 4. R ¹ and R ² are preferably condensed benzene rings having a hydroxyl group, and more preferably x and y are 1 and z is 0.

上記式（２）～（４）において、ａ、ｂ、ｃ、ｄ、ｅ、ｆは、１～４の整数を表し、Ｒ^６、Ｒ^７、Ｒ^８、Ｒ^９、Ｒ^１０、Ｒ^１１は、それぞれ独立して、水素、水酸基、アルコキシ基、カルボキシル基、スルホン酸基、アミノ基、ハロゲン、ニトロ基及びアルキル基からなる群から選択される。Ｒ^６、Ｒ^７、Ｒ^８、Ｒ^９、Ｒ^１０、Ｒ^１１は、水酸基であることがより好ましい。 The fluorescein compound obtained by the production method of the present invention is more preferably selected from the group of compounds represented by the following formulas (2) to (4). The fluorescein compound may be one kind of compound or a mixture of a plurality of kinds of compounds among the compounds represented by the following formulas (2) to (4).

In the above equations (2) to (4), a, b, c, d, e, and f represent integers of 1 to 4, and R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ are. , Each independently selected from the group consisting of hydrogen, hydroxyl group, alkoxy group, carboxyl group, sulfonic acid group, amino group, halogen, nitro group and alkyl group. It is more preferable that R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ , and R ¹¹ are hydroxyl groups.

The fluorescein compound obtained in the present invention is more preferably a compound selected from the group of compounds represented by the following formulas (5) to (7). The fluorescein compound may be one kind of compound or a mixture of a plurality of kinds of compounds among the compounds represented by the following formulas (5) to (7).

Hereinafter, a specific description will be given with reference to examples. The chemical purity of the fluorescein compounds obtained in the present specification was measured by the following method.
(Chemical purity)
The fraction of the peak area of the fluorescein compound (HPLC area%) was measured by liquid chromatography under the following conditions (LC-10Vp manufactured by Shimadzu Corporation) and used as the chemical purity.
Column: Mightysil RP18GP, 4.6 x 150 mm (5 μm)
Column temperature: 40 ° C
Mobile phase: (A) 5 mmol / L aqueous solution of tetrabutylammonium hydrogen sulfate
(B) Methanol
Gradient 0 minutes (A): (B) = 65:35
20 minutes (A): (B) = 35:65
22 minutes (A): (B) = 35:65
Flow rate: 1 mL / min
Injection volume: 5 μL
Detection: UV254nm
Analysis time: 30 minutes Sample preparation: 0.01 g of sample was weighed and diluted to about 25 mL of methanol.
However, it is not limited to these analysis conditions as long as the same results as the analysis results based on the above analysis conditions can be obtained.

(Example 1)
6.41 g (0.04 mol) of 1,6-dihydroxynaphthalene, 2.96 g (0.02 mol) of phthalic anhydride, and methanesulfonic acid in a 100 mL four-necked flask equipped with a thermometer, a cooling tube, and a stirrer. (MeSO _3H ) 15.4 g (0.16 mol) and 25 g of acetic acid were charged, and the inside of the system was replaced with nitrogen. The temperature of this mixed solution was raised to 120 ° C. with stirring, and a synthetic reaction of naphthofluorescein was carried out. After heating and stirring for 24 hours, naphthofluorescein in the reaction solution was analyzed by HPLC, and the chemical purity of naphthofluorescein was 86%.

The obtained reaction solution was cooled to 30-40 ° C. to precipitate naphthofluorescein crystals. The precipitated crystals were filtered, washed with a mixed solution of acetic acid and water, and then dried to obtain 6.6 g of naphthofluorescein as reddish purple crystals. The obtained naphthofluorescein had a chemical purity of 99.0% and a yield of 76%.

(Example 2)
In Example 1, the reaction was carried out in the same manner as in Example 1 except that 7.7 g (0.08 mol) of methanesulfonic acid and 32.7 g of acetic acid were used. The naphthofluorescein obtained by filtration, slurry washing and drying had a chemical purity of 99% and a yield of 58%.

(Example 3)
In Example 1, the reaction was carried out in the same manner as in Example 1 except that 3.8 g (0.04 mol) of methanesulfonic acid and 36.5 g of acetic acid were used. The naphthofluorescein obtained by filtration, slurry washing and drying had a chemical purity of 97% and a yield of 60%.

(Example 4)
In Example 1, the reaction was carried out in the same manner as in Example 1 except that the amount of acetic acid used was 12.5 g. The naphthofluorescein obtained by filtration, slurry washing and drying had a chemical purity of 98% and a yield of 70%.

(Example 5)
In Example 1, the reaction was carried out in the same manner as in Example 1 except that 15.6 g (0.16 mol) of sulfuric acid was used instead of methanesulfonic acid as an acidic catalyst. The naphthofluorescein obtained by filtration, slurry washing and drying had a chemical purity of 96% and a yield of 50%.

(Example 6)
The operation of filtering the precipitated crystals was carried out in the same manner as in Example 1. The filtered crystals were washed with 15.0 g (0.25 mol) of acetic acid to obtain a wet cake containing the crystals. A 100 mL four-necked flask equipped with a thermometer, a condenser and a stirrer was charged with 22.0 g (4.3 weight times / naphthofluorescein) of N-methylpyrrolidone (NMP) obtained by washing. The inside of the system was replaced with nitrogen. The temperature of this mixture was raised to 80 ° C. while stirring, and stirring was continued for 30 minutes. Then, the mixture was cooled to room temperature over 2 hours, and 5.2 g of water (1.0 weight times / naphthofluorescein) was added dropwise at 30 ° C. over 1 hour to precipitate naphthofluorescein in thin purple crystals. The crystals were filtered, washed with 2-propanol and dried to give 3.6 g of fluorescein as pale purple crystals. The obtained naphthofluorescein had a chemical purity of 99.7% and a yield of 50%.

(Example 7)
The operation of filtering the precipitated crystals was carried out in the same manner as in Example 1. The filtered crystals were washed with 15.0 g (0.25 mol) of acetic acid to obtain a wet cake containing the crystals. Wet cake obtained by washing in a 200 mL four-necked flask equipped with a thermometer, a condenser and a stirrer, 74.4 g of water (8.6 weight times / fluorescein), 111.6 g of 2-propanol (IPA). (12.9 weight times / fluorescein) was charged, and the inside of the system was replaced with nitrogen. The temperature of this mixture was raised to 60 ° C. while stirring, and stirring was continued for 30 minutes. After that, 2-propanol was distilled off at 60 ° C. and a reduced pressure of 250 torr for 3.7 hours, and fluorescein in red crystals was precipitated. The crystals were filtered, slurry washed with water, the filtered cake was washed with 2-propanol, and then dried to obtain 5.1 g of naphthofluorescein as red crystals. The obtained naphthofluorescein had a chemical purity of 99% and a yield of 59%.
The reaction conditions and evaluation results of Examples 1 to 7 are summarized in Table 1.

(Comparative Example 1)
In a 100 mL four-necked flask equipped with a thermometer, a condenser and a stirrer, 10.0 g (0.063 mol) of 1,6-dihydroxynaphthalene, 4.6 g (0.031 mol) of phthalic anhydride, and zinc chloride 4. 2 g (0.030 mol) was charged and the inside of the system was replaced with nitrogen. The temperature of this mixed solution was raised to 120 ° C. while stirring, and a fluorescein synthesis reaction was carried out, but the reaction was interrupted because the progress of the reaction could not be confirmed.

(Comparative Example 2)
4.77 g (0.03 mol) of 1,6-dihydroxynaphthalene, 2.22 g (0.015 mol) of phthalic anhydride, 57 of methanesulfonic acid in a 100 mL four-necked flask equipped with a thermometer, a condenser and a stirrer. .66 g (0.60 mol) was charged and the inside of the system was replaced with nitrogen. The temperature of this mixed solution was raised to 120 ° C. with stirring, and a synthetic reaction of naphthofluorescein was carried out. After heating and stirring for 13 hours, the reaction solution was analyzed by naphthofluorescein HPLC, and the chemical purity of fluorescein was 85%.

The obtained reaction solution was added dropwise to 70 g of pure water at 10 ° C. Since heat generation was observed at this time, it was slowly dropped to precipitate fluorescein crystals. The precipitated crystals were filtered, washed with pure water, and dried to obtain 5.6 g of a black powdered fluorescein compound. The obtained fluorescein compound had a chemical purity of 82% and a yield of 87%.

(Comparative Example 3)
The reaction was carried out in the same manner as in Example 1 except that methanesulfonic acid was not used in Example 1. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC. The formation of naphthofluorescein in the reaction solution could not be confirmed.

(Comparative Example 4)
In Example 1, the reaction was carried out in the same manner as in Example 1 except that toluene was used 7.8 times by weight of 1,6-dihydroxynaphthalene instead of acetic acid as the reaction solvent and the reaction temperature was set to 70 ° C. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the formation of naphthofluorescein in the reaction solution could not be confirmed.

(Comparative Example 5)
In Comparative Example 4, the reaction was carried out in the same manner as in Comparative Example 4 except that isopropyl alcohol (IPA) was used instead of toluene as the reaction solvent. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the formation of naphthofluorescein in the reaction solution could not be confirmed.

(Comparative Example 6)
In Comparative Example 4, the reaction was carried out in the same manner as in Comparative Example 4 except that tetrahydrofuran (THF) was used instead of toluene as the reaction solvent. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the formation of naphthofluorescein in the reaction solution could not be confirmed.

(Comparative Example 7)
In Comparative Example 4, the reaction was carried out in the same manner as in Comparative Example 4 except that isopropyl acetate was used instead of toluene as the reaction solvent. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the naphthofluorescein in the reaction solution was 4%, which did not lead to isolation.

(Comparative Example 8)
In Comparative Example 4, the reaction was carried out in the same manner as in Comparative Example 4 except that acetonitrile was used instead of toluene as the reaction solvent. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the formation of naphthofluorescein in the reaction solution could not be confirmed.

(Comparative Example 9)
In Comparative Example 4, the reaction was carried out in the same manner as in Comparative Example 4 except that acetic anhydride was used instead of toluene as the reaction solvent. After heating and stirring for 24 hours, the reaction solution was analyzed by HPLC, but the formation of naphthofluorescein in the reaction solution could not be confirmed.
The reaction conditions and evaluation results of Comparative Examples 1 to 9 are summarized in Table 2.

Claims (3)

A method for producing a fluorescein compound by reacting a compound having a phenolic hydroxyl group with an acid anhydride compound in the presence of an acidic catalyst, wherein the compound having a phenolic hydroxyl group is 1,6-dihydroxynaphthalene, and the reaction solvent is The step of carrying out the reaction using a solvent containing acetic acid , the step of isolating the fluorescein compounds from the reaction solution of the compound having a phenolic hydroxyl group and the acid anhydride compound by cold crystallization, the isolated fluoresceins. The following formula (5), which comprises a step of dissolving the compound in an aprotonic solvent and crystallizing it in a poor solvent, or purifying the compound by washing the slurry with a protonic polar solvent, having a chemical purity of 95% or more. ), A method for producing a high-purity fluorescein compound.

The method for producing a fluorescein compound according to claim 1, wherein the chemical purity is 98% or more. The method for producing a fluorescein compound according to claim 1 or 2 , wherein the acidic catalyst is a protonic acid.