JP4597573B2 - Compound, method for producing compound and method for detecting apoptotic cell - Google Patents

Description

  The present invention relates to a novel compound, a method for producing the same, and a method for detecting apoptotic cells.

Various fluorescent coloring methods are effectively used to detect the shape and various states of cells. For example, fluorescent antibodies in which an antibody against a specific antigen is fluorescently labeled, or fluorescent dyes that stain cells in a specific state are widely used.
In recent years, research on apoptosis, which is one of important life phenomena as well as pre-programmed cell death, has been advanced. Apoptosis is a phenomenon different from cell death due to external damage (necrosis), and it is necessary to distinguish it from necrosis in order to identify the phenomenon of apoptosis. On the other hand, it has been confirmed that zinc ions are involved in the apoptosis phenomenon. For this reason, based on this finding, a zinc-sensitive fluorescent compound is used as a probe to accurately distinguish apoptosis from necrosis. In addition, since zinc is an essential element for living organisms, it is known that zinc is deeply involved in various life phenomena.

  As such a zinc fluorescent probe capable of fluorescent coloration by zinc ions, those shown below such as dansylamidodethylcyclen are widely used (for example, see Non-Patent Documents 1 to 4 and Patent Document 1). ).

Proc. Natl. Acad. Sci. USA, 2003, 100 (7), 3731 J. Am. Chem. Soc., 2003, 125, 1778-1787 J. Am. Chem. Soc., 2003, 125, 3889-3895 J. Am. Chem. Soc., 2004, 126, 712-713 JP 2000-239272 A

  However, since there are various ions in addition to zinc ions in living organisms, in order to detect small amounts of zinc ions effectively, they are highly sensitive and selective for zinc ions under physiological conditions in a short time. It is necessary to form a complex by reacting with, and to strongly fluoresce. When cells are targeted, blood cells and the like often show weak alkalinity at pH 7.4, but some cells such as eosinophils have a lower pH. Similarly, when used as a fluorescent probe for various cells, it is required that complex formation occurs in as wide a pH range as possible and that the fluorescence intensity of only the compound before complex formation is as low as possible. Furthermore, it is required to selectively stain cells in the apoptotic process in which such zinc ions are deeply involved.

Therefore, an object of the present invention is to provide a compound and a fluorescent probe that can detect zinc ions well in a wide pH range.
Another object of the present invention is to satisfactorily detect only apoptotic cells.

  The compounds of the present invention are compounds represented by the following general formulas (I) and (II).

[In the formulas (I) and (II), R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, a halogen atom, a sulfonylamide group, a sulfonylalkyl group, a lower alkyl group or XNR ^7. (X represents SO ₂ or halogen, R ⁷ represents an alkyl group having 1 to 3 carbon atoms); R ⁸ , R ⁹ and R ¹⁰ each independently represents a hydrogen atom or a lower alkyl group; ˜q each independently represents 2 or 3, r represents 1 or 2, and Y ⁻ represents a halogen anion, a nitrate ion, a sulfate ion or a carboxylate ion. ]

The method for producing a compound represented by the general formula (II) of the present invention is characterized by including disposing the compound represented by the general formula (I) in the presence of zinc ions.
Furthermore, the method for detecting apoptotic cells of the present invention comprises a step of adding the compound represented by the general formula (I) to the target cell, a step of irradiating the cell with excitation light for emitting fluorescence, and fluorescence staining. And a step of detecting the apoptotic cells formed.

According to the present invention, the compound represented by the general formula (I) is a compound represented by the general formula (II) which forms a complex in a short time with high selectivity to zinc ions and exhibits strong fluorescence intensity. Is generated. Since these characteristics of these compounds are observed in a wide pH range, zinc ions can be detected well in a wide pH range.
In addition, by using the above novel compound, apoptotic cells in which zinc ions are present can be detected well.

  The compound of the present invention is a compound represented by the following general formula (I) or a salt thereof.

In the above general formula (I), R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, halogen atom, sulfonylamide group, sulfonylalkyl group, lower alkyl group or XNR ⁷ (X is Represents SO ₂ or halogen, and R ⁷ represents an alkyl group having 1 to 3 carbon atoms). The lower alkyl group herein is preferably an alkyl group having 1 to 6 carbon atoms, and particularly preferably a methyl group from the viewpoint of ease of synthesis. Moreover, Cl, Br, and I can be mentioned as a halogen concerning X in a formula. X is preferably SO ₂ .

Among these, from the viewpoint of synthesis, R ¹ , R ² and R ⁴ are all hydrogen atoms, and R ³ is XNR ⁷ (X represents SO ₂ or halogen, and R ⁷ represents a methyl group). It is particularly preferable that both of them are provided.

Moreover, in general formula (I), R <^8> , R < ⁹ > and R < ¹⁰ > show a hydrogen atom or a lower alkyl group each independently. Here, as the lower alkyl group, those described above are applicable. From the viewpoint of ease of synthesis, R ⁸ , R ⁹ and R ¹⁰ are preferably all hydrogen atoms. In the general formula (I), m to q are each independently 2 or 3, preferably 2, and r represents 1 or 2, preferably 1.

  The compound of general formula (I) may be a salt. Examples of the salt include hydrochloride, oxalate, and oxalate.

  A particularly preferable compound as the compound corresponding to the general formula (I) is a compound represented by the following formula (III).

  The compound of the present invention can be synthesized, for example, as follows.

  1,4,7-tris (tert-butoxycarbonyl) -cyclene [1] (3Boc-cyclene) and quinolinol derivative 2 are reacted to form [3] to deprotect all protecting groups including the Boc group. To obtain the compound [4] of the present invention.

  The compound represented by the general formula (II) of the present invention is a complex formed by the compound of the present invention represented by the above general formula (I) and a zinc ion. Since the compound of the above general formula (I) has 8-quinolinol, it has high reactivity with zinc ions, and in the presence of zinc ions, quinolinol oxygen and nitrogen, and four nitrogen atoms of the cyclic polyamine moiety become zinc ions. Coordinates to form a complex easily. The compound in which the complex is formed (formula (II)) emits fluorescence more strongly than the compound (ligand) represented by formula (I). For this reason, the compound of the formula (II) formed by the compound of the formula (I) and the zinc ion is used as a sensitive zinc fluorescent probe that can be effectively detected even with a small amount of zinc. be able to.

In the above formula (II), R ¹ , R ² , R ³ , R ⁴ and R ⁵ are as defined for the above general formula (I), including the preferred examples. In the formula, Y ⁻ represents a halogen anion, a nitrate ion (NO ₃ ⁻ ), a sulfate ion or a carboxylate ion.

The compound represented by the general formula (II) can be produced by a method including disposing the compound of the above formula (I) in the presence of zinc ions.
Arranging in the presence of zinc ions means that the compound of formula (I) may be added to a system in which zinc ions may be present, or zinc ion is added to the system in which the compound of formula (I) is present. A system that may exist may be added. The system in which zinc ions may exist includes not only the case where zinc ions actually exist, but also the case where zinc ions are generated in the system by a predetermined reaction.

  Arranging in the presence of zinc ions can include, for example, adding the compound of formula (I) to a cell culture system that releases zinc ions, in particular, zinc ions are generated in the early stages of apoptosis. Therefore, it can be added in the presence of cells undergoing apoptosis.

  In order to place the compound of formula (I) in the presence of zinc ions, an aqueous solution of the compound is prepared and added to a solution in which zinc ions may be present, or zinc ions may be present. What is necessary is just to add the aqueous solution of the compound prepared above to the solution with the property.

  Accordingly, the present invention includes a method for detecting apoptotic cells. The method for detecting apoptotic cells includes a step of adding a compound of the above formula (I) to a target cell, a step of irradiating the cells with excitation light for emitting fluorescence, and a step of detecting fluorescently stained apoptotic cells. And are included.

The compound of the general formula (I) may be added to the target cell by adding an aqueous solution of the compound of the present invention to the target cell culture system.
In addition, irradiation with excitation light may be performed in accordance with a method usually used for fluorescent staining. In general, apoptotic cells can be easily detected by adding excitation light after the compound is added to the cells and then detecting the fluorescence of the zinc fluorescent probe of the present invention after irradiation. For the fluorescent color development of the zinc fluorescent probe, the conditions for normal color development of the fluorescent probe may be applied as it is. For example, the zinc fluorescent probe may be irradiated with excitation light of 330 to 385 nm. For example, in the compound represented by the above formula (II), fluorescence of 528 nm can be developed by irradiating with excitation light of 330 nm (quantum emission efficiency (Φ) = 0.04).

  For detection of apoptotic cells that have been fluorescently stained, the conditions usually used for fluorescent staining can be applied as they are. In particular, since the zinc fluorescent probe of the present invention can easily form a complex by reacting with zinc ions in an equal amount in a wide pH range, the detection time is preferably 5 to 30 minutes, depending on the cell type. Can be 30 minutes. In addition, the pH at the time of detection can be satisfactorily detected, but it is preferably pH 3.5 or more, particularly preferably pH 4.0 to pH 9.0. A pH of 3.5 or less is not preferable because the efficiency of complex formation decreases.

  Moreover, you may include the process of adding the other fluorescent dye which dye | stains a dead cell to an object cell simultaneously or continuously with the addition of the compound of this invention. Thereby, apoptotic cells can be detected more reliably. Examples of fluorescent dyes that can be used in combination in this way include fluorescent dyes known in the art, such as propidium iodide.

  Since the compound of the formula (I) of the present invention can be used in a wide pH range, it can be used without particular limitation for cells used for detection of apoptotic cells. For example, HeLa cells and HL60 cells can be mentioned, and in particular, cells in the early stage of apoptosis can be effectively detected.

  Examples of the present invention will be described below, but the present invention is not limited thereto. Further,% in the examples is based on weight (mass) unless otherwise specified.

[Example 1]
Synthesis of Compound A method for synthesizing the compound of the present invention will be described below using typical compounds as examples. The compound numbers in the examples correspond to the compound numbers in the following scheme.

Synthesis of 8-benzenesulfonyloxy-2-methylquinoline-5- (N, N-dimethyl) sulfonamide [2] 8-hydroxy-2-methylquinoline-5- (N, N-dimethyl) sulfonamide [1] (1.7 g, 6.4 mmol) was dissolved in 13 ml of dichloromethane and cooled to 0 ° C. with ice, triethylamine (780 mg, 7.7 mmol, 1.2 eq) was added, and benzenesulfonyl chloride (1.3 g, 7.1 mmol, 1. 1 eq) was added dropwise over 1 hour. The mixture was further stirred at room temperature for 1.5 hours, added with 5 ml of water, stirred for 0.5 hours, and extracted with an aqueous potassium carbonate solution and dichloromethane. The dichloromethane layer was dehydrated with potassium carbonate and purified by silica gel chromatography (developing solvent: AcOEt / n-hexane = 1: 4) to obtain [2] as a colorless transparent amorphous. (2.5 g, 6.1 mmol, 96%)
¹ H NMR (CDCl ₃ ) δ = 8.93 (1H, J = 8.7 Hz, d), 8.10 (1H, J = 8.3 Hz, d), 8.01 (2H, J = 8.3 Hz) , D), 7.73 (1H, J = 8.3 Hz, d), 7.62 (1H, J = 7.6, 1.4 Hz, dd), 7.48 (2H, J = 8.0, 1.6 Hz, dd), 7.35 (1 H, J = 8.9 Hz, d), 2.81 (6 H, s), 2.55 (3 H, s)

Synthesis of 8-benzenesulfonyloxy-5- (N, N-dimethyl) sulfonamido-2-bromomethylquinoline [3] [2] (1.5 g, 3.7 mmol) was dissolved in 190 ml of distilled carbon tetrachloride. Thereafter, N-bromosuccinimide (NBS) (380 mg, 2.6 mmol, 0.6 eq) and 2,2′-azobis-2-methylpropionitrile (AIBN) (catalytic amount) were added in small portions and refluxed for 8 hours. After cooling to room temperature, the solvent was distilled off under reduced pressure, and the residue was dissolved in dichloromethane and washed with a saturated aqueous solution of sodium carbonate / sodium thiosulfate = 1: 1. The aqueous layer was extracted with dichloromethane, and the dichloromethane layers were combined and dried over anhydrous sodium sulfate. The solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: AcOEt / n-hexane = 1: 5) to obtain [3] as a colorless transparent amorphous. (250 mg, 0.52 mmol, 14%).
¹ HNMR (CDCl ₃ ) δ = 9.05 (1H, J = 9.0 Hz, d), 8.18 (1H, J = 8.3 Hz, d), 8.00 (2H, J = 8.5 Hz, d), 7.80 (1H, J = 8.3 Hz, d), 7.67 (1H, J = 8.9 Hz, d), 7.65 (1H, J = 7.4), 7.52 ( 2H, J = 7.8 Hz, t), 4.42 (2H, s), 2.83 (6H, s)

1- (8-Benzenesulfonyloxy-5- (N, N-dimethyl) sulfonamido-quinolinylmethyl) -4,7,10-tris (tert-butoxycarbonyl) -1,4,7,10-tetraazacyclododecane Synthesis of [5] 3Boc-cyclen [4] (160 mg, 0.34 mmol) (Kimura, E .; Aoki, S .; Koike, T .; Shiro, M.. J. Am. Chem. Soc. 1997, 119 , 3068-3076) and [3] (200 mg, 0.41 mmol, 1.2 eq) were dissolved in 50 ml of acetonitrile and stirred at 60 ° C. for 1 day in the presence of sodium carbonate (60 mg, 0.6 mmol, 1.7 eq). . After allowing to cool to room temperature and filtering off unwanted materials, the solvent was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (developing solvent: MeOH / CH ₂ Cl ₂ = 1: 80) to obtain [5] as a pale yellow Obtained as amorphous. (260 mg, 0.29 mmol, 93%)
¹ HNMR (CDCl ₃ ) δ = 8.96 (1H, J = 8.9 Hz, d), 8.11 (1H, J = 8.0 Hz, d), 8.00 (2H, J = 7.1) 1.2 Hz, dd), 7.65 (1H, J = 4.4, t), 7.61 (2H, J = 8.3, d), 7.53 (2H, J = 8.0 Hz, t) ), 3.96 (2H, s), 2.34-3.56 (13H, br), 2.79-2.83 (10H, br), 1.42-1.54 (35H, br).

1- (8-hydroxy-5- (N, N-dimethyl) sulfonamido-quinolinylmethyl) -4,7,10-tris (tert-butoxy-carbonyl) -1,4,7,10-tetraazacyclododecane [ Synthesis of 6] [5] (1.0 g, 1.1 mmol) was dissolved in 40 ml of methanol, 35 ml of 28% aqueous ammonia solution was added, and the mixture was refluxed for 5 days. The solvent was removed, and the residue was purified by silica gel chromatography (developing solvent: MeOH / CH ₂ Cl ₂ = 1: 100) to obtain [6] as a pale yellow amorphous. (820 mg, 1.1 mmol, 97%)

¹ H-NMR (CD ₃ OD) δ = 9.01 (1H, J = 9.0 Hz, d), 8.10 (1H, J = 8.5 Hz, d), 7.75 (1H, J = 8 .5Hz, d), 7.20 (1H, J = 8.3 Hz, d), 4.12 (2H, s), 3.31-3.64 (10H, br), 3.30 (5H, s) ), 2.68-2.77 (10H, br), 0.91-1.49 (30H, br)
¹³ C-NMR (CD ₃ OD) δ = 159.6, 159.2, 157.9, 157.5, 157.4, 140.9, 139.2, 135.4, 134.0, 126.2 , 125.8, 123.0, 120.7, 115.4, 110.2, 97.3, 95.6, 95.5, 81.3, 81.2, 59.1, 57.7, 57 .5, 57.4, 56.1, 56.0, 37.8, 29.1, 28.9, 17.8, 17.6, 17.5, 17.3, 17.2, 17.1 , 16.9, 11.1

Synthesis of 1- (8-hydroxy-5- (N, N-dimethyl) sulfonamido-quinolinylmethyl) -1,4,7,10-tetraazacyclododecane tetrahydrochloride salt [7] [6] (500 mg, 0. 68 mmol) was dissolved in 60 ml of methanol, 1N hydrochloric acid (15 ml, 22 eq) was added under ice cooling, and the mixture was stirred for 1 hour, and further stirred at 60 ° C. for 2 days. The solvent was distilled off under reduced pressure and recrystallization from water and ethanol gave [7] as yellow needles (390 mg, 0.61 mmol, 89%).
¹ HNMR (D ₂ O) δ = 9.00 (1H, J = 9.0, d), 8.17 (1H, J = 8.6 Hz, d), 7.66 (1H, J = 9.0) , D), 7.34 (1H, J = 3.3 Hz, d), 4.24 (1H, s), 3.08-3.37 (16H, br), 2.81 (6H, s)
¹³ CNMR (D ₂ O) δ = 161.2, 159.8, 141.0, 137.8, 135.7, 128.3, 126.3, 123.7, 113.7, 98.7, 60 .4, 52.3, 47.3, 45.3, 44.9, 39.6
Elemental analysis Theoretical value C: 37.95%, H: 6.74%, N: 13.02%, S: 4.97
Measurement C: 37.50%, H: 7.23%, N: 13.21%, S: 5.39

Synthesis of 1- (8-hydroxy-5- (N, N-dimethyl) sulfonamido-quinolinylmethyl) -1,4,7,10-tetraazacyclododecane Zn (NO ₃ ) complex Synthesis of [8] [7] (40 mg, 0.062 mmol) was dissolved in 10 ml of water, and zinc perchlorate hexahydrate (27 mg, 0.072 mmol) dissolved in 3 ml of water was added little by little. The pH was adjusted to 7.5 with an aqueous sodium hydroxide solution, and unnecessary substances were removed by filtration, followed by concentration under reduced pressure at room temperature to obtain [8] as yellow cuboid crystals. (15 mg, 0.025 mmol, 40%)
¹ HNMR (D ₂ O) δ = 9.00 (1H, J = 9.2, d), 8.09 (1H, J = 8.8 Hz, d), 7.65 (1H, J = 8.8) D), 6.96 (1H, J = 8.8 Hz, d), 4.32 (1H, s), 3.25-2.78 (16H, m), 2.73 (6H, s)
Elemental analysis Theoretical value C: 40.01%, H: 5.20%, N: 14.00%, S: 5.34%
Measurement value C: 39.92%, H: 5.19%, N: 13.89%, S: 5.48%

  X-ray crystal structure analysis of a one-to-one complex of quinolinol cyclen [7] and zinc ion synthesized in Example 1 was performed. The result is shown in FIG. This proved that the hydroxyl group of the quinolinol part was deprotonated and coordinated to the zinc ion.

[Example 2]
Fluorescence properties of the compound The quinolinol cyclen [7] synthesized in Example 1 was examined for fluorescence properties.
5 μM quinolinol cyclen [7] was dissolved in a 10 mM HEPES aqueous solution (pH 7.4, I = 0.1 (NaNO ₃ )), and the fluorescence spectrum at each wavelength with 338 nM as the excitation wavelength was measured with a fluorescence spectrophotometer (Japan). (Manufactured by Spectroscopic Co.). The results are shown in FIG. 2 and FIG.

  As shown in FIG. 2, in the fluorescence spectrum of quinolinol cyclen before complex formation, almost no peak was observed from 400 nm to 700 nm (Φ = 0.002). On the other hand, when a complex was formed with zinc ions, a fluorescence spectrum having a maximum intensity at 512 nm was shown (Φ = 0044). In addition, as shown in FIG. 3, when the excitation wavelength is 338 nm and the fluorescence wavelength is 512 nm, the intensity of fluorescence color development increases depending on the concentration of zinc ions, and is the maximum at the same amount. At this time, the fluorescence intensity increased to 17 times that before the complex formation.

  Therefore, it was revealed that the quinolinol cyclene of this example can form a complex with zinc ions at a ratio of 1: 1, and the fluorescence intensity increases up to 17 times at a fluorescence wavelength of 512 nm (excitation wavelength of 338 nm). Further, since the fluorescence intensity at 512 nm changes depending on the amount of zinc ions, the concentration of zinc ions can be measured quantitatively.

[Example 3]
Zinc Selectivity (1) Selectivity for Various Metal Ions Next, the zinc selectivity of the compound of this example was evaluated.
Add 5 μM of the quinolinol cyclen of Example 1 to 100 mM HEPES (pH 7.5, I = 0.1 (NaNO ₃ )) containing various metal ions (5 μM or 5 mM), excitation wavelength 338 nm, fluorescence wavelength The fluorescence intensity was measured in the same manner as described above at 512 nm. The results are shown in FIG.

  As shown in FIG. 4, the fluorescence intensity increases up to 17 times in the presence of zinc ions, whereas ions that are present in a living body such as calcium ions and magnesium ions, and various metals such as aluminum ions and silver ions. It was shown that the fluorescence intensity does not increase with respect to ions.

(2) Comparison with dansyl cyclen In addition, with respect to zinc ions and copper ions, dansyl cyclen (dansylamidoethylcyclen; manufactured by Dojin Chemical Co., Ltd.), which is an existing zinc fluorescent probe and widely used, is shown in this example. Comparison with 1 quinolinol cyclen. The fluorescence characteristics of the dansylcyclene zinc complex are found to have an excitation wavelength of 323 nm and a fluorescence wavelength of 528 nm, and the dissociation constant for zinc ions is 8 pM (log K _app = 11.1) at pH 7.4.

  Zinc ions or copper ions were added by 1 μM each to 10 mM HEPES (pH 7.5) containing 5 μM quinolinol cyclen, and the fluorescence spectrum was measured in the same manner as described above. The results are shown in FIG.

As shown in FIG. 5 (A), in the quinolinol cyclen of this example, the fluorescence intensity at 512 nm is hardly enhanced with respect to copper ions, but a complex is formed with respect to zinc ions, which is 17 times higher. The fluorescence intensity was enhanced up to. On the other hand, as shown in FIG. 5 (B), dansyl cyclen showed only an enhancement effect up to 5 times the selectivity for zinc ions at a fluorescence wavelength of 528 nm.
Therefore, the quinolinol cyclen of this example not only has high selectivity to zinc ions, but also can exhibit fluorescence intensity that is three times or more stronger than conventional zinc fluorescent probes, in order to detect zinc ions with high accuracy. It proved to be an effective zinc fluorescent probe.

[Example 4]
pH-dependent complex formation Normal cells have a pH of around 7, but some cell types have an acidic or alkaline pH. For this reason, the stability as a fluorescent probe when a complex was formed was examined in various pH ranges.

The dansyl cyclen used in Example 3 or the quinolinol cyclen of this example was dissolved in a pH buffer solution of pH 3 to pH 12 at 5 μM, respectively, and the fluorescence spectrum was measured at 25 ° C.
The results are shown in FIG. The quinolinol cyclen of this example is shown in FIG. 6 (A) and the dansyl cyclen is shown in FIG. 6 (B). In each, the zinc fluorescent probe in which the complex is formed is shown by a solid line, and the one before complex formation Is indicated by a broken line.

As shown in FIG. 6 (A), in the quinolinol cyclen of this example, only about 25% of a complex is formed at pH 3, but a complex is rapidly formed when the pH is increased. It was found that 100% of the complex formed a zinc fluorescent probe. The dissociation constant (K _d ) of the quinolinol cyclene of this example with respect to zinc ions is 1 fM or less at pH 7.4 (K _d = 8 fM at pH 6.2) (log K _app > 15), and the complex formation rate (t _{1 / 2} ) was 30 msec. For this reason, if it is pH 4 or more, the quinolinol cyclene and zinc ion of a present Example will form a complex reliably and rapidly by 1: 1, Therefore A zinc ion can be measured quantitatively.

On the other hand, as shown in FIG. 6 (B), dansyl cyclen started to form a complex with zinc ions from pH 5 and formed almost 100% complex at pH 7.4. For this reason, at about pH 7 or less, a complex may not be formed even if zinc ions are present, and it is difficult to measure zinc ions quantitatively.
Further, when the respective K _d values at pH 7.4, which is the pH of blood cells, are compared, the quinolinol cyclen of this example is 1 fM or less, the dansyl cyclen is 8 fM, and the quinolinol cyclen of this example is. It was revealed that the zinc can form a complex with zinc more rapidly.
Furthermore, in FIG. 6 (A) and FIG. 6 (B), the area | region shown in gray is the area | region which can quantify the zinc ion of the quinolinol cyclene and dansyl cyclen of a present Example, respectively. It can be seen that the quinolinol cyclen of this example has a wider pH range in which zinc ions can be quantified.

  Therefore, since the quinolinol cyclen of this example can form a complex with zinc ions even at an acidic pH, it can be used for quantitative detection of zinc ions in a wide pH range.

[Example 5]
Detection of apoptotic cells After culturing HeLaS3 cells (Cell Resource Center for Biomedical Research, Tohoku University) in HBSS medium supplemented with 10% fetal bovine serum for 1 day, TNFα (1 mg / ml) and actinomycin D (ActD, 0 The medium was changed to HBSS medium containing 3 μg / ml) and incubated for 6 hours to induce apoptosis.

  To the cells after incubation, quinolinol cyclen and propidium iodide (PI, manufactured by Aldrich) of Example 1 were added at concentrations of 100 μM and 30 μM, respectively, and incubated for 10 minutes, followed by fluorescence microscopy (Carl Zeiss Co., Ltd.). Made). Quinolinol cyclene was excited by irradiating 300-350 nm excitation light, while PI was excited by irradiating 460-490 nm excitation light. The results are shown in FIG.

  7A to 7D show stained images in which the same visual field is double-stained. In HeLaS3 cells, apoptosis is induced by TNFα and ActD, and the field of view includes completely dead cells, late-apoptotic cells, early-apoptotic cells, and living cells (A). Of these, the quinolinol cyclen of the Example stained cells in the early stages of apoptosis and did not stain dead cells (B). On the other hand, PI stains both dead cells and late apoptotic cells (C), but not early apoptotic cells. Therefore, by double staining with quinolinol cyclen and PI of Example, cells in the early stage of apoptosis could be easily detected (D).

  Thus, by using the compound of the present invention, it is possible to obtain a zinc fluorescent probe capable of detecting zinc ions with high selectivity over a wide pH range. By using this zinc fluorescent probe, the zinc ions can be quantitatively detected. It is possible to detect and efficiently detect apoptotic cells.

It is the figure (crystal structure) which showed that the compound (quinolinol cyclene) concerning a present Example has produced | generated the one-to-one complex compound with the zinc ion. It is a graph which shows the fluorescence characteristic of the compound (quinolinol cyclene) concerning a present Example. It is the graph which showed the change of the fluorescence accompanying the quinolinol cyclen concerning a present Example producing | generating a one-to-one complex with a zinc ion. It is a graph explaining the selectivity with respect to various metal ions of the quinolinol cyclen concerning a present Example. (A) is the graph which showed the reactivity with respect to the zinc ion or copper ion of the quinolinol cyclen (5 micromol) concerning a present Example, (B) showed the reactivity with respect to the zinc ion or copper ion of dansyl cyclen. It is a graph. (A) is a graph which shows the change of the fluorescence intensity when changing pH in the presence or absence of zinc ion for quinolinol cyclen (5 μM) according to this example, and (B) is dansyl cyclen. It is the graph which showed the change of the fluorescence intensity when changing pH about (5 micromol) in the presence and absence of zinc ion. (A) shows a microscopic image (× 400) of HeLaS3 cells in which apoptosis was induced, (B) is a stained image with quinolinol cyclen according to this example of (A), (C) is an iodine image of (A). Stained image with propidium iodide (PI), (D) is a double-stained image with quinolinol cyclen and PI according to this example.

Claims (10)

A compound represented by the following general formula (I) or a salt thereof.
[In the formula (I), R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, halogen atom, sulfonylamide group, sulfonylalkyl group, lower alkyl group or XNR ⁷ (X is SO ₂ or halogen, R ⁷ represents an alkyl group having 1 to 3 carbon atoms); R ⁸ , R ⁹ and R ¹⁰ each independently represent a hydrogen atom or a lower alkyl group; Independently represents 2 or 3; r represents 1 or 2; ] ^2. The compound according to claim ¹ , wherein R ¹ , R ² and R ⁴ are all hydrogen atoms. The compound according to claim 1 or 2, wherein R ³ is XNR ⁷ (wherein X represents SO ₂ or halogen, and R ⁷ is a methyl group). Wherein R ¹ , R ² and R ⁴ are all hydrogen atoms, and R ³ is XNR ⁷ (wherein X represents SO ₂ and R ⁷ is a methyl group). The described compound. The compound represented by the following general formula (II).
[In the formula (II), R ¹ , R ² , R ³ , R ⁴ and R ⁵ are each independently a hydrogen atom, a halogen atom, a sulfonylamide group, a sulfonylalkyl group, a lower alkyl group or XNR ⁷ (X is SO ₂ or halogen, R ⁷ represents an alkyl group having 1 to 3 carbon atoms); R ⁸ , R ⁹ and R ¹⁰ each independently represent a hydrogen atom or a lower alkyl group; Independently represents 2 or 3; r represents 1 or 2; Y ⁻ represents a halogen anion, a nitrate ion, a sulfate ion or a carboxylate ion. ] Formula (II), R ^1, R ^2, and R ⁴ is The compound of claim 5, wherein the both hydrogen atoms. In the formula (II), R ³ is XNR ⁷ (wherein X represents SO ₂ or halogen, R ⁷ is a methyl group) The compound of claim 5, wherein the a. In the formula (II), R ¹ , R ² and R ⁴ are all hydrogen atoms, R ³ is XNR ⁷ (wherein X represents SO ₂ and R ⁷ is a methyl group), Y ⁻ is 6. The compound according to claim 5, wherein the compound is nitrate ion (NO ₃ ⁻ ). A method for producing a compound represented by formula (II) according to claim 5,
Disposing the compound according to any one of claims 1 to 4 in the presence of zinc ions,
Manufacturing method. A method for detecting apoptotic cells, comprising:
Adding the compound according to any one of claims 1 to 4 to a target cell;
Irradiating a cell with excitation light for emitting fluorescence;
Detecting fluorescently stained apoptotic cells;
A method for detecting apoptotic cells.