JP6675125B2 - pH-dependent fluorescent compound - Google Patents

Description

  The present invention relates to a compound that emits fluorescence in a pH-dependent manner and a precursor compound thereof.

Fluorescent dyes are widely used from basic research to clinical examination as an alternative to conventional tracer technology using radioactive substances because of their high sensitivity and safe handling. Many fluorescent dyes have been discovered so far, but these are roughly classified into two types based on their functions: an “Always ON” type (Patent Document 1) and an “ON / OFF Switch” type (Non-Patent Document 1). You.
Always ON type is a dye that is hardly affected by environmental changes and always emits constant fluorescence. By labeling probes that recognize target molecules such as nucleic acids, proteins, antibodies, and small molecule ligands, tracking of target molecules can be performed. to enable. On the other hand, a probe that has caused non-specific binding or a free probe that does not bind to a target also emits fluorescence, so that the background signal is likely to be high, and its use in vivo requiring a large amount of administration is limited.
The ON / OFF Switch type is a dye whose light emission and quenching are regulated by the concentration gradient of a target molecule, and a typical example is a dye sensitive to ion concentration such as H ⁺ ion (pH) and Ca ²⁺ ion. However, these dyes have a threshold in the concentration of the measurement target, and light emission / quenching is adjusted above and below the threshold. Therefore, it is impossible to measure a target molecule having a reference concentration range like a normal biological component. Dyes targeting enzymes such as β-galactosidase also fall into this category, but their use is limited due to their irreversible fluorescence emission / quenching.

Also, recent studies have shown that cancer cells are more acidic than normal cells. This is thought to be due to abnormally faster metabolism of cancer cells than normal cells, improvement in ATP production ability, increase in expression of H ⁺ pumps and renewal of functions. That is, it does not give fluorescence at around pH 7.4, which is the pH of normal cells and blood, and around 1.5 to 1.0, which is the pH of the stomach occupying the center of the upper body and occupying a large volume. There is a demand for a fluorescent dye that provides an extremely unique property of emitting fluorescence only in a weakly acidic region.

JP 2012-219258 A

Angew. Chem. Int. Ed. 2014, 53, 6085-6089

  An object of the present invention is to provide a compound that emits fluorescence in a pH-dependent manner and a precursor compound thereof.

As a result of intensive studies to solve the above problems, the present inventors have found a compound that emits strong fluorescence only in a specific pH range and a precursor compound thereof, and have completed the present invention. That is, the present invention is as follows.
[1] Formula (I ′):

(In the formula, R represents a hydrogen atom or an acyl group, and R ¹ and R ² each independently represent a hydrogen atom, an amino group, or a carboxyl group.)
(Hereinafter may be abbreviated as compound (I '));
[2] the compound of [1], wherein R is a hydrogen atom;
[3] the compound of [1], wherein R is acetyl;
[4] the compound of any one of [1] to [3], wherein R ¹ and R ² are both hydrogen atoms;
[5] The compound according to any one of [1] to [3], wherein R ¹ is an amino group and R ² is a hydrogen atom;
[6] The compound according to any one of [1] to [3], wherein R ¹ is a carboxyl group and R ² is a hydrogen atom.

Further, the present invention relates to the following.
[1A] Formula (I):

(In the formula, R represents a hydrogen atom or an acyl group.)
(Hereinafter may be abbreviated as compound (I));
[2A] the compound of [1A], wherein R is a hydrogen atom;
[3A] The compound of [1A], wherein R is acetyl.

The compound of the present invention is useful as a fluorescent compound which can be said to be a third type and a precursor compound thereof, which emits fluorescence only when the concentration of the measurement object is within a specific region. The measurement target is pH (H ⁺ ion). When the compound of the present invention is dissolved in water, no fluorescence is emitted in the range of strong basicity of pH 14 to neutrality of pH 7, but when the pH is lowered to 6 or less, the pH gradually decreases. And gives maximum fluorescence at around pH 4 (when an amino group is introduced, the pH giving the maximum fluorescence intensity shifts to a neutral region (6.5 to 7.0)). When the pH is further lowered, the fluorescence gradually decreases, and when the pH becomes 2 or less, the solution becomes non-fluorescent. Furthermore, the compound of the present invention is activated in a weakly acidic region, and can be applied to a technique for distinguishing between a weakly acidic region and a neutral region in a cell or tissue, or a weakly acidic region and a strongly acidic region. Conceivable.

2 μM solution of compound (I) (R = hydrogen atom) obtained in Example 1 (solvent: data of pH 1 is 0.1 M hydrochloric acid, pH 0.5 to pH 12.0 in increments of 0.5) The data shows the results obtained by irradiating a 25 mM phosphate buffer, pH 14 data with a 1 M aqueous sodium hydroxide solution) with 315 nm excitation light, and photographing the emitted fluorescence with a digital camera. 1 μM solution of compound (I) (R = hydrogen atom) obtained in Example 1 (solvent: data of pH 1 is 0.1 M hydrochloric acid, pH 2.0, 2.5, 7.0, 7.5, The data at 8.0, 9.0, 10.0 and pH 12.0 are 25 mM phosphate buffer, and the data at pH 0.1 from pH 3.0 to pH 6.5 are 50 mM phthalate buffer, pH 0.1. Data No. 14 shows the fluorescence intensity of a 1 M aqueous sodium hydroxide solution) at an excitation wavelength of 488 nm and a fluorescence wavelength of 528 nm with a change in pH. 1 shows an excitation / fluorescence spectrum of a 1 μM solution (solvent: 50 mM phthalate buffer) of compound (I) (R = hydrogen atom) obtained in Example 1 at pH 4.5. FIG. 2 shows a graph of pH and fluorescence intensity of a 1 μM solution (solvent: 50 mM phthalate buffer) of compound (I) (R = hydrogen atom) obtained in Example 1. FIG. FIG. 2 is an ORTEP diagram showing the molecular structure of compound (I) (R = acetyl) obtained in Example 2. The result of observing the cell staining using the compound (I) (R = acetyl) obtained in Example 2 with a fluorescence microscope is shown. The result of observing the multiple staining of the cell using the compound (I) (R = acetyl) obtained in Example 2, Hoechst 33342 and Acidifluor Orange by a confocal laser fluorescence microscope is shown. The result of observing the double staining of the cells using the compound (I) (R = acetyl) obtained in Example 2 and Acidifluor Orange using an inverted fluorescence microscope is shown. 5 μM solution of the hydrochloride salt of the compound (I ′) (R = hydrogen atom, R ¹ = amino group, R ² = hydrogen atom) obtained in Example 3 (data of a solvent: pH 1 is 0.1 M hydrochloric acid, pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8. The data at 0, 8.5, 9.0, 9.5 and 12.0 are 25 mM phosphate buffer, and the data at pH 14 is 0.1 M aqueous sodium hydroxide solution) at an excitation wavelength of 484 nm and an emission wavelength of 534 nm. 4 shows the fluorescence intensity accompanying a pH change. 1 μM solution of compound (I ′) (R = hydrogen atom, R ¹ = carboxyl group, R ² = hydrogen atom) obtained in Example 4 (solvent: pH 1, data is 0.1 M hydrochloric acid, pH 2.0 , 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10 0.0, 11.0 and 12.0 are 25 mM phosphate buffer, pH 14 data is 0.1 M aqueous sodium hydroxide solution). Show.

Hereinafter, embodiments of the present invention will be described in detail.
The compound of the present invention has the formula (I ′):

(In the formula, R represents a hydrogen atom or an acyl group, and R ¹ and R ² each independently represent a hydrogen atom, an amino group, or a carboxyl group.)
It is represented by
Also, the compound of the present invention has the formula (I):

(In the formula, R represents a hydrogen atom or an acyl group.)
It is represented by
Examples of the “acyl group” represented by R include a C _1-6 alkyl-carbonyl group (eg, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2-dimethylpropanoyl, hexanoyl, heptanoyl), C _6-14 aryl-carbonyl group (eg, benzoyl, 1-naphthoyl, 2-naphthoyl), C _7-16 aralkyl-carbonyl group (eg, phenylacetyl, phenylpropionyl) And C _1-6 alkyl-carbonyl groups (eg, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl, Hexanoyl and heptanoyl) are preferable, and acetyl is more preferable. Is more preferable.
R represents a hydrogen atom or a C _1-6 alkyl-carbonyl group (eg, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl, 2-methylbutanoyl, 2,2-dimethylpropanoyl) Noyl, hexanoyl, heptanoyl) are preferable, and a hydrogen atom or acetyl is more preferable.
As R ¹ and R ² , an embodiment in which R ¹ and R ² are both hydrogen atoms, an embodiment in which R ¹ is an amino group and R ² is a hydrogen atom, and an embodiment in which R ¹ is a carboxyl group and R ^{1 An} embodiment in which ² is a hydrogen atom is preferred.

Next, a method for producing the compound of the present invention will be described.
The compound (I ′) of the present invention is obtained by using the compound (1 ′) as a starting material.

(Wherein R ¹ and R ² are as defined above)
Can be produced in the same manner as in the method for producing the compound (I) of the present invention described below or a method analogous thereto, or by the method described in Examples.

The compound (I ′) in which R ¹ and / or R ² is an amino group is a compound (1 ′) in which the amino group is protected by a protective group known in the art, and the obtained amino group is protected. The compound (I ′) obtained by deprotecting the compound (I ′) by a method known per se or using a compound in which R ¹ and / or R ² is a nitro group as a precursor of the compound (1 ′), ^{by 1} and / or R ² is reduced by a method known per se a compound that is a nitro group (I '), it can also be produced.

  In addition, compound (I) of the present invention can be produced by the method shown in the following scheme, a method analogous thereto, or a method described in Examples.

(Step 1)
1,2,3,4-benzenetetracarboxylic acid (mellophanic acid) (1) is heated, if necessary, in a solvent inert to the reaction or in a powder state to obtain an acid anhydride (2). It is a process.
1,2,3,4-benzenetetracarboxylic acid (mellophanic acid) (1) is available as a commercial product, and can also be produced according to a method known per se or a method analogous thereto.
Examples of the "solvent" include ethers such as diethyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane, hydrocarbons such as benzene, toluene, cyclohexane and hexane, N, N-dimethylformamide, N, N Amides such as -dimethylacetamide and 1-methyl-2-pyrrolidone; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane; nitriles such as acetonitrile and propionitrile; dimethylsulfoxide and the like Sulfoxides, nitrogen-containing aromatic hydrocarbons such as pyridine, lutidine, and quinoline, acid anhydrides such as acetic anhydride, and solvents such as water or a mixed solvent thereof are preferable.
The reaction temperature varies depending on the type of the solvent, but is generally 0 to 180 ° C, preferably 140 to 150 ° C, and the reaction time is generally 1 to 48 hours, preferably 6 to 24 hours.

(Step 2)
This is a step of reacting the acid anhydride (2) with the resorcinol (3) in the presence of an acid, if necessary, in a solvent inert to the reaction to obtain a compound (I) (R = hydrogen atom).
The amount of resorcinol (3) to be used is generally 2 to 8 equivalents, preferably 3 to 5 equivalents, relative to acid anhydride (2).
Examples of the “acid” include mineral acids (hydrochloric acid, hydrobromic acid, sulfuric acid, etc.), carboxylic acids (acetic acid, trifluoroacetic acid, trichloroacetic acid, etc.), sulfonic acids (methanesulfonic acid, toluenesulfonic acid, etc.), A Lewis acid (aluminum chloride, tin chloride, zinc bromide, etc.) is used, and two or more kinds may be mixed as necessary.
The amount of the “acid” to be used is generally 4-800 equivalents, preferably 40-80 equivalents, relative to the acid anhydride (2), and can be used as a solvent.
Examples of the "solvent" include ethers such as diethyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane, hydrocarbons such as benzene, toluene, cyclohexane and hexane, N, N-dimethylformamide, N, N Amides such as -dimethylacetamide and 1-methyl-2-pyrrolidone; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane; nitriles such as acetonitrile and propionitrile; dimethylsulfoxide and the like Sulfoxides, nitrogen-containing aromatic hydrocarbons such as pyridine, lutidine and quinoline, and solvents such as water, or a mixed solvent thereof are preferred.
The reaction temperature varies depending on the type of the acid and the solvent, but is usually 0 to 120 ° C, preferably 80 to 100 ° C, and the reaction time is usually 1 to 120 hours, preferably 24 to 72 hours.

(Step 3)
In this step, the hydroxyl group of compound (I) (R = hydrogen atom) is acylated using an acylating agent in a solvent inert to the reaction, if necessary, to obtain compound (I) (R = acyl group). .
As the "acylating agent", for example, carboxylic acid halides, carboxylic anhydrides and the like are used.
The amount of the "acylating agent" to be used is generally 4 to 800 equivalents, preferably 4 to 40 equivalents, relative to compound (I) (R = hydrogen atom), and can be used as a solvent.
Examples of the "solvent" include ethers such as diethyl ether, tetrahydrofuran, dioxane and 1,2-dimethoxyethane, hydrocarbons such as benzene, toluene, cyclohexane and hexane, N, N-dimethylformamide, N, N Amides such as -dimethylacetamide and 1-methyl-2-pyrrolidone; halogenated hydrocarbons such as dichloromethane, chloroform, carbon tetrachloride and 1,2-dichloroethane; nitriles such as acetonitrile and propionitrile; dimethylsulfoxide and the like Sulfoxides, nitrogen-containing aromatic hydrocarbons such as pyridine, lutidine and quinoline, and solvents such as water, or a mixed solvent thereof are preferred.
The reaction temperature varies depending on the type of the acylating agent and the solvent, but is usually 0 to 180 ° C, preferably 20 to 140 ° C, and the reaction time is usually 1 to 24 hours, preferably 1 to 3 hours. .

The compounds (I ′) and (I) (R = hydrogen atom) of the present invention emit strong fluorescence only in a weakly acidic region (pH 3.0 to 6.5), and strongly acidic region (pH 1.0 to 2). .5) and in the neutral to strongly basic region (pH 7.0 to 14.0), only weak fluorescence is emitted.
The compounds (I ′) and (I) (R = acyl group) of the present invention are precursor compounds of the compounds (I ′) and (I) (R = hydrogen atom), and themselves emit fluorescence. Instead, the compound is deacylated and converted into the compounds (I ′) and (I) (R = hydrogen atom), thereby emitting strong fluorescence only in the weakly acidic region as described above.

  Compounds (I ′) and (I) (R = hydrogen atom) of the present invention can be converted into compounds (I ′) and (I) (R = acyl group) by acylating the hydroxyl group. Thereby, the lipophilicity is improved, and it is easy to permeate the cell membrane and be taken into the cells. Compounds (I ′) and (I) (R = acyl group) taken up into cells are deacylated by an enzyme (esterase) present in the cytoplasm, and compounds (I ′) and (I) (R = hydrogen) Atom), and emits strong fluorescence in a weakly acidic region.

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
The following measuring instruments were used for various measurements.
Superconducting nuclear magnetic resonance apparatus Varian UNITYplus500
Mass spectrometer JMS-700 manufactured by JEOL
High Brightness X-ray Single Crystal Structure Analyzer Varimax Saturn / 1200S manufactured by RIGAKU
UV-Visible Spectrophotometer JASCO V-630BIO
Spectrofluorimeter JASCO FP-8200
Inverted fluorescence microscope Leica DM IRB
Confocal Laser Microscope LSM710 manufactured by Carl Zeiss

Example 1
(1) Synthesis of Compound (I) (R = hydrogen atom) 4.2 g of melophanoic acid was dissolved in 100 mL of tetrahydrofuran, and the solvent was distilled off under reduced pressure while keeping in an ice bath. The obtained powder was heated at 140 ° C. for 24 hours. After returning to room temperature under reduced pressure, 7.3 g of resorcinol and 66 mL of methanesulfonic acid were added, and the mixture was stirred at 80 ° C. for 3 days. After the temperature of the reaction solution was returned to room temperature, it was gradually added to 700 mL of ice-cooled water while stirring. The precipitated solid was collected by filtration, washed with water, and freeze-dried to obtain 12.4 g of a dark red powder. This powder was suspended in a small amount of methanol and heated under reflux with stirring. After returning to room temperature and collecting the powder by filtration, the obtained powder was further recrystallized from methanol to obtain 7.2 g (yield 74%) of the target compound as an orange powder. The analysis results of the synthesized compound (I) (R = hydrogen atom) are shown below.
¹ H-NMR (500 MHz, DMSO-d6) δ 8.34 (d, 1H, J = 8.1 Hz), 7.63 (d, 1H, J = 8.1 Hz), 6.82 (d, 2H, J = 8.6 Hz), 6.695 (d, 2H, J = 2.4 Hz), 6.687 (d, 2H, J = 2.4 Hz), 6.59 (dd, 2H, J = 2.4, 8.6 Hz), 6.57 (dd, 2H, J = 2.4, 8.6 Hz) , 6.43 (d, 2H, J = 8.6 Hz); ¹³ C-NMR (126 MHz, DMSO-d6) δ 167.3, 164.5, 160.0, 159.7, 152.0, 151.9, 149.7, 132.0, 128.8, 128.6, 127.0, 121.2, 112.9, 112.6, 108.6, 102.5, 102.3; FAB-HRMS (m / z) calcd for C ₃₄ H ₁₉ O ₁₀ : 587.0973; found: 587.0981 [M + H] ⁺

(2) Fluorescent Property The fluorescent property of the compound (I) obtained in the above (1) was evaluated.
2 μM solution of compound (I) (solvent: data of pH 1 is 0.1 M hydrochloric acid, data from pH 2.0 to pH 12.0 in increments of 0.5 is 25 mM phosphate buffer, data of pH 14 is (1M aqueous sodium hydroxide solution) was irradiated with 315 nm excitation light, and the emitted fluorescence was photographed with a digital camera. The result is shown in FIG. From FIG. 1, it can be seen that no fluorescence was emitted in the strongly acidic region and the neutral to strongly basic region, and fluorescence was emitted only in the weakly acidic region.
In addition, a 1 μM solution of compound (I) (solvent: data of pH 1 is 0.1 M hydrochloric acid, pH 2.0, 2.5, 7.0, 7.5, 8.0, 9.0, 10.0 And data at pH 12.0 are for 25 mM phosphate buffer, data for pH 3.0 to pH 6.5 in 0.1 increments are for 50 mM phthalate buffer, and data for pH 14 are for 1 M aqueous sodium hydroxide. FIG. 2 shows the fluorescence intensity accompanying the pH change at the excitation wavelength of 488 nm and the fluorescence wavelength of 528 nm. FIG. 2 shows that the maximum fluorescence intensity was obtained at pH 4.2 to 4.5.
FIG. 3 shows an excitation / fluorescence spectrum of a 1 μM solution of the compound (I) (solvent: 50 mM phthalate buffer) at pH 4.5. FIG. 3 shows that the absorption maxima are 460 nm and 484 nm. Since these absorption maxima almost coincide with the excitation light (458 nm and 488 nm) of the Ar laser, the compound (I) is considered to be optimal for fluorescence observation of cells and tissues using a laser fluorescence microscope.
Further, FIG. 4 shows a graph of the pH and the fluorescence intensity of a 1 μM solution of the compound (I) (solvent: 50 mM phthalate buffer). FIG. 4 shows that a good linear relationship was obtained between the pH change and the fluorescence intensity between pH 4.5 and 6.0. From this, it is considered that compound (I) can be applied as an intracellular pH sensor.

Example 2
(1) Synthesis of compound (I) (R = acetyl) A mixture of 1.17 g of compound (I) (R = hydrogen) and 8 mL of acetic anhydride was stirred at 100 ° C. for 20 hours. After the temperature of the reaction solution was returned to room temperature, it was poured into a mixed solution of 50 mL of dichloromethane and 200 mL of a saturated aqueous solution of sodium hydrogen carbonate with vigorous stirring. When the generation of gas disappeared, the solution was transferred to a separating funnel, and after removing the aqueous layer, the organic layer was washed twice with water and once with saturated saline. The obtained organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was purified by silica gel column chromatography (dichloromethane / ethyl acetate = 195/5 to 95/5). The fraction containing the target compound was collected, the solvent was distilled off to a small amount under reduced pressure, ethyl acetate was added, and the mixture was cooled with ice. The precipitated powder was collected by filtration, washed with ice-cooled ethyl acetate, and dried under reduced pressure to obtain 1.14 g (yield 76%) of the target compound as a white powder. The analysis results of the synthesized compound (I) (R = acetyl) are shown below.
¹ H-NMR (400 MHz, DMSO-d6) δ 8.48 (d, 1H, J = 8.1 Hz), 7.91 (d, 1H, J = 8.1 Hz), 7.32 (d, 2H, J = 2.4 Hz), 7.31 (d, 2H, J = 2.4 Hz), 7.24 (d, 2H, J = 8.7 Hz), 6.99 (dd, 2H, J = 2.4, 8.7 Hz), 6.97 (dd, 2H, J = 2.4, 8.7 Hz) , 6.79 (d, 2H, J = 8.7 Hz), 2.29 (s, 6H), 2.28 (s, 6H); ¹³ C-NMR (126 MHz, DMSO-d6) δ 168.7, 166.8, 164.3, 158.4, 152.4, 152.2, 151.0, 150.8, 149.0, 132.9, 129.0, 128.8, 127.7, 120.7, 118.8, 118.3, 115.0, 114.8, 110.7, 110.3, 82.8, 81.1, 20.9, 20.8; FAB-HRMS (m / z) calcd for C ₄₂ H ₂₇ O ₁₄ : 755.1395; found: 755.1400 [M + H] ⁺

(2) Molecular Structure The molecular structure of the compound (I) (R = acetyl) obtained in the above (1) was determined by single crystal X-ray structure analysis. Tables 1 to 3 show crystallographic data, diffraction X-ray intensity measurement data, and precise structural analysis data of the compound (I) (R = acetyl) .CH ₃ CO ₂ C ₂ H ₅ .

  The molecular structure of compound (I) (R = acetyl) is shown in the ORTEP diagram of FIG. FIG. 5 shows that the bonding position of the two xanthene skeletons is at the meta position.

(3) Cell staining Cell staining was performed using the compound (I) (R = acetyl) obtained in the above (1).
HeLa cells were seeded on a cell culture dish (35 mm diameter), and 89% (v / v) Dulbecco's modified Eagle medium, 10% (v / v) fetal bovine serum, and 1% (v / v) penicillin-streptomycin-amphotericin The cells were cultured for 3 to 4 days at 37 ° C. in a 5% CO ₂ atmosphere in 2 mL of a cell culture medium (hereinafter referred to as a cell culture solution) composed of the B mixed solution. After washing the subconfluent HeLa cells with a phosphate buffered saline, 2 mL of a cell culture solution containing 5 μM of the compound (I) (R = acetyl) is added, and the cells are incubated at 37 ° C. in a 5% CO ₂ atmosphere. After culturing for 24 hours, the cells were observed with an inverted fluorescence microscope using a blue filter.
FIG. 6 shows the results of observing the stained cells with a fluorescence microscope. Compound (I) (R = acetyl) that has permeated the cell membrane is deacetylated by esterase present in the cytoplasm, and is converted to compound (I) (R = hydrogen atom). No fluorescence was observed in the cytoplasm where the pH was neutral (〜7.4), and strong fluorescence was observed only in the lysosome (around pH 5), which is an acidic organelle.
In addition, multiple staining of cells was performed using the compound (I) (R = acetyl) obtained in the above (1).
HeLa cells were seeded on a glass bottom dish (35 mm in diameter), and cultured in 2 mL of a cell culture solution at 37 ° C. in a 5% CO ₂ atmosphere for 3 to 4 days. After washing the subconfluent HeLa cells with phosphate buffered saline, 2 mL of a cell culture solution containing 1 μM of compound (I) (R = acetyl), 1 μM of Hoechst 33342, and 1 μM of Acidifluor Orange is added. After culturing at 37 ° C. for 24 hours in a 5% CO ₂ atmosphere, the cells were observed by exciting with an argon laser (405 nm, 488 nm and 543 nm) using a confocal laser microscope.
Hoechst 33342 (1 μM dimethyl sulfoxide solution) that stains the cell nucleus blue, Acidifluor Orange (1 μM aqueous solution) that stains lysosomes orange, and compound (I) (R = acetyl) (1 μM dimethyl sulfoxide solution) were added to Hela cells. After culturing at 37 ° C. for 24 hours, the cells were observed with a confocal laser fluorescence microscope (wavelengths: 405 nm, 488 nm and 543 nm). FIG. 7 shows the result. From FIG. 7, it can be seen that compound (I) (R = acetyl) emits strong fluorescence in lysosomes and is applicable to multiple staining of cells.
In addition, cell staining was performed under strongly acidic conditions using the compound (I) (R = acetyl) obtained in the above (1).
HeLa cells were seeded on a glass bottom dish (35 mm in diameter), and cultured in 2 mL of a cell culture solution at 37 ° C. in a 5% CO ₂ atmosphere for 3 to 4 days. After washing the subconfluent HeLa cells with phosphate buffered saline, 2 mL of a cell culture solution containing 25 μM of compound (I) (R = acetyl) and 5 μM of Acidifluor Orange was added, and 5% CO ₂ was added. The cells were cultured at 37 ° C. for 24 hours in an atmosphere. After acquiring an image with an inverted fluorescence microscope using a blue filter and a green filter, 1 mL of 0.2 M hydrochloric acid was added to the glass bottom dish, and immediately observed using the blue filter and the green filter.
FIG. 8 shows the result. As shown in FIG. 8, the compound (I) (R = acetyl), although HeLa cells began to detach from the dish under the influence of a strong acid, maintained fluorescence derived from lysosomes in the cells, and stained cells even under strongly acidic conditions. Has been shown to be possible. On the other hand, Acidifluor Orange exhibits a property as a general acid-sensitive dye in which the fluorescence intensity increases from around neutral to around pH 3 and maintains the maximum fluorescence at pH 3 or less. The dye dissolved in the cell emits light and cells cannot be observed.

Example 3
(1) Synthesis of hydrochloride of compound (I ′) (R = hydrogen atom, R ¹ = amino group, R ² = hydrogen atom) (A) 1,4-dimethyl-7-oxabicyclo [2,2,1 Synthesis of hepta-5-ene-2,3-dicarboxylic anhydride (1):

  To a solution of 2,5-dimethylfuran (12.7 g) in diethyl ether (13 mL), maleic anhydride (12.9 g) was added with stirring under a nitrogen atmosphere. After the maleic anhydride was completely dissolved, the reaction solution was further stirred for 3 hours. The reaction solution was cooled in an ice bath, and the deposited precipitate was collected by filtration, washed with ice-cooled diethyl ether, and dried in vacuo to give the target compound 1 as white to pale yellow needle crystals (15.3 g, yield 66%).

  (B) Synthesis of 3,6-dimethylphthalic anhydride (2):

  Compound 1 (15.3 g) was dissolved little by little in ice-cooled concentrated sulfuric acid (56.4 ml) such that the temperature of concentrated sulfuric acid did not exceed 10 ° C. After complete addition of Compound 1, the reaction solution was further stirred under ice cooling for 2 hours. The reaction solution was poured into 300 g of ice, and the deposited precipitate was collected by filtration, washed with ice-cooled water, and lyophilized to give Target Compound 2 as a pale yellow powder (7.7 g, yield 33%). Obtained.

  (C) Synthesis of 3,6-dimethyl-4-nitrophthalic anhydride (3):

Fuming nitric acid (7.4 mL) was added dropwise to an ice-cooled solution of Compound 2 (7.7 g) in concentrated sulfuric acid (40 mL) such that the temperature of concentrated sulfuric acid did not exceed 10 ° C. The reaction solution was poured into 150 g of ice, and the deposited precipitate was collected by filtration, washed with ice-cooled water, and lyophilized to give the target compound 3 as a pale yellow powder (8.6 g, yield 89%). Obtained.
¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.06 (s, 1H), 2.85 (s, 3H), 2.79 (s, 3H)
FAB-HRMS (m / z): calcd for C ₁₀ H ₈ NO ₅ : 222.0397; found: 222.0402 [M + H] ⁺

  (D) Synthesis of nitromellophanic acid (4):

A solution of potassium permanganate (66.9 g) in water (250 mL) was heated to 80 ° C., and compound 3 was added little by little with stirring, and the reaction was continued at 80 ° C. One day later, potassium permanganate (11.2 g) was added, water was added until the total amount became 400 mL, and the reaction was continued at 80 ° C. One day later, potassium permanganate (11.2 g) was further added, water was added until the total amount became 400 mL, and the reaction was continued at 80 ° C. Three days after the start of the reaction, the reaction solution was cooled, methanol (20 mL) was added, and the mixture was stirred for 3 hours. The precipitated black precipitate was removed by filtration, concentrated hydrochloric acid was added to the filtrate until foaming stopped, and the mixture was heated under reflux for 1 hour. After water was distilled off under reduced pressure, it was freeze-dried. The residue was heated to reflux with 100 mL of acetic acid and the supernatant was carefully separated while hot. This extraction operation was repeated three times. The supernatant was combined, the solvent was distilled off under reduced pressure, and the residue was extracted with 50 mL of tetrahydrofuran twice after vacuum drying. The extracts were combined, the solvent was distilled off under reduced pressure, and the residue was dried under vacuum. Dichloromethane was added to the residue, the suspension was stirred at room temperature for 1 day, the precipitate was collected by filtration, washed with dichloromethane, and dried in vacuo to give the target compound 4 as a white powder (10.2 g, yield 48%). As obtained.
¹ H-NMR (400 MHz, DMSO-d6) δ 8.46 (s)
FAB-HRMS (m / z): calcd for C ₁₀ H ₄ NO ₁₀ : 297.9841; found: 297.9839 [MH] ^-

  (E) Synthesis of compound 6:

Compound 4 (3.0 g) and resorcinol (4.4 g) were added to methanesulfonic acid (26 mL), and the mixture was stirred at 90 ° C. for 2 days. The reaction solution was poured into 250 mL of cold water, and the resulting precipitate was collected by filtration, washed with cold water, and freeze-dried to obtain a crude product of Compound 5 as an orange powder (1.74 g). This was heated to reflux with acetic anhydride (20 mL) for 1 day. After evaporating the solvent of the reaction solution under reduced pressure, the residue was purified twice by silica gel column chromatography (first time: dichloromethane / ethyl acetate = 98/2 → 95/5, second time; dichloromethane / ethyl acetate = 98) / 2 → 96/4) to obtain the target compound 6 as a pale yellow powder (1.71 g, yield: 21%).
¹ H-NMR (400 MHz, DMSO-d6) δ 9.23 (s, 1H), 7.41 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 2.4 Hz, 2H), 7.31 (d, J = 2.4 Hz, 2H), 7.02 (dd, J = 2.4, 8.8 Hz, 2H), 6.95 (dd, J = 2.4, 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 2.29 (s, 6H), 2.27 (s, 6H)
FAB-HRMS (m / z): calcd for C ₄₂ H ₂₆ NO ₁₆ : 800.1246; found: 800.1242 [M + H] ⁺

  (F) Synthesis of compound 5:

To a suspension of compound 6 (568 mg) in methanol (14 mL) was added a 1 M aqueous sodium hydroxide solution (14 mL), and the mixture was stirred at 50 ° C for 1 day. Only the methanol fraction of the reaction solution was distilled off using an evaporator, and 5M hydrochloric acid was added to the remaining solution. The resulting precipitate was collected on ice and collected by filtration, washed with ice-cooled water, and lyophilized to give the target compound 5 as an orange powder (440 mg, yield 98%).
¹ H-NMR (400 MHz, DMSO-d6) δ 10.19 (s, 2H), 10.18 (s, 2H), 9.03 (s, 1H), 6.99 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 2.4 Hz, 2H), 6.67 (s, 2H), 6.57 (dd, J = 2.4, 8.8 Hz, 2H), 6.49 (s, 4H)
FAB-HRMS (m / z): calcd for C ₃₄ H ₁₈ NO ₁₂ : 632.0824; found: 632.0814 [M + H] ⁺

  (G) Synthesis of hydrochloride salt of compound 7:

Compound 5 (950 mg) and sodium hydrosulfide (841 mg) were sequentially added to a solution of sodium sulfide 9-hydrate (2.0 g) in water (30 mL), and the mixture was stirred at room temperature until all the reagents were dissolved. Stirred at C for 1 day. Sodium hydrosulfide (120 mg) was added, and the mixture was further stirred at 100 ° C. for one day. 5 M hydrochloric acid was added to the reaction solution, and the mixture was heated under reflux for 3 hours. The solvent of the reaction solution was distilled off under reduced pressure and freeze-dried. A small amount of methanol was added to the residue, the mixture was cooled with ice while stirring, the insolubles were removed by filtration, and the insolubles were further washed with cold methanol. The filtrates were combined, the solvent was distilled off under reduced pressure, and the residue was dried under vacuum to obtain the hydrochloride of the target compound 7 as a dark red powder (1.0 g, quantitative).
¹ H-NMR (400 MHz, DMSO-d6) δ 7.46 (s, 1H), 6.86 (d, J = 8.3 Hz, 2H), 6.75 (bs, 2H), 6.69 (bs, 2H), 6.62 (bs, 6H)
FAB-HRMS (m / z): calcd for C ₃₄ H ₂₀ NO ₁₀ : 602.1082; found: 602.1092 [M + H] ⁺

(2) Fluorescent Property The fluorescent property of the compound (I ′) obtained in the above (1) was evaluated.
5 μM solution of the hydrochloride salt of compound (I ′) (solvent: data of pH 1 is 0.1 M hydrochloric acid, pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5 The data of 0.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 12.0 are 25 mM phosphate buffer. FIG. 9 shows the fluorescence intensity associated with a pH change at an excitation wavelength of 484 nm and a fluorescence wavelength of 534 nm for 0.1M sodium hydroxide aqueous solution (data of pH 14). FIG. 9 shows that the maximum fluorescence intensity was obtained at pH 6.5 to 7.0.

Example 4
(1) Synthesis of compound (I ′) (R = hydrogen atom, R ¹ = carboxyl group, R ² = hydrogen atom) (A) Synthesis of compound 2 ′:

Benzenepentacarboxylic acid (894 mg) was suspended in tetrahydrofuran, and the powder was pulverized by ultrasonic waves, and then the solvent was distilled off under reduced pressure. The obtained powder was heated at 140 ° C. for 24 hours. After returning to room temperature under reduced pressure, resorcinol (1.32 g) and methanesulfonic acid (10 mL) were added, and the mixture was stirred at 90 ° C. for 2 days. After returning the reaction solution to room temperature, it was gradually added to ice-cooled water (100 mL) with stirring. The precipitated solid was collected by filtration, washed with water, and freeze-dried to obtain a dark red powder (1.88 g). This was heated to reflux with acetic anhydride (14 mL) for 1 day. After evaporating the solvent of the reaction solution under reduced pressure, the residue was purified three times by silica gel column chromatography (first and second times; dichloromethane / methanol = 95/5 → 93/7 → 90/10, third time; ethyl acetate). / Methanol = 100/0 → 95/5 → 90/10) to obtain the target compound 2 ′ as a pale yellow powder (1.6 g, yield 67%).
¹ H-NMR (400 MHz, DMSO-d6) δ 8.81 (s, 1H), 7.29 (s, 2H), 7.26 (d, J = 8.3 Hz, 2H), 7.14 (s, 2H), 6.98 (d, J = 8.3 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H), 6.61 (d, J = 8.3 Hz, 2H), 2.29 (s, 6H), 2.26 (s, 6H)
FAB-HRMS (m / z): calcd for C ₄₃ H ₂₇ O ₁₆ : 799.1294; found: 799.1302 [M + H] ⁺

  (B) Synthesis of compound 1 ':

To a suspension of compound 2 ′ (800 mg) in methanol (4 mL) was added a 5 M aqueous sodium hydroxide solution (4 mL), and the mixture was stirred at 50 ° C. for 1 day. Only the methanol fraction of the reaction solution was distilled off using an evaporator, and 5M hydrochloric acid was added to the remaining solution. The resulting precipitate was collected on ice and collected by filtration, washed with ice-cooled water and freeze-dried. This powder was suspended in a small amount of methanol and heated under reflux with stirring. After returning to room temperature and collecting the powder by filtration, the obtained powder was dissolved in a small amount of a 5M aqueous sodium hydroxide solution, and 5M hydrochloric acid was added. The resulting precipitate was collected by filtration with ice cooling, washed with ice-cooled water, and lyophilized to give the target compound 1 ′ as an orange powder (518 mg, yield 79%).
¹ H-NMR (400 MHz, DMSO-d6) δ 10.18 (bs, 4H), 8.66 (s, 1H), 6.62 (bs, 6H), 6.49 (bs, 6H)
FAB-HRMS (m / z): calcd for C ₃₅ H ₁₉ O ₁₂ : 631.0871; found: 631.0891 [M + H] ⁺

(2) Fluorescent Property The fluorescent property of the compound (I ′) obtained in the above (1) was evaluated.
1 μM solution of compound (I ′) (solvent: pH 1 was 0.1 M hydrochloric acid, pH 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, The data for 5.5, 6.0, 6.5, 7.0, 8.0, 9.0, 10.0, 11.0 and 12.0 are 25 mM phosphate buffer and the data for pH 14 are 0. FIG. 10 shows the fluorescence intensity associated with a pH change at an excitation wavelength of 488 nm and a fluorescence wavelength of 528 nm for a .1M sodium hydroxide aqueous solution. FIG. 10 shows that the maximum fluorescence intensity was obtained at pH 4.5.

  The compound of the present invention is useful as a compound that emits strong fluorescence only in a specific pH range and a precursor compound thereof.

Claims (6)

Formula (I ′):
(In the formula, R represents a hydrogen atom or an acyl group, and R ¹ and R ² each independently represent a hydrogen atom, an amino group, or a carboxyl group.)
A compound represented by the formula:   The compound according to claim 1, wherein R is a hydrogen atom.   2. The compound according to claim 1, wherein R is acetyl. The compound according to any one of claims 1 to 3, wherein R ¹ and R ² are both hydrogen atoms. The compound according to any one of claims 1 to 3, wherein R ¹ is an amino group and R ² is a hydrogen atom. The compound according to any one of claims 1 to 3, wherein R ¹ is a carboxyl group and R ² is a hydrogen atom.