JP4441606B2 - Sulfonate compound and fluorescent probe using the same - Google Patents

Description

  The present invention relates to a sulfonate compound and a fluorescent probe using the same.

  It has become clear that various diseases are caused by the increased generation of reactive oxygen species in vivo. Therefore, in vivo dynamic analysis of reactive oxygen species is indispensable for elucidating the etiology and pathology. In the analysis of reactive oxygen species, a bioimaging method using a fluorescent probe plays a central role. Since the generation of superoxide is at the source of all reactive oxygen species generation, its analysis is very important. However, only hydroethidine (HE) (Scheme 1 below) exists as a fluorescent probe for superoxide. (For non-patent documents 1 to 10 etc., refer to, for example, HE.)

  Furthermore, since the fluorescence mechanism of HE is based on an oxidation reaction by superoxide, there is a problem in specificity (selectivity). That is, as a result of many of the active oxygen species acting as an oxidant, the oxidation of HE 3 is also caused by other active oxygen species, and the degree is in the order of peroxynitrite> hydroxyl radical> superoxide> hydrogen peroxide. HE is also known to be oxidized by cytochrome c. Therefore, it is considered that the fluorescence response obtained using HE should be used as an indicator of “total amount of in-vivo oxidizing agent containing active oxygen species”, not superoxide generation amount. On the other hand, nitro blue tetrazolium (NBT) is a probe based on the reducing power of superoxide. NBT is reduced by superoxide and converted to the blue dye diformazan. However, the absorption probe NBT has problems such as being reduced by various reductases such as NOS, and diformazan produced being converted to NBT by disproportionation and oxidation, as well as flow cytometry and confocal. It cannot be used for advanced fluorescence analysis methods such as laser microscopes. Under such circumstances, development of a fluorescent probe that responds to superoxide with high selectivity is highly desired from the viewpoint of cell physiology.

  On the other hand, assay methods such as acetylcholinesterase (AChE) assay are important in the fields of biochemistry and medicine. In such an assay method, a compound capable of detecting a compound having a mercapto group (thiol group) (mercapto group detection compound) is considered useful. So far, several compounds having the ability to detect mercapto groups have been actually developed, and they can be roughly classified into the following three types of types 1 to 3. That is, [type 1] is a fluorescent compound introduction reagent, [type 2] is a mercapto group labeling agent, and [type 3] is a coloring reagent based on a reaction with a mercapto group. Examples of these compounds belonging to types 1 to 3 and an outline of the mercapto group detection mechanism are shown in the following scheme 2.

  Reagents belonging to type 1 such as compound 9 are used exclusively for the purpose of synthesizing fluorescently labeled proteins and nucleic acids, but have the following disadvantages when used for AChE assays and the like. That is, first, the compound 9 is a fluorescent compound like the product 10. Therefore, when the AChE assay or AChE inhibitory activity is measured using 9, it is complicated because the separation operation of 9 and 10 is always required after the enzymatic reaction. In addition, maleimide, which is a reaction site with a mercapto group in 9, reacts with other nucleophiles such as amines and alcohols, and thus has a difficulty in specificity (selectivity).

  Next, reagents belonging to type 2 such as compound 11 are exclusively used as labeling agents for separation analysis. Type 2 reagent 11 is non-fluorescent and reacts with a mercapto group to give 12 which is a fluorescent dye. Therefore, unlike 9, the reagent separation operation after the reaction is not necessary. However, since 11 also reacts with various nucleophiles to give fluorescent compounds similar to 12, it still has difficulty in specificity for use in AChE assays and the like. However, reacting with various nucleophiles is advantageous as a labeling agent for these nucleophiles, and therefore 11 is useful as a labeling agent in HPLC analysis of amines and thiols.

  Type 3 reagent 13 (Ellman's reagent) is the reagent actually used in the AChE assay. However, 13 has a problem of low sensitivity due to poor stability in an aqueous solution, that is, a large blank response.

  In addition to these types 1 to 3, as examples of compounds that selectively react with thiols, deprotection reactions of sulfonamide compounds 15 with thiols have also been reported (Non-patent Document 11). The aromatic nucleophilic substitution reaction in Compound 15 proceeds more smoothly with a thiol compound than with an amino compound, that is, in the case of an amino compound, a long reaction time is required with a large excess over 15. It has been reported. However, since compound 15 and its product do not exhibit fluorescence, it cannot be used as a thiol detection compound.

As described above, the development of a new mercapto group detection compound to replace Compound 13 etc. is awaited from the viewpoint of utilizing AChE assay using acetyl thiocholine as a substrate as a more general technique. .
M. Nakano, Cell. Mol. Neurobiol. 1998, 18, 565-579 CL Murrant, MB Reid, Microsc. Res. Tech. 2001, 55, 236-248 MM Tarpey, I. Fridovich, Circ. Res. 2001, 89, 224-236 T. Munzel, IB Afanas'ev, AL Klescchyov, DG Harrison, Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1761-1768 MD Esposti, Methods, 2002, 26, 335-340 G. Rothe, G. Valet, J. Leukoc. Biol. 1990, 47, 440-448 WO Carter, PK Narayanan, JP Robinson, J. Leukoc. Biol. 1994, 55, 253-258 VP Bindokas, J. Jordan, CC Lee, RJ Miller, J. Neurosci. 1996, 16, 1324-1336 AB Al-Mehdi, H. Shuman, AB Fisher, Am. J. Pysiol. 1997, 272, L294-L300 L. Benov, L. Sztejnberg, I. Fridovich, Free Radic. Biol. Med. 1998, 25, 826-831 Fukuyama, T., et al., Tetrahedron Lett., 1997, 38, 5831-5834

  Accordingly, an object of the present invention is to provide a novel organic compound that can be used for a fluorescent probe that responds to a superoxide or mercapto compound with high selectivity.

  In order to solve the above-described problems, the present invention provides a sulfonate compound having a structure represented by the following general formula (I).

In the formula (I), the atomic group A-O is an atomic group that forms a fluorescent compound by breaking a covalent bond with the sulfonyl group, and the atomic group B-SO ₃ -bonded to the atomic group A is 1 B may be a ring substituted with one or more electron-withdrawing groups, and the electron-withdrawing group is at least selected from the group consisting of a halogenated alkyl group, a nitro group, and a cyano group. Including one, B may be the same or different when there are a plurality.

  The sulfonic acid ester compound of the present invention has a structure represented by the above general formula (I), so that, for example, it does not respond to hydroxyl radicals and hydrogen peroxide, but responds only to superoxide. It can be used for applications such as a fluorescent probe or a fluorescent probe that responds with high selectivity to a mercapto compound. Furthermore, the fluorescent probe using the sulfonate compound of the present invention has a very low response to not only intracellular reactive oxygen species other than superoxide, such as hydroxyl radical and hydrogen peroxide, but also other physiologically active substances. Alternatively, it can be used as a fluorescent probe that responds highly selectively to a mercapto compound. The other physiologically active substances include, for example, reducing compounds such as ascorbic acid and 1,4-hydroquinone, nucleophilic compounds such as glucose, propylamine and diethylamine, and cytochrome P450 reductase + NADPH system and diaphorase + NADH system And the like. In the present invention, the “mercapto compound” refers to all compounds having a mercapto group (—SH group), and the atom to which the mercapto group is bonded may be a carbon atom or any other atom such as nitrogen, It may be phosphorus or the like.

  First, the characteristics of the compound of the present invention in superoxide detection will be described.

  The conventional fluorescent probe HE is converted into a fluorescent compound by an oxidation reaction. Therefore, there is a problem regarding specificity as described above. On the other hand, there is no fluorescent probe that uses a fluorescence mechanism based on a reduction reaction, but there is NBT as an absorption probe. In the case of NBT, a simple reduction reaction is used as the fluorescence mechanism, so there is an inverse reaction, that is, a conversion reaction of the resulting dye to NBT. On the other hand, the fluorescence mechanism in the compound of the general formula (I) is not based on a simple redox reaction but based on a desulfonylation reaction induced by, for example, an aromatic nucleophilic substitution reaction. That is, for example, as shown in Scheme 4 below. However, Scheme 4 is merely an example and does not limit the present invention.

  Therefore, according to the compound of the present invention represented by the general formula (I), it is considered that not only the problem in HE can be avoided, but also the reverse reaction in NBT does not occur. The present inventors are the first to design and develop a fluorescent probe based on such a fluorescence reaction.

  A more specific mechanism of the reaction of Scheme 4 is presumed as shown in the following Scheme 5, for example. However, Scheme 5 shows only an example of a mechanism that can be estimated, and does not limit the present invention.

  Next, the characteristics of the sulfonic acid ester compound of the present invention in mercapto compound detection will be described.

  The sulfonic acid ester compound of the present invention belongs to type 3 as a mercapto group detection compound, but has the following characteristics as compared with existing type 1-3 reagents.

  The fluorescence mechanism of the mercapto compound-responsive fluorescent probe of the present invention is presumed to be based on, for example, an aromatic nucleophilic substitution reaction according to Scheme 6 below. However, Scheme 6 shows only an example of a mechanism that can be estimated, and does not limit the present invention.

  Fluorescence due to such a reaction has not been reported for sulfonic acid ester compounds such as the compounds of the present invention. And the compound of this invention responds with high specificity and high sensitivity with respect to a mercapto compound compared with an amino compound etc. by being represented by the said general formula (I). Furthermore, since the fluorescent compound is formed by cleaving the covalent bond between the atomic group AO and the sulfonyl group, the mercapto compound can be detected by a simple operation without separating the compound after the reaction. Is possible. Therefore, it can be expected that the construction of an assay system that cannot be achieved by the conventional reagent for detecting a mercapto compound can be performed using the compound of the present invention.

  Hereinafter, embodiments of the present invention will be described.

  The atomic group B in the formula (I) is a ring substituted with one or more electron-withdrawing groups as described above, but is an aromatic ring or heteroaromatic ring substituted with one or more electron-withdrawing groups. Is preferred. That is, if the ring forming the atomic group B is an aromatic ring or a heteroaromatic ring, it is expected that the detection ability when used for a superoxide detection probe or a mercapto compound detection probe is higher. That is, for example, the reaction is considered to be an aromatic nucleophilic substitution reaction rather than a simple nucleophilic substitution reaction as in the above-mentioned schemes 5 and 6, so that it can be expected to be more excellent in detectability.

  In the atomic group B in the formula (I), the electron withdrawing group is at least one selected from the group consisting of a linear or branched alkyl halide group having 1 to 6 carbon atoms, a nitro group, and a cyano group. Preferably, from the viewpoint of detection ability for superoxide and mercapto compounds, it is more preferable to include at least one of a nitro group and a trifluoromethyl group, and it is more preferable to include a nitro group. In addition, the atomic group B may be further substituted with an electron withdrawing group other than a halogenated alkyl group, a nitro group, and a cyano group, for example, a halogen atom, etc. It may be further substituted with a group such as methyl group, isopropyl group, methoxy group and the like. In the atomic group B, the ring is a benzene ring, naphthalene ring, anthracene ring, pyrene ring, pyridine ring, pyrrole ring, thiophene ring, furan ring, benzopyridine ring, benzopyrrole ring, benzothiophene ring, and benzofuran. More preferably, it is at least one selected from the group consisting of rings.

  The atomic group B includes 2,4-dinitrophenyl group, 4-nitrophenyl group, 2-nitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2-nitro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4-nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, 4-nitro-2,6- The sensitivity of the fluorescent probe is at least one selected from the group consisting of a dimethylphenyl group, a 2-nitro-4-chlorophenyl group, a 4-nitro-2-chlorophenyl group, and a 2-nitro-4-isopropylphenyl group And from the viewpoint of specificity (selectivity) for superoxide and mercapto compounds, 2,4-dinitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2- Tro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4-nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, At least one selected from the group consisting of 4-nitro-2,6-dimethylphenyl group, 2-nitro-4-chlorophenyl group, 4-nitro-2-chlorophenyl group, and 2-nitro-4-isopropylphenyl group More preferably, it is particularly preferably a 2,4-dinitrophenyl group, but various other groups are possible.

  When used as a probe for detecting a specific substance, it is preferable to keep the response to other substances as low as possible from the viewpoint of specificity (selectivity). However, responding to a substance other than a specific substance does not exclude the possibility of being used as a probe for detecting the specific substance, and by appropriately setting measurement conditions, etc. It can be sufficiently used for the detection of. The sulfonic acid ester compound of the present invention can be obtained by appropriately designing the molecule within the range shown in the formula (I) to obtain a sulfonic acid ester compound having high specificity for either the superoxide or the mercapto compound. The specificity (selectivity) of the fluorescent probe using the sulfonic acid ester compound of the present invention is not particularly limited, but is preferably as follows, for example. That is, the fluorescence response to the superoxide or mercapto compound is preferably 10 times or more than the fluorescence response to hydrogen peroxide, more preferably 20 times or more, and particularly preferably 100 times or more. The upper limit of the fluorescence response to the superoxide or mercapto compound is not particularly limited, but is, for example, 1000 times or less the fluorescence response to hydrogen peroxide. Further, the fluorescence response to the superoxide or mercapto compound is preferably 10 times or more, and 20 times or more of the fluorescence response to hydroxyl radical, glucose, ascorbic acid, 1,4-hydroquinone, propylamine, or diethylamine. More preferably, it is particularly preferably 100 times or more, and the upper limit is not particularly limited. For example, it is 1000 times or less of the fluorescence response to any of these substances. The fluorescence response to the superoxide or mercapto compound is preferably 4 times or more, more preferably 5 times or more, and even more preferably 10 times or more when compared with the fluorescence response to cytochrome P450 reductase + NADPH system or diaphorase + NADH system. 20 times or more is particularly preferable, and the upper limit value is not particularly limited. For example, it is 100 times or less of the fluorescence response to any of these systems. These are values obtained by comparing the superoxide or mercapto compound and each of the above substances in equimolar amounts, the measurement temperature is 37 ° C., the excitation wavelength is 485 ± 20 nm, and the emission wavelength is 530. It shall be ± 20 nm. However, the measurement conditions using the fluorescent probe of the present invention are not limited to this, and any measurement conditions are possible.

When the sulfonic acid ester compound of the present invention is used as a probe for superoxide detection, from the viewpoint of specificity (selectivity), there are a plurality of atomic groups B-SO ₃ − bonded to the atomic group A in the formula (I). In this case, it is more preferable that the electron withdrawing group in the atomic group B in the formula (I) includes at least one of a nitro group and a trifluoromethyl group. More preferably, it contains a nitro group. The atomic group B includes 2,4-dinitrophenyl group, 4-nitrophenyl group, 2-nitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2-nitro-4-methoxyphenyl group, 4 -Nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4-nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, 4-nitro-2,6-dimethyl More preferably, it is at least one selected from the group consisting of a phenyl group, a 2-nitro-4-chlorophenyl group, a 4-nitro-2-chlorophenyl group, and a 2-nitro-4-isopropylphenyl group, 4-dinitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2-nitro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4-D B-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, 4-nitro-2,6-dimethylphenyl group, 2-nitro-4-chlorophenyl group, 4-nitro-2-chlorophenyl group, And at least one selected from the group consisting of 2-nitro-4-isopropylphenyl groups, and more preferably 2,4-dinitrophenyl groups. In addition, when a sulfonate ester compound having one atomic group B—SO ₃ − bonded to the atomic group A in the formula (I) is used for a superoxide detection probe, from the viewpoint of superoxide selectivity, In the atomic group B in the formula (I), the electron withdrawing group preferably contains only one nitro group. Examples of such an atomic group B include a 2-nitrophenyl group, a 4-nitrophenyl group, or a group derived therefrom as described above. However, sulfonic acid ester compounds other than these in the present invention can naturally be used for the fluorescent probe for detecting superoxide.

In addition, when the sulfonate compound of the present invention is used for a probe for detecting a mercapto compound, from the viewpoint of specificity (selectivity), an atomic group B-SO bonded to the atomic group A in the formula (I) is used. It is preferable that ₃ − is one. In this case, from the viewpoint of higher specificity and mercapto compound detection ability, in the atomic group B in the formula (I), the electron withdrawing group preferably includes a nitro group, and includes a plurality of nitro groups. It is particularly preferred. For example, in the above formula (I), the atomic group B is 2,4-dinitrophenyl group, 4-nitrophenyl group, 2-nitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2- Nitro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4-nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, At least one selected from the group consisting of 4-nitro-2,6-dimethylphenyl group, 2-nitro-4-chlorophenyl group, 4-nitro-2-chlorophenyl group, and 2-nitro-4-isopropylphenyl group And more preferably 2,4-dinitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2-nitro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2 -D B-4-Methylphenyl group, 4-nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, 4-nitro-2,6-dimethylphenyl group, 2-nitro-4-chlorophenyl group , 4-nitro-2-chlorophenyl group, and 2-nitro-4-isopropylphenyl group are more preferable, and 2,4-dinitrophenyl group is particularly preferable. . However, sulfonic acid ester compounds other than these can naturally be used for the probe for detecting a mercapto compound.

  In the atomic group A-O in the formula (I) in the sulfonate compound of the present invention, for example, when the O atom is directly bonded to an aromatic ring or a heteroaromatic ring, It is further preferable from the viewpoint of higher detectability and the like. In the sulfonate compound of the present invention, the fluorescent compound formed by cleaving the covalent bond between the atomic group AO and the sulfonyl group includes fluorescein, resorufin, 7-hydroxycoumarin, 1-naphthol, 2- Naphthol, 1-hydroxyanthracene, 2-hydroxyanthracene, 9-hydroxyanthracene, 1-hydroxypyrene, 1-hydroxyacridine, 2-hydroxyacridine, 9-hydroxyacridine, 2-hydroxyquinolone, 4-hydroxyquinolone, 5-hydroxy It is preferably quinolone, 6-hydroxyquinolone, 8-hydroxyquinolone, 4-hydroxy-7-nitro-2-oxa-1,3-diazole or a derivative thereof, but is not limited to these, and any fluorescence Compound is It is a function. In addition, when the fluorescent compound is 7-hydroxycoumarin or a 7-hydroxycoumarin derivative other than 7-hydroxy-4- (trifluoromethyl) coumarin, the atomic group B is 2,4- Dinitrophenyl group, 2-nitro- (4-trifluoromethyl) phenyl group, 2-nitro-4-methoxyphenyl group, 4-nitro-2-methoxyphenyl group, 2-nitro-4-methylphenyl group, 4- Nitro-2-methylphenyl group, 2-nitro-4,6-dimethylphenyl group, 4-nitro-2,6-dimethylphenyl group, 2-nitro-4-chlorophenyl group, 4-nitro-2-chlorophenyl group, Or 2-nitro-4-isopropylphenyl group is more preferable. The fluorescein derivative is a derivative in which at least one of the 2-position, 4-position, 5-position and 7-position of fluorescein is substituted with a linear or branched alkyl group having 1 to 6 carbon atoms, or halogen, The hydroxycoumarin derivative is more preferably a derivative in which the 4-position of 7-hydroxycoumarin is substituted with a linear or branched alkyl group having 1 to 6 carbon atoms or a trifluoromethyl group. Specific examples of particularly preferable compounds as the fluorescent compound formed by cleaving the covalent bond between the atomic group AO and the sulfonyl group include, for example, fluorescein, 2,7-dichlorofluorescein, and 2,7-difluorofluorescein. 4,5-difluorofluorescein, 2,4,5,7-tetrafluorofluorescein, 2,7-dimethylfluorescein, 4,5-dimethylfluorescein, 2,4,5,7-tetramethylfluorescein, 2,7- Diisopropylfluorescein, 2,7-di-t-butylfluorescein, 2,7-dimethoxyfluorescein, 2,4-difluoro-5,7-dimethylfluorescein, resorufin, 2,8-dichlororesorufin, 7-hydroxy-4- (Trifluoromethyl) coumarin, and 7- A Dorokishi 4-methyl coumarin.

  Next, the sulfonic acid ester compound of the present invention is more preferably represented by any of the following formulas (i) to (iv), for example.

In formulas (i) to (iv),
X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or halogen, which may be the same or different,
X ³ is a linear or branched alkyl group having 1 to 6 carbon atoms, or a trifluoromethyl group,
R ¹ and R ³ are each a hydrogen atom, a nitro group, a methyl group, a chloro group or a methoxy group, and may be the same or different;
R ² and R ⁴ are each a hydrogen atom, a nitro group, a trifluoromethyl group, a methyl group, an isopropyl group, a chloro group or a methoxy group, and may be the same or different;
And at least one of R ¹ and R ² is a nitro group, and at least one of R ³ and R ⁴ is a nitro group, and in formula (iv), X ³ is a straight chain having 1 to 6 carbon atoms or In the case of a branched alkyl group, R ¹ and R ² are both groups other than hydrogen atoms.

When used for a probe for detecting a mercapto compound, it is more preferably represented by any one of the above formulas (ii) to (iv), and both R ¹ and R ² are nitro groups.

  As a particularly preferable example of the sulfonic acid ester compound of the present invention, for example, there is a compound represented by any one of the following formulas 1 and 3-8.

In formulas 1 and 8, X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a methyl group or a halogen, which may be the same or different. In formulas 4, 5 and 6, R ¹ is hydrogen. An atom, a nitro group, a methyl group, a chloro group or a methoxy group, R ² is a hydrogen atom, a nitro group, a trifluoromethyl group, a methyl group, an isopropyl group, a chloro group or a methoxy group, and R ¹ and R ^At least one of ² is a nitro group.

  Among these, when used as a probe for superoxide detection, a sulfonate compound represented by any one of the following formulas 1a to 1d, 3, 4a to 4e, 5a, 5b, 6a and 6b is particularly preferable. When used for a compound detection probe, a sulfonate compound represented by any one of the following formulas 5a, 6a, 7, 8a, 8b and 8c is particularly preferred.

  The method for producing the sulfonic acid ester compound of the present invention represented by the general formula (I) is not particularly limited, and a known method for producing a sulfonic acid ester compound and the like can be appropriately used. ) And a compound of the following formula (III) can be produced by a production method comprising a step of combining them.

In formula (III), X ⁵ is halogen, and A and B are the same as those in formula (I). Moreover, as said compound of Formula (II), the said fluorescent compound etc. can be used, for example, A preferable fluorescent compound is as above-mentioned, for example. That is, as the compound of the formula (II), for example, fluorescein represented by the following formula and its derivatives, resorufin, 7-hydroxy-4- (trifluoromethyl) coumarin, 7-hydroxy-4-methylcoumarin, etc. Although there is a pigment, it is not limited to these, and various compounds can be used.

  In the present invention, “halogen” refers to any halogen element, and examples thereof include fluorine, chlorine, bromine and iodine. The halogenated alkyl group is not particularly limited. For example, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group are each halogenated. Trifluoromethyl group is particularly preferred.

  When isomers such as tautomers, stereoisomers, and optical isomers exist in the compound represented by the formula (I), these isomers are also included in the compound of the present invention. Furthermore, when the compound of the above formula (I) and other compounds according to the present invention can form a salt, the salt is also included in the compound of the present invention. The salt is not particularly limited, and may be, for example, an acid addition salt or a base addition salt. Further, the acid forming the acid addition salt may be an inorganic acid or an organic acid, and the base forming the base addition salt may be an inorganic base. An organic base may be used. Although it does not specifically limit as said inorganic acid, For example, a sulfuric acid, phosphoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, etc. are mention | raise | lifted. The organic acid is not particularly limited, and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. The inorganic base is not particularly limited, and examples thereof include ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, carbonates and hydrogen carbonates, and more specifically, for example, Examples thereof include sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydroxide and calcium carbonate. The organic base is not particularly limited, and examples thereof include ethanolamine, triethylamine, and tris (hydroxymethyl) aminomethane.

  The method for producing the salt of the compound of the present invention is not particularly limited, and for example, it can be produced by a method such as appropriately adding the above acid or base to the compound of the present invention by a known method.

  The fluorescent probe of the present invention exhibits high selectivity for superoxide and mercapto compounds by including the sulfonate compound of the present invention. Therefore, the fluorescent probe of the present invention is excellent in sensitivity and accuracy, and the fluorescent probe of the present invention, or the superoxide detection method or mercapto compound detection method using the same is suitable for application to, for example, a bioimaging method. Therefore, the sulfonate compound of the present invention is also suitable for uses such as clinical analysis reagents.

  In addition, the structure of the sulfonic acid ester compound of the present invention is not limited depending on its use, but it is preferable to select and use the structure appropriately according to the use. When the compound of the present invention is used for a superoxide detection fluorescent probe or a mercapto compound detection probe, preferred compounds for each use are as described above from the viewpoint of specificity (selectivity). By including these compounds of the present invention, the fluorescent probe of the present invention can have high specificity (selectivity) and detectability with respect to superoxide or mercapto compounds.

  The fluorescent probe of the present invention is suitable for uses such as bioimaging as described above, but can be used for various other applications. The use of the fluorescent probe for detecting superoxide of the present invention is not particularly limited. For example, an assay method using blotting (transcription), a superoxide dismutase quantification method, a method for measuring the superoxide elimination ability of functional foods or medicines, clinical analysis It is suitable for a method and a method for screening a medicine. In addition, the use of the fluorescent probe for detecting a mercapto compound of the present invention is not particularly limited. For example, in a kinase assay method, an acetylcholinesterase assay method, an assay method using blotting (transcription), a clinical analysis method, and a pharmaceutical screening method. Is suitable. Hereinafter, these methods will be described more specifically.

  The assay method using blotting (transcription) is an assay method including a step of detecting a substance blotted (transcribed) on a support using the fluorescent probe of the present invention. The operation and the like are not particularly limited, and any known method can be appropriately used except that the substance is reacted with the fluorescent probe of the present invention on the support. The support is not particularly limited, and known materials such as agarose gel, polyacrylamide gel, nitrocellulose membrane, and nylon membrane can be used. For example, by appropriately designing a kit including the fluorescent probe of the present invention and the support, it can be used in the assay method using the blotting.

  Next, the superoxide quantification method using the fluorescent probe for detecting superoxide of the present invention comprises a fluorescent response after reacting the fluorescent probe for detecting superoxide of the present invention with superoxide, and the reaction includes superoxide dismutase. This is done by comparing the case in the presence of the sample with the case in the absence. For example, the superoxide dismutase concentration in the human or animal body fluid is measured by performing the superoxide dismutase quantification method using a human or animal body fluid or a sample extracted from the body fluid as the superoxide dismutase-containing sample. can do. Furthermore, the superoxide dismutase quantification method can be applied to, for example, measurement of superoxide scavenging ability of functional foods or pharmaceuticals. That is, a step of measuring a superoxide dismutase concentration in a body fluid of a human or animal using the superoxide dismutase quantification method, a step of administering a functional food or a medicine to the human or animal, The superoxide scavenging ability of the functional food or medicine can be measured by a method comprising a step of measuring the superoxide dismutase concentration in the human or animal body fluid using an oxide dismutase assay method. For example, by performing assays on humans or animals using these measurement methods, it is possible to screen for drugs for the treatment, prevention or alleviation of symptoms related to superoxide-related diseases. A kit suitable for these measurement methods can be obtained by appropriately designing a kit containing the superoxide detection probe of the present invention.

  Next, the kinase assay method using the probe for detecting a mercapto compound of the present invention includes, for example, a step of thiophosphorylating a substrate using a kinase and γ-S-ATP, and the thiophosphorylated substrate is converted to the mercapto of the present invention. Detecting using a compound detection probe. For example, a protein can be used as the substrate. That is, when a protein is phosphorylated by a kinase and ATP, if γ-S-ATP is used instead of ATP, the protein can be thiophosphorylated. A kinase assay method can be performed by detecting the thiophosphorylated protein with the probe for detecting a mercapto compound of the present invention. The kinase assay method preferably further comprises a step of separating the kinase from the sample using an antibody. A kit suitable for this kinase assay method can be obtained, for example, by appropriately designing a kit containing the mercapto compound detection probe of the present invention, γ-S-ATP and a substrate, and preferably further comprises a kinase antibody.

  Next, an acetylcholinesterase assay method using the fluorescent probe for detecting a mercapto compound of the present invention includes, for example, a step of generating thiocholine by reaction of acetylthiocholine and acetylcholinesterase, and the thiocholine for detecting a mercapto compound of the present invention. Detecting using a fluorescent probe. This acetylcholinesterase assay kit can be obtained by appropriately designing a kit containing the fluorescent probe for detecting a mercapto compound of the present invention and acetylthiocholine.

  The general kinase assay and acetylcholinesterase (AChE) assay are described below, and the advantages of the kinase assay and acetylcholinesterase (AChE) assay of the present invention are described.

  In the case of an enzyme such as AChE or kinase, the amount of activity rather than the amount expressed is closely related to the life phenomenon. The development of these simple enzyme activity assays is very important from a clinical point of view and also very significant from the viewpoint of new drug development. That is, the enzyme activity measurement method can be used not only as a diagnostic method for a disease state for a certain disease, but also as a screening method for enzyme inhibitors that serve as seeds for new drugs. Regardless of whether it is used for diagnostic purposes or screening methods, the assay method is preferably an environmentally friendly technique that is simple in operation and can be measured in a short time.

One of the assays have been used conventionally AChE or kinase, there is a radio assay using labeled substrate acetyl choline ⁽³ H-labeled) or ATP ⁽³² P-labeled) with RI. Although this method has the advantage of high sensitivity, it uses RI, so there is a problem from the viewpoint of environmental impact. As an alternative method for AChE assay, Ellman's reagent is used, but there are problems with sensitivity as described above.

  Furthermore, an enzyme immunoassay has been conventionally used as a non-radioactive technique for the kinase assay. These operating procedures have the following two types. That is, (1) phosphorylation of immobilized substrate → antigen-antibody reaction with phosphorylated substrate by enzyme-modified antibody → washing → color development or chemiluminescence by enzyme reaction → detection, and (2) phosphorylation of immobilized substrate → biotin modification Antigen-antibody reaction with phosphorylated substrate by antibody → washing → complex formation with avidin-modifying enzyme → washing → color development or chemiluminescence by enzyme reaction → detection. These methods have the problem that a modified antibody specific to the phosphorylated substrate is necessarily required.

  The advantages of the kinase assay and the acetylcholinesterase (AChE) assay of the present invention will be described as follows. That is, first, the operation is simple. Specifically, in the AChE assay, the enzyme reaction and the fluorescence reaction can be performed simultaneously, and in the kinase assay, the measurement can be performed by a simple operation of performing the fluorescence reaction after the enzyme reaction. It can also be measured using only the substrate and the probe. Furthermore, it can be expected that the detection sensitivity of the measurement method using the fluorescent probe for detecting a mercapto compound of the present invention can be comparable to that of chemiluminescence.

  By conducting an assay on humans or animals using the acetylcholinesterase assay method or kinase assay method of the present invention, for example, screening for drugs for the treatment, prevention or alleviation of symptoms related to acetylcholine or kinase-related diseases is performed. You can also. The acetylcholine- or kinase-related disease is not particularly limited, and examples thereof include cancer, Alzheimer's disease, hypertension, angina pectoris, arteriosclerotic diseases such as myocardial infarction, inflammatory diseases such as arthritis, and various metabolic diseases. Disease etc. are mentioned.

  Various clinical analysis methods can be carried out by assaying humans or animals using the superoxide detection probe and mercapto compound detection probe of the present invention as described above. For this reason, the sulfonate compound of the present invention is suitable for clinical analysis reagents as described above, and for clinical analysis suitable for various applications by appropriately setting a kit containing this clinical analysis reagent. A kit is obtained.

(Example)
Hereinafter, the present invention will be described in more detail with reference to examples. However, this embodiment is merely an example of the present invention, and the present invention is not limited to this.

  In this example, the compounds 1a to 1d, 3, 4a, 4b, 5a, 5b, 6a and 6b were synthesized, and the function of these compounds as a superoxide detection fluorescent probe was evaluated. In addition, application tests to cell lines were also conducted.

(Measurement conditions, etc.)
The nuclear magnetic resonance (NMR) spectrum was measured using EX-270 (trade name) manufactured by JEOL (270 MHz at ¹ H measurement) as a measuring instrument. Chemical shifts are expressed in parts per million (ppm). Tetramethylsilane (TMS) was used for the internal standard of 0 ppm. Coupling constants (J) are shown in hertz and the abbreviations s, d, t, q, m and br are singlet, doublet, triplet, quadruple, respectively. Represents a quartet, a multiplet, and a broad line. Mass spectrometry (MS) was performed by high resolution (HRMS) / fast atom bombardment method (FAB) using JMS-700 (trade name) manufactured by JEOL. Infrared absorption (IR) spectrum was performed by KBr method using VALOR-III (trade name) manufactured by JASCO Corporation. Elemental analysis was performed using CHN CORDER MT-5 (trade name) manufactured by Yanaco. The melting point was measured using MP-S3 (trade name) manufactured by Yanaco Corporation. Unless otherwise indicated, fluorescence intensity measurements were made using the CytoFluor II multiwell fluorescence plate reader (trade name) manufactured by PerSeptive Biosystems, USA, with excitation and emission filters at 485 ± 20 and 530 ± 25 nm, respectively. It was done by setting. For column chromatography separation, silica gel (manufactured by Merck, silica gel 60 (trade name)) was used. All chemicals were reagent grade and were purchased from any of Aldrich, Lancaster, Tokyo Kasei, Nacalai Tesque, and Wako Pure Chemical unless otherwise indicated.

(Synthesis)
[Synthesis of 1a ~ 1d]
Compounds 1a to 1d were all synthesized by the same method. That is, first, fluorescein 2a to 2d corresponding to each was prepared. Specifically, 2a and 2b are commercially available products, and 2c and 2d are W.-C.Sun, KR Gee, DH Klaubert, RP Haungland, J. Org. Chem. 1997, 62, 6469-6475. Synthesized by the method described. Next, 2,6-lutidine (5.0 mL) was added to a suspension of this fluorescein (1.0 g) and 2,4-dinitrobenzenesulfonyl chloride (2.2 eq) in dichloromethane (20 mL) at 0 ° C. The resulting mixture was stirred at room temperature for 4-6 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL × 2) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane) to obtain the desired product. The yields and instrumental analysis values of compounds 1a to 1d are shown below.

1a: 1.4 g (59%) pale yellow powder. Melting point. 131-135 ° C. ¹ H-NMR (270 MHz, d ₆ -DMSO, TMS): δ = 9.11 (d, ⁴ J _{H, H} = 2.3 Hz , 2H; aromatic ring), 8.62 (dd, ³ J _{H, H} = 8.6 Hz, ⁴ J _{H, H} = 2.3 Hz, 2H; aromatic ring), 8.36 (d, ³ J _{H, H} = 8.6 Hz, 2H; Aromatic ring), 8.05 (d, ³ J _{H, H} = 7.3 Hz, 1H; aromatic ring), 7.85-7.73 (m, 2H, aromatic ring), 7.39-7.36 (m, 3H; aromatic ring), 7.00-6.98 (m, 4H; aromatic ring). FTIR (KBr): ν = 1770 (CO, s), 1557 (NO ₂ , s), 1541 (NO ₂ , s) cm ^-1 . FAB HRMS calculated C ₃₂ H ₁₇ N ₄ O ₁₇ S ₂ (MH ⁺ ): 793.0030; found: 793.0017.
1b: 1.0 g (47%) pale yellow powder. Melting point. 199-202 ° C. ¹ H-NMR (270 MHz, d ₆ -DMSO, TMS): δ = 9.12 (d, ⁴ J _{H, H} = 2.1 Hz , 2H; aromatic ring), 8.66 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.1 Hz, 2H; aromatic ring), 8.43 (d, ³ J _{H, H} = 8.7 Hz, 2H; Aromatic ring), 8.05 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 7.86-7.74 (m, 2H, aromatic ring), 7.59 (s, 2H; aromatic ring), 7.43 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 7.23 (s, 2H; aromatic ring). FTIR (KBr): ν = 1773 (CO, s), 1558 (NO ₂ , s), 1541 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₃₂ H ₁₄ Cl ₂ N ₄ O ₁₇ S ₂ : C 44.61, H 1.64, N 6.50; Found: C 44.54, H 1.82, N 6.27. FAB HRMS calculated C ₃₂ H ₁₅ Cl ₂ N ₄ O ₁₇ S ₂ (MH ⁺ ): 860.9251; found: 860.9221.
1c: 1.5 g (67%) white crystals. Melting point. 140-146 ° C (recrystallized from ethyl acetate). ¹ H-NMR (270 MHz, d ₆ -DMSO, TMS): δ = 9.13 (d, ⁴ J _{H, H} = 2.0 Hz, 2H; aromatic ring), 8.66 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.0 Hz, 2H; aromatic ring), 8.43 (d, ³ J _{H, H} = 8.7 Hz, 2H; aromatic ring), 8.04 (d, ³ J _{H, H} = 7.3 Hz, 1H; aromatic ring), 7.85-7.32 (m, 2H, aromatic ring), 7.58 (d, ⁴ J _{H, F} = 6.3 Hz, 2H; aromatic ring), 7.43 (d, 3J _{H, H} = 7.4 Hz, 1H; aromatic ring), 7.13 (d, ³ J _{H, F} = 10.2 Hz, 2H; aromatic ring). FTIR (KBr ): ν = 1774 (CO, s), 1557 (NO ₂ , s), 1542 (NO ₂ , s) cm ^-1 .Elemental analysis value (%) Calculated value C ₃₂ H ₁₄ F ₂ N ₄ O ₁₇ S ₂・ C ₄ H ₈ O ₂ : C 47.17, H 2.42, N 6.11; Actual value: C 47.20, H 2.47, N 6.03.FAB HRMS calculated value C ₃₂ H ₁₅ F ₂ N ₄ O ₁₇ S ₂ (MH ⁺ ): 828.9842; found: 828.9847.
1d: 1.1 g (51%) white crystals. Melting point. 155-160 ° C (recrystallized from ethyl acetate-hexane). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.74 (d, ⁴ J _{H, H} = 2.1 Hz, 2H; aromatic ring), 8.63 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.1 Hz, 2H; aromatic ring), 8.40 (d, ³ J _{H, H} = 8.7 Hz, 2H; aromatic ring), 8.09 (d, ³ J _{H, H} = 6.9 Hz, 1H; aromatic ring), 7.83-7.72 (m, 2H, aromatic ring), 7.21 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 6.52 (dd, ³ J _{H, F} = 9.3 Hz, ⁴ J _{H, F} = 2.1 Hz, 2H; aromatic ring). FTIR (KBr): ν = 1775 (CO, s ), 1558 (NO ₂ , s), 1542 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₃₂ H ₁₂ F ₄ N ₄ O ₁₇ S ₂ : C 44.45, H 1.64, N 6.48 ; Found: C 44.35, H 1.64, N 6.24. Calculated FAB HRMS C ₃₂ H ₁₃ F ₄ N ₄ O ₁₇ S ₂ (MH ⁺ ): 864.9653; Found: 864.9625.

[Composition of 3]
To a suspension of tetrafluorofluorescein 2d (1.0 g) and 2-nitro-4- (trifluoromethyl) benzenesulfonyl chloride (2.2 eq) in dichloromethane (20 mL) was added 2,6-lutidine (5.0 mL) at 0 ° C. added. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL x 2) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane) to obtain the desired product. The yield and instrumental analysis value of this compound are shown below.

3: 2.1 g (92%) white crystals. Melting point. 138-142 ° C (recrystallized from ethyl acetate). ¹ H-NMR (270 MHz, d ₆ -DMSO, TMS): δ = 8.84 (s, 2H; Aromatic ring), 8.47 (d, ³ J _{H, H} = 8.4 Hz, 2H; aromatic ring), 8.40 (d, ³ J _{H, H} = 8.4 Hz, 2H; aromatic ring), 8.05 (d, ³ J _{H, H} = 6.9 Hz, 1H; aromatic ring), 7.86-7.74 (m, 2H, aromatic ring), 7.46 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 7.15 (dd, ³ J _{H, F} = 10.2 Hz, ⁴ J _{H, F} = 2.1 Hz, 2H; aromatic ring). FTIR (KBr): ν = 1776 (CO, s), 1557 (NO ₂ , s) cm ^-1 . Elemental analysis (% ) Calculated value C ₃₄ H ₁₂ F ₁₀ N ₂ O ₁₃ S ₂・ C ₄ H ₈ O ₂ : C 45.70, H 2.02, N 2.81; Actual value: C 45.56, H 1.82, N 2.73.FAB HRMS calculated value C ₃₄ H ₁₃ F ₁₀ N ₂ O ₁₃ S ₂ (MH ⁺ ): 910.9699; found: 910.9674.

[Synthesis of 4a and 4b]
Both 4a and 4b were synthesized by the same method. Specifically, first, a suspension of tetrafluorofluorescein 2d (2.0 g) and 2-nitro-4- (trifluoromethyl) benzenesulfonyl chloride or 4-nitrobenzenesulfonyl chloride (1.1 eq) in a suspension of dichloromethane (20 mL) at 0 ° C. 2,6-lutidine (1.1 eq) was added. Next, the obtained mixed liquid was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane-acetone = 20: 1) to obtain the desired product. The yields and instrumental analysis values of compounds 4a and 4b are shown below.

4a: 0.72 g (22%) yellow powder. Melting point. 118-135 ° C (recrystallized from ethyl acetate-hexane). ¹ H-NMR (270 MHz, CD ₃ CN, TMS): δ = 8.36 (d, ⁴ J _{H, H} = 1.2 Hz, 1H; aromatic ring), 8.32 (d, ³ J _{H, H} = 8.2 Hz, 1H; aromatic ring), 8.19-7.99 (m, 1H; aromatic ring), 7.82-7.70 (m , 2H; aromatic ring), 7.27-7.24 (m, 1H, aromatic ring), 6.68 (dd, ³ J _{H, F} = 10.1, ³ J _{H, H} = 2.4 Hz, 1H; aromatic ring), 6.49 (dd, ³ J _{H, F} = 10.1 Hz, ⁴ J _{H, F} = 2.4 Hz, 2H; aromatic ring). FTIR (KBr): ν = 3208 (OH, br), 1766 (CO, s), 1557 (NO ₂ , s) cm ^-1 .FAB HRMS calculated C ₂₇ H ₁₁ F ₇ NO ₉ S (MH ⁺ ): 658.0043; found: 658.0040.
4b: 0.6 g (21%) yellow powder. Melting point. 245-250 ° C (recrystallized from ethyl acetate-hexane). ¹ H-NMR (270 MHz, CD ₃ CN, TMS): δ = 8.45-8.40 (m , 2H; aromatic ring), 8.24-8.20 (m, 2H; aromatic ring), 8.02-7.99 (m, 1H; aromatic ring), 7.82-7.70 (m, 2H; aromatic ring), 7.27-7.23 (m, 1H , Aromatic ring), 6.64 (dd, ³ J _{H, F} = 10.1, ³ J _{H, H} = 2.3 Hz, 1H; aromatic ring), 6.47 (dd, ³ J _{H, F} = 10.9 Hz, ⁴ J _{H, F} = 2.3 Hz, 2H; aromatic ring)). FTIR (KBr): ν = = 3183 (OH, br), 1747 (CO, s), 1538 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₂₆ H ₁₁ F ₄ NO ₉ S: C 52.98, H 1.88, N 2.38; Found: C 53.01, H 2.15, N 2.23.FAB HRMS calculated C ₂₆ H ₁₂ F ₄ NO ₉ S (MH ⁺ ) : 590.0169; Found: 590.0161.

[Synthesis of 5a and 5b]
Both 5a and 5b were synthesized by the same method. Specifically, first, a suspension of resorufin sodium salt (2.0 g) corresponding to 5a or 5b in a pyridine (20 mL) suspension at −40 ° C. with 2,4-dinitrobenzenesulfonyl chloride or 2-nitro-4- (trifluoro Methyl) benzenesulfonyl chloride (1.1 eq) was added. Next, the obtained mixed liquid was stirred at −40 to −20 ° C. for 4 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL x 2) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane-acetone = 20: 1) to obtain the desired product. The yield and instrumental analysis values are shown below.

5a: 0.9 g (24%) orange crystals. Melting point. 213-215 ° C (recrystallized from benzene). ¹ H-NMR (270 MHz, [D] ₆ DMSO, TMS): δ = 9.13 (s, 1H; Aromatic ring), 8.62 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 8.34 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 7.90 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 7.55 (d, ³ J _{H, H} = 9.8 Hz, 1H; aromatic ring), 7.47 (s, 1H, aromatic ring), 7.25 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 6.85 (d, ³ J _{H, H} = 9.8 Hz, 1H; aromatic ring), 6.29 (s, 1H; aromatic ring). FTIR (KBr): ν = 1622 (CO, s ), 1558 (NO ₂ , s), 1517 (NO ₂ , s) cm ^-1 .Elemental analysis (%) Calculated C ₁₈ H ₉ N ₃ O ₉ S: C 48.76, H 2.05, N 9.48, S 7.23 ; Actual value: C 48.88, H 2.27, N 9.26, S 7.17.
5b: 1.2 g (30%) orange crystals. Melting point. 204-207 ° C (recrystallized from ethyl acetate). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.35 (d, ³ J _{H, H} = 8.1 Hz, 1H; aromatic ring), 8.15 (s, 1H; aromatic ring), 8.01 (d, ³ J _{H, H} = 8.1 Hz, 1H; aromatic ring), 7.81 (d, ³ J _{H, H} = 8.4 Hz, 1H; aromatic ring), 7.42 (d, ³ J _{H, H} = 9.7 Hz, 1H; aromatic ring), 7.27-7.23 (m, 2H, aromatic ring), 6.87 (dd, ³ J _{H, F} = 9.7, ³ J _{H, H} = 2.0 Hz, 1H; aromatic ring), 6.33 (d, ⁴ J _{H, H} = 2.0 Hz, 1H; aromatic ring). FTIR (KBr): ν = 1647 (CO, s), 1561 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₁₉ H ₉ F ₃ N ₂ O ₇ S: C 48.93, H 1.95, N 6.01; Found: C 48.75, H 2.05, N 5.76. Calculated FAB HRMS C ₁₉ H ₁₀ F ₃ N ₂ O ₇ S (MH ⁺ ): 467.0161; Found: 467.0149.

[Synthesis of 6a and 6b]
Both 6a and 6b were synthesized by the same method. That is, first, 7-hydroxy-4- (trifluoromethyl) coumarin (1.0 g) and 2,4-dinitrobenzenesulfonyl chloride or 2-nitro-4- (trifluoromethyl) benzenesulfonyl chloride (1.1 eq) Triethylamine (1.1 eq) was added to a suspension of dichloromethane (20 mL) at 0 ° C. Next, the obtained mixed liquid was stirred at room temperature for 1 hour. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane) to obtain the desired product. The yield and instrumental analysis values are shown below.

6a: 1.9 g (95%) white crystals. Melting point. 123-124.5 ° C (recrystallized from benzene). ¹ H-NMR (270 MHz, [D] ₆ -DMSO, TMS): δ = 9.13 (d, ⁴ J _{H, H} = 2.3 Hz, 1H; aromatic ring), 8.64 (dd, ³ J _{H, H} = 8.7 Hz, ⁴ J _{H, H} = 2.3 Hz, 1H; aromatic ring), 8.35 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 7.79 (dd, ³ J _{H, H} = 8.9 Hz, ³ J _{H, H} = 1.5 Hz, 1H; aromatic ring), 7.54 (d, ⁴ J _{H, H} = 2.5 Hz, 1H, aromatic ring), 7.32 (dd, ³ J _{H, H} = 8.9, ⁴ J _{H, H} = 2.5 Hz, 1H; aromatic ring), 7.15 (s, 1H; aromatic ring). FTIR (KBr): ν = 1751 (CO, s), 1558 (NO ₂ , s), 1542 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated value C ₁₆ H ₇ F ₃ N ₂ O ₉ S: C 41.75 , H 1.53, N6.09; Found: C 41.74, H 1.63, N 5.92.FAB HRMS calculated C ₁₆ H ₈ F ₃ N ₂ O ₉ S (MH ⁺ ): 460.9903; Found: 460.9888.
6b: 2.0 g (95%) white crystals. Melting point. 134.5-136 ° C (recrystallized from ethyl acetate-hexane). ¹ H-NMR (270 MHz, [D] ₆ -DMSO, TMS): δ = 8.82 ( s, 1H; aromatic ring), 8.33 (d, ⁴ J _{H, H} = 1.5 Hz, 2H; aromatic ring), 7.83 (dd, ³ J _{H, H} = 8.9 Hz, ⁴ J _{H, H} = 1.8 Hz, 1H ; Aromatic ring), 7.57 (t, ⁴ J _{H, H} = 2.2 Hz, 1H; aromatic ring), 7.34 (dt, ³ J _{H, H} = 8.9 Hz, ⁴ J _{H, H} = 2.2 Hz, 1H, aromatic ring ), 7.15 (s, 1H; aromatic ring). FTIR (KBr): ν = 1752 (CO, s), 1557 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₁₇ H ₇ F ₆ NO ₇ S: C 42.25, H 1.46, N 2.90; Found: C 42.24, H 1.55, N 2.72.FAB HRMS calculated C ₁₇ H ₈ F ₆ NO ₇ S (MH ⁺ ): 483.9926; Found: 483.9925 .

(Evaluation as a superoxide selective fluorescent probe)
When the reactivity with superoxide was investigated about compound 1a-1d, all reacted with superoxide and showed a fluorescence response, but it turned out that 1d is especially excellent in points, such as a sensitivity. Therefore, next, the performance as a superoxide-selective fluorescent probe was evaluated mainly using the compound 1d as a test compound, and a test in a cell system was also conducted. Hereinafter, these tests will be described.

[1-1. Reactivity to superoxide]
The reactivity of compound 1d with superoxide was tested as follows. That is, Compound 1d (0.25 mmol) was reacted with KO ₂ (5 eq) in a mixed solution (1: 1) of DMSO and 10 mM HEPES buffer at pH 7.4 at room temperature for 10 minutes. The fluorescence derived from 2d was confirmed. Examination of the product showed that 1d was completely consumed and an additional 0.24 mmol of 2,4-dinitrophenol was isolated. The product was identified by comparing ¹ H-NMR, IR and mass spectra with commercially available compounds. Although this reaction mechanism is not clear, it is estimated as shown in the following scheme 7, for example. Since the amount of 2,4-dinitrophenol generated was almost 1 equivalent to 1d despite the large excess of KO ₂ , the second elimination reaction (conversion reaction from 17d to 2d) It seems that it is not caused by the reaction with superoxide but rather by another mechanism. However, Scheme 7 is merely an example of a mechanism presumed under the above conditions for Compound 1d, and does not limit the present invention.

[1-2. Superoxide selectivity (1)]
First, when the performance of the compound 1d as a superoxide selective fluorescent probe was tested, it was found that 1d has an excellent specificity for superoxide. FIG. 1 shows the test results. This figure is a graph showing the results of tracing fluorescence emission by reacting compound 1d with H ₂ O ₂ or enzymatically generated superoxide, the horizontal axis is the reaction time (seconds), and the vertical axis is the fluorescence intensity. (Au). In the figure, (a) is in the presence of HPX and XO, (b) is in the presence of HPX, XO and SOD, (c) is anaerobic under the presence of HPX, XO and SOD, and (d) is H ₂ O. The results when reacted with ₂ only are shown. Hereinafter, the test results of FIG. 1 will be described in more detail.

  First, as shown in FIG. 1 (a), 1d significantly increased the fluorescence response in a short time in response to superoxide generated from the hypoxanthine (HPX) / xanthine oxidase (XO) system. It was found that it has high sensitivity. More specifically, compound 1d (16 μM), xanthine oxidase (XO, 0.01 U / mL), and hypoxanthine (HPX, 40 μM) at 37 ° C. in HEPES buffer (pH 7.4, 10 mM, 2.5 mL). ) Followed by an excitation wavelength of 511 nm and an emission wavelength of 531 nm, a fluorescence response derived from the formation of compound 2d (Scheme 4 above) was observed, and FIG. The progressive curve shown was obtained. In addition, when the test was conducted in the same manner except that superoxide dismutase (SOD) (40 U / mL) was further added, the fluorescence response was significantly inhibited as shown in FIG. ) Was confirmed to be caused by superoxide.

As shown in FIG. 1 (d), the reaction between compound 1d and H ₂ O ₂ (40 μM) caused only a negligible increase in fluorescence intensity. That is, it was shown that 1d hardly responds to hydrogen peroxide and has excellent specificity for superoxide.

Further, from the results shown in FIG. 1 (c), it can be seen that the compound 1d has an advantage as compared with nitro blue tetrazolium (NBT) which is a conventional superoxide detector. Specifically, it is as follows. NBT is used for probes for spectrophotometric measurement and the like because it has the property of being reduced by superoxide O ₂ ⁻ . However, NBT has a big problem that it is reduced not only by superoxide but also by reductase present in biological systems. Therefore, the compounds according to 1d O ₂ ^- whether, detection has a similar problem, compound 1d under anaerobic conditions, XO, was tested by an enzymatic reaction using a HPX and SOD, in FIG. 1 (c) As shown, the production of 1d to 2d due to the XO reductant was very slow. This result is in sharp contrast to the observation that the reduction of NBT to formazan by the xanthine-XO system reduced more rapidly under anaerobic conditions than under aerobic conditions. Thus, fluorescence-based detection of O ₂ ⁻ using compound 1d has been shown to eliminate or significantly reduce complications due to intracellular reductase.

[1-3. Superoxide selectivity (2)]
Furthermore, reactivity with H ₂ O ₂ and other various nucleophiles, reducing agents, etc. was also tested for all of the compounds 1a to 1d under conditions different from the above. More specifically, XO-HPX, XO-HPX-SOD, H ₂ O ₂ , ascorbic acid, 1,4-hydroquinone, propylamine, diethylamine, glucose, esterase, cytochrome P450 reductase + NADPH and diaphorase + The fluorescence response was measured for each of the NADH systems. The operation is as follows. That is, first, 1a to 1d and each of the above reagents were dissolved in 10 mM HEPES at pH 7.4 to prepare a solution. Next, a 96-well microplate is prepared, and each well is filled with one of the solutions 1a to 1d (25 μM, 170 μL), and further, 30 μL of the blank solution or the reagent to be reacted is added. 30 μL of solution was added. Final concentrations in the wells were 21.3 μM for 1a-d, 13.1 mU / mL for XO, HPX, H ₂ O ₂ , ascorbic acid, 1,4-hydroquinone, propylamine, diethylamine, glucose, NADPH and NADH was adjusted to 50 μM each, cytochrome P450 reductase to 68 mU / mL, diaphorase to 65 mU / mL, and esterase to 0.5 U / mL. And after making it react at 37 degreeC for 10 minutes as it was in the well, the fluorescence response was measured. For fluorescence intensity measurement, JASCO FP-750 spectrofluorometer (trade name) is equipped with JASCO ETC-272 Peltier thermostatted single cell holder (trade name) or SpectraMax GeminiEM fluorescence plate reader (trade name) manufactured by Molecular Devices. The excitation and emission wavelengths were measured at 1485, 515 nm for 1a, 1b and 1c and 511 and 540 nm for 1d, respectively. As a result, as shown in Table 1 below, it was confirmed that any of 1a to 1d showed excellent response to superoxide from the results for XO-HPX and XO-HPX-SOD systems. However, it did not respond at all to H ₂ O ₂ , ascorbic acid, 1,4-hydroquinone, propylamine, diethylamine, and glucose, and only a negligibly small response to esterase. The cytochrome P450 reductase + NADPH system and the diaphorase + NADH system were also found to have no response that would affect the detection of superoxide. 1a, 1b, 1c, and 1d show a cathodic response near -0.5 V vs. SCE in a voltammetric measurement with CH ₃ CN-H ₂ O (3: 1) containing 0.1 M Et ₄ NClO ₄ Although it was also expected that superoxide detection was inhibited by reduction with intracellular reductase other than oxide, contrary to that expectation, it showed high selectivity for superoxide as described above.

(Table 1) Comparison of fluorescence response of 1a, 1b, 1c or 1d to various biological reactants or enzyme systems

Reagent or enzyme system Fluorescence response
1a 1b 1c 1d
Blank 10 10 10 10
XO-HPX 70 288 797 554
XO-HPX-SOD 12 22 48 60
H ₂ O ₂ 10 11 11 11
Ascorbic acid 10 11 11 11
1,4-hydroquinone 10 11 10 11
Propylamine 10 10 10 10
Diethylamine 11 10 10 11
Glucose 10 10 10 10
Esterase 17 20 23 15

(* The numerical values in Table 1 above are relative fluorescence intensity values calculated with the fluorescence response intensity at blank time set to 10.)

[2. Quantitative]
The sensitivity and quantification in the fluorescence response of the probe (compound 1d) in the HPX / XO system was tested by a 96 well microplate assay. Specifically, a probe solution was prepared by diluting 1d DMSO solution (10 mM) 400 times with HEPES (pH 7.4, 10 mM), and this probe solution (150 μL) was added to HPX HEPES solution (10 μL). ) And XO in HEPES solution (0.26 U / mL, 10 μL) and allowed to stand at 37 ° C. for 10 minutes, and then the fluorescence intensity was measured. When the measurement was performed with various changes in the HPX concentration, the detection limit value of the HPX concentration was 5.0 pmol (RSD, n = 8; 2.7%), and it was found that this value was compatible with cell-level analysis. It was. Furthermore, since a good linear calibration curve was obtained when HPX was between the detection limit value and 10.0 nmol, the fluorescence response of 1d was relative to the amount of O ₂ ⁻ · generated by the enzymatic reaction of HPX and XO. It was confirmed to show good dependence. The correlation coefficient of this linear calibration curve was 1.000, and the slope was 0.58 au / pmol.

  Further, when the quantitative property was tested in the same manner as described above except that the fluorescence intensity measuring instrument and the assay conditions were changed, it was confirmed that excellent quantitative property was also exhibited. That is, for the assay conditions, the 1d DMSO solution concentration was 5 mM, and this was diluted 400 times with HEPES (pH 7.4, 10 mM) to prepare the probe solution, and the probe solution usage was 180 μL. The procedure was the same as above except that. For fluorescence intensity measurement, JASCO FP-750 spectrofluorometer (trade name) is equipped with JASCO ETC-272 Peltier thermostatted single cell holder (trade name) or SpectraMax GeminiEM fluorescence plate reader (trade name) manufactured by Molecular Devices. It was done. As a result, the detection limit value of HPX concentration is 1.0 pmol (RSD, n = 8; 2.9%), and the correlation coefficient is 0.9993 and the slope is 0.80 au / pmol between HPX and 2.0 nmol. A straight calibration curve was obtained. The reason why the slope of the linear calibration curve is different from the above is that the unit of the fluorescence intensity is an arbitrary unit and the measuring instrument is different.

[3. Application to cell systems]
It is known that neutrophils are stimulated with phorbol esters to produce superoxide. When the possibility of 1d as a fluorescent probe to measure this phenomenon was tested using neutrophils stimulated with phorbol myristate acetate (PMA), 1d was able to detect superoxide with high sensitivity even in cell lines. It was revealed. The specific test method is as follows.

First, whole blood provided by healthy volunteers was heparinized, added to a monopoly separation solution (Mono-Poly resolving medium) manufactured by Dainippon Pharmaceutical Co., Ltd., and centrifuged. After separation, the cells are washed twice with PBS (−), resuspended at a concentration of 1 × 10 ⁶ cells or 1 × 10 ⁵ cells / mL with PBS (+), and cooled on ice until use. Stored under conditions. PBS is phosphate buffered saline, that is, phosphate buffered saline, PBS (+) refers to PBS containing 0.54 mM CaCl ₂ and 1.22 mM MgSO ₄ , and PBS (-) includes them Not PBS. The cell suspension (100 μL) thus obtained, probe solution (25 μM in PMS (+), 50 μL), PMA solution (0.64 μM in PBS (+), 50 μL) or blank solution ( PBS (+), 50 μL) was added to a 96-well flat bottom microtiter plate (Asahi Techno Glass Co., Ltd.). The fluorescence intensity was immediately measured (measured at time 0) and then incubated at 37 ° C. for 120 minutes. During the incubation, changes in the fluorescence intensity of the cells were measured every 30 minutes. All measurements were performed using the CytoFluor II multiwell fluorescence plate reader (trade name) manufactured by PerSeptive Biosystems, USA, with the excitation and emission filters set to 485 ± 20 and 530 ± 25 nm, respectively. It was.

The result of the test is shown in the graph of FIG. The figure shows the results in the presence or absence of PMA when the number of cells in each well is 1.0 × 10 ⁴ or 1.0 × 10 ⁵ , respectively. The horizontal axis represents incubation time (minutes), and the vertical axis represents fluorescence intensity (au). As can be seen from the figure, the assay with compound 1d clearly showed that PMA-stimulated neutrophils released O ₂ ⁻ . An increase in fluorescence intensity was also observed in unstimulated cells, which is believed to be due to neutrophil activation due to the interaction between the tissue culture plate surface and the cells used, according to previous studies. ing.

In addition, by changing the fluorescence intensity measuring device, ie, JASCO FP-750 spectrofluorometer (trade name), JASCO ETC-272 Peltier thermostatted single cell holder (trade name) or Molecular Devices SpectraMax GeminiEM fluorescence plate reader When the test was performed in the same manner as described above except that (trade name) was used and the change in fluorescence intensity was measured every 15 minutes, the same result was confirmed. The result is shown in the graph of FIG. The number of cells in each well is 1.0 × 10 ⁵ , and the horizontal axis is the incubation time (minutes). The vertical axis represents the fluorescence intensity (au), and is expressed as the average value ± standard deviation of the actual measurement values obtained for 8 wells. The numerical values of the fluorescence intensities are different between FIGS. 2 and 3 because the unit is an arbitrary unit and the measuring instrument is different.

Furthermore, the toxicity of compound 1d to neutrophils was evaluated by estimating cell viability by trypan blue staining. That is, neutrophils (1.0 × 10 ⁵ cells) were either placed in a glass test tube coated with human serum albumin under the conditions where both compound 1d (6.25 μM) and PMA (0.16 μM) were present. Incubation was carried out at 37 ° C. for 120 minutes in the presence of only one, as well as in the absence of either, and the cell viability after incubation relative to before incubation was measured. As a result, in the presence of neither 1d nor PMA, the cell viability after incubation was 98% compared to that before incubation. In contrast, the cell viability after incubation was 95% in the presence of 1 only, 93% in the presence of PMA alone, and 97% in the presence of both 1 and PMA. That is, it can be seen that almost no neutrophil death occurred under any condition. Thus, it was confirmed that not only PMA but also Compound 1d did not show toxicity to neutrophils in the 120-minute incubation.

The above results also show that reductive deprotection from compound 1d to 2d is effectively induced by O ₂ ⁻ ·, so that compound 1d is released from PMA-stimulated neutrophils O ₂ ⁻ -It has been shown that it functions as a novel fluorescent probe for detection and does not damage cells during incubation. The probe and its homologues, cells derived with the O ₂ ^- to facilitate measurement of,, not mitochondria only, dynamic capabilities of oxidative stress expressed by phagocytic cells (phagocytes) and vascular cells (vascular cells) ( It can be expected to help elucidate dynamic functions.

[4. Test for other compounds]
As with compound 1d, 1a to 1c, 5a and 6a containing 2,4-dinitrobenzenesulfonyl group were tested for their performance as fluorescent probes for superoxide detection. It became clear that it was converted to a pigment. Further, 3, 4a, 5b and 6b introduced with 4-trifluoromethyl-2-nitrobenzenesulfonyl group instead of 2,4-dinitrobenzenesulfonyl group, and 4b introduced with 4-nitrobenzenesulfonyl group are also superoxide. It was also found that it was deprotected and converted to a fluorescent dye. Therefore, any compound consisting of a combination of a phenolic dye and 2,4-dinitrobenzenesulfonyl group, 4-trifluoromethyl-2-nitrobenzenesulfonyl group or 4-nitrobenzenesulfonyl group is also used as a fluorescent probe for superoxide detection. It is considered that it can be used. For example, as shown in Scheme 8 below. Furthermore, the present invention is not limited to these combinations, and it is suitable for the intended purpose by freely designing the compound of the present invention within the scope of the general formula (I) and utilizing the fluorescence mechanism developed by the present inventors. It can be expected that a compound for fluorescent probe having characteristics can be freely designed and developed. As the use of the compound of the present invention, for example, a wide variety of uses are conceivable, such as application to a micro coloring technique using superoxide.

  In this example, in addition to the compound synthesized in Example 1, sulfonic acid ester compounds represented by the formulas 4c to 4e, 7 and 8a to c were synthesized. These compounds were tested for functionality as a fluorescent probe for detecting a mercapto compound. Unless otherwise indicated, JASCO FP-750 spectrofluorometer (trade name), JASCO ETC-272 Peltier thermostatted single cell holder (trade name) or Molecular Devices SpectraMax GeminiEM fluorescence plate reader (product) Name) was used and measured. The other measurement conditions are the same as in Example 1.

[Synthesis of 4c]
First, a suspension of tetrafluorofluorescein (2.0 g) and 2-nitrobenzenesulfonyl chloride (1.1 eq) in dichloromethane (20 mL) was prepared. The suspension was then cooled to 0 ° C. and 2,6-lutidine (1.1 eq) was added at that temperature. The resulting mixture was stirred at room temperature for 4 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL) and saturated brine (200 mL), and dried over magnesium sulfate. Further, the solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel column chromatography (dichloromethane-acetone = 20: 1) to obtain the target compound 4c. The yield and instrumental analysis value of this compound are shown below.

4c: 0.66 g (22%) Dark yellow crystals. Melting point. 234-246 ° C (recrystallized from ethyl acetate-hexane). 1H-NMR (270 MHz, [D] ₆ DMSO, TMS): δ = 11.33 (s , 1H, COOH), 8.27 (t, ³ J _{H, H} = 8.9 Hz, 2H; aromatic ring), 8.17 (t, ³ J _{H, H} = 7.7 Hz, 1H; aromatic ring), 8.05-7.97 (m, 2H; aromatic ring), 7.85-7.73 (m, 2H; aromatic ring), 7.43 (d, ³ J _{H, H} = 7.4 Hz, 1H, aromatic ring), 7.02 (d, 3JH, F = 10.1 Hz, 1H; Aromatic ring), 6.59 (d, ³ J _{H, F} = 10.9 Hz, 2H; aromatic ring). FTIR (KBr): ν = 3254 (OH, br), 1752 (CO, s), 1553 (NO ₂ , s ) cm ^-1 .FAB HRMS calculated C ₂₆ H ₁₂ F ₄ NO ₉ S (MH +): 590.0169; Found: 590.0167.

[Synthesis of 4d and 4e]
4d was obtained in the same manner as the synthesis of 4c except that 2-nitro-4-methoxybenzenesulfonyl chloride was used in place of 2-nitrobenzenesulfonyl chloride. Further, 4e was obtained in the same manner as in the synthesis of 4c except that 4-nitro-2-methoxybenzenesulfonyl chloride was used in place of 2-nitrobenzenesulfonyl chloride. The yields and instrumental analysis values are shown below.

4d: 0.46 g (15%) Dark orange powder. Melting point. 111-125 ° C. ¹ H-NMR (270 MHz, CD ₃ CN, TMS): δ = 8.07-8.01 (m, 2H; aromatic ring), 7.79 -7.68 (m, 2H; aromatic ring), 7.36 (s, 2H; OH and aromatic ring), 7.20-7.16 (m, 2H; aromatic ring), 6.41 (dd, ³ J _{H, F} = 9.5, ³ J _{H , H} = 2.2 Hz, 1H; aromatic ring), 6.34 (dd, ³ J _{H, F} = 10.2 Hz, ⁴ J _{H, F} = 2.0 Hz, 1H; aromatic ring), 3.99 (s, 3H; OCH ₃ ). FTIR (KBr): ν = = 3227 (OH, br), 1755 (CO, s), 1552 (NO ₂ , s) cm ^-1 . Elemental analysis (%) Calculated C ₂₆ H ₁₁ F ₄ NO ₉ S : C 52.98, H 1.88, N 2.38; found: C 53.01, H 2.15, N 2.23.FAB HRMS calculated C ₂₇ H ₁₄ F ₄ NO ₁₀ S (MH ⁺ ): 620.0275; found: 620.0280.
4e: 0.52 g (17%) Dark orange powder. Melting point. 127-144 ° C. 1H-NMR (270 MHz, CD ₃ CN, TMS): δ = 8.10-8.05 (m, 2H; aromatic ring), 7.95- 7.90 (m, 2H; aromatic ring), 7.79-7.68 (m, 2H; aromatic ring), 7.18 (d, ³ J _{H, H} = 6.9 Hz ,, 1H; aromatic ring), 6.39 (dd, ³ J _{H, F} = 9.6, ³ J _{H, H} = 2.0 Hz, 1H; aromatic ring), 6.33 (dd, ³ J _{H, F} = 10.3 Hz, ⁴ J _{H, F} = 1.9 Hz, 1H; aromatic ring), 4.12 (s _{, 3H; OCH 3) FTIR (} KBr):.. ν = 3222 (OH, br), 1763 (CO, s), 1538 (NO 2, s) cm -1 FAB HRMS calcd C ₂₇ H ₁₄ F ₄ NO ₁₀ S (MH ⁺ ): 620.0275; found: 620.0280.

[Synthesis of 7]
First, a suspension of 4-methyl-7-hydroxycoumarin (1.0 g) in 2 dichloromethane (20 mL) was prepared, cooled to 0 ° C., and triethylamine (1.2 eq) at that temperature. ) Was added. This was stirred for 5 minutes before 2,4-dinitrobenzenesulfonyl chloride (1.2 eq) was further added. The resulting mixture was stirred at room temperature for 4-6 hours. The reaction mixture was diluted with dichloromethane (200 mL), washed with 1M hydrochloric acid (200 mL) and saturated brine (200 mL), and dried over magnesium sulfate. The residue obtained by evaporating the solvent under reduced pressure was purified by silica gel column chromatography (dichloromethane-acetone = 20: 1) to obtain the target compound 7. The yield and instrumental analysis value of this compound are shown below.

7: 2.2 g (95%) white needles. Melting point. 188-190 ° C (recrystallized from benzene). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ = 8.75 (d, ⁴ J _{H , H} = 2.0 Hz, 1H; aromatic ring), 8.45 (dd, ³ J _{H, H} = 8.6 Hz, ⁴ J _{H, H} = 2.0 Hz, 1H; aromatic ring), 8.16 (d, ³ J _{H, H} = 8.6 Hz, 1H; aromatic ring), 7.75 (d, ³ J _{H, H} = 8.6 Hz, 1H; aromatic ring), 7.21-7.16 (m, 2H; aromatic ring), 6.31 (s, 1H; aromatic ring), 2.41 (S, 3H; CH ₃ ). FTIR (KBr): ν = 1732 (CO, s), 1560 (NO ₂ , s), 1537 (NO ₂ , s) cm ^-1 . FAB HRMS calculated C ₁₆ H ₁₁ N ₂ O ₉ S (MH ⁺ ): 407.0185; found: 407.0184.

[Synthesis of 8a-c]
Compounds 8a-c were synthesized in the same manner as the synthesis of Compound 7, except that fluorescein derivatives (1.0 g) corresponding to the structures of 8a-c were used instead of 4-methyl-7-hydroxycoumarin, respectively. . The yield and instrumental analysis value of these compounds are shown below.

8a: 0.63 g (37%) yellow solid. Melting point. 125-147 ° C. ¹ H-NMR (270 MHz, CD ₃ OD, TMS): δ 8.87 (d, ⁴ J _{H, H} = 2.8 Hz, 1H; Aromatic ring), 8.44 (dd, ³ J _{H, H} = 9.2 Hz, ⁴ J _{H, H} = 2.8 Hz, 1H; aromatic ring), 8.03-7.98 (m, 1H; aromatic ring), 7.81-7.69 (m, 2H; aromatic ring), 7.33-7.16 (m, 3H; aromatic ring), 6.90-6.81 (m, 2H; aromatic ring), 6.69-6.55 (m, 3H; aromatic ring). FTIR (KBr): ν = 3438 (OH, br) 1736 (CO, s), 1539 (NO ₂ , s) cm ^-1 .FAB HRMS calculated C ₂₆ H ₁₅ N ₂ O ₁₁ S (MH ⁺ ): 563.0397; found: 563.0396.
8b: 0.75 g (46%) yellow solid. Melting point. 131-156 ° C. ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ 8.70 (s, 1H; aromatic ring), 8.58 (d, ³ J _{H, H} = 8.4 Hz, 1H; aromatic ring), 8.32 (d, ³ J _{H, H} = 8.4 Hz, 1H, aromatic ring), 8.06 (d, ³ J _{H, H} = 6.8 Hz, 1H; aromatic ring) , 7.71-7.66 (m, 2H; aromatic ring), 7.15 (d, ³ J _{H, H} = 6.8 Hz, 1H; aromatic ring), 7.05 (s, 1H; aromatic ring), 6.66 (s, 1H; aromatic ring ), 6.63 (s, 1H; aromatic ring), 6.47 (s, 1H; aromatic ring), 5.67 (s, 1H; OH), 2.06 (S, 6H; CH ₃ x 2). FTIR (KBr): ν = 3318 (OH, br), 1735 (CO, s), 1557 (NO ₂ , s), 1542 (NO ₂ , s) cm ^-1 .FAB HRMS calculated C ₂₈ H ₁₉ N ₂ O ₁₁ S (MH ⁺ ) : 590.0710; Found: 599.0698.
8c: 0.62 g (38%) yellow solid. Melting point. 143-158 ° C (recrystallized from CHCl ₃ ). ¹ H-NMR (270 MHz, CDCl ₃ , TMS): δ 8.67 (d, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 8.55 (dd, ³ J _{H, H} = 8.6 Hz, ⁴ J _{H, H} = 2.1 Hz, 1H; aromatic ring), 8.25 (d, ³ J _{H, H} = 8.6 Hz , 1H, aromatic ring), 8.01 (d, ³ J _{H, H} = 7.1 Hz, 1H; aromatic ring), 7.72-7.60 (m, 2H; aromatic ring), 7.16 (d, ³ J _{H, H} = 7.4 Hz , 1H; aromatic ring), 6.73 (d, ³ J _{H, H} = 7.4 Hz, 1H; aromatic ring), 6.60 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 6.55 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 6.48 (d, ³ J _{H, H} = 8.7 Hz, 1H; aromatic ring), 5.50 (s, 1H; OH), 2.44 (S, 3H; CH ₃ .), 2.38 (S, 3H ; CH 3) FTIR (KBr): ν = 3266 (OH, br), 1735 (CO, s), 1558 (NO 2, s), 1541 (NO 2, s) cm - . ¹ FAB HRMS calcd _{C 28 H 19 N 2 O 11} S (MH +): 590.0710; Found: 599.0705.

(Evaluation as a mercapto compound responsive fluorescent probe)
[1. Specificity for mercapto compounds]
For compounds 1d, 5a, 6a, 7 and 8a, [1-3. Under the same conditions as the superoxide selectivity (2)], the reactivity with glutathione (GSH), which is a kind of superoxide and thiol, was tested. FIG. 4 shows the result. The vertical axis represents the fluorescence intensity and is expressed in% of control. As shown in the figure, it was found that 1d was excellent in superoxide selectivity (specificity), and 5a, 6a, 7 and 8a were excellent in mercapto compound selectivity although they also showed a response to superoxide. In addition, it was confirmed that compounds 8b and 8c also responded to glutathione with high sensitivity. From these facts, 7-hydroxycoumarin, resorufin, and fluorescein, which are fluorescent compounds having a phenolic hydroxyl group, and O-2,4-dinitrobenzenesulfonylated derivatives of these derivatives are compared with mercapto compounds such as glutathione and cysteine. Therefore, it seems to be particularly preferable as a probe that responds with high sensitivity in a short time. Further, for 5a, 6a, 7, 8a, 8b and 8c, the response to each compound shown in Table 1 ([1-3. Superoxide selectivity (2)] of Example 1) is the same as that of Example 1. As a result of testing under the condition, it did not show a response at all. That is, it was found that the compounds 5a, 6a, 7, 8a, 8b, and 8c hardly respond to a reactive agent other than a mercapto compound such as H ₂ O ₂ or an amine, and thus the selectivity to the mercapto compound was extremely high.

[2. Evaluation as mercapto compound responsive fluorescent probe]
Next, the performance of the compound 6a and the compound 8a as a mercapto compound responsive fluorescent probe was evaluated.

Glutathione (GSH) and cysteine, which are thiols, were used to evaluate the quantitative properties of compounds 6a and 8a with respect to mercapto compounds. That is, first, an EtOH solution (10 mM) of compound 6a was prepared, which was further diluted 500-fold with a HEPES buffer solution of pH 7.4 to prepare a probe solution containing compound 6a. Next, add 200 μL of this probe solution and 10 μL of various concentrations of glutathione or cysteine solution (dissolved in HEPES buffer at pH 7.4) to each well of a 96-well microplate and leave at 37 ° C for 10 minutes. And reacted. Further, a probe solution containing 8a was prepared in the same manner except that 8a was used in place of compound 6a, and this probe solution was also reacted with a thiol solution in the same manner as the probe solution containing 6a. And after reaction, the fluorescence response was measured about each well. The measurement was performed at λ _ex = 383 nm and λ _em = 500 nm for 6a and at λ _ex = 483 nm and λ _em = 515 nm for 8a. Here, λ _ex represents the excitation wavelength, and λ _em represents the emission wavelength.

  FIG. 5 shows the measurement result of the fluorescence response. FIG. 5A is a graph showing the measurement results for 6a, and FIG. 5B is a graph showing the measurement results for 8a. As shown in the figure, when 6a was used, the detection limits of GSH and cysteine were both 7 pmol, and a good calibration curve was obtained in the concentration range of 7 pmol to 1000 pmol. For GSH, the slope = 0.37 au / pmol and the correlation coefficient (r) = 0.9998, and for cysteine, the slope = 0.66 au / pmol, r = 0.9995. On the other hand, when 8a was used, the detection limits of GSH and cysteine were 2 and 1 pmol, respectively, and a good calibration curve was obtained in the concentration range of 1000 pmol from the detection limit. For GSH, the slope = 4.25 au / pmol, correlation coefficient (r) = 1.0000, and for cysteine, the slope = 4.82 au / pmol, r = 0.9999.

  From these results, it was shown that 6a and 8a can be used as mercapto compound-responsive fluorescent probes. Specifically, it can be seen from the value of the correlation coefficient (r) that the sample has very good quantitativeness, and the magnitude of the slope has high sensitivity. In particular, the sensitivity of 8a was high.

  Furthermore, the fluorescent probe containing 6a or 8a has a very short reaction time, and 6a and 8a itself is not fluorescent, and only the dye produced in the reaction is fluorescent. It was characteristic that the separation operation was unnecessary.

  Furthermore, when the above fluorescent probe containing 6a or 8a was reacted under the same conditions except that another nucleophile containing a hydroxyl group or an amino group was used instead of thiol, no fluorescence was confirmed. It was done. That is, it was found that these fluorescent probes do not respond to hydroxyl groups, amino groups, etc., and have high specificity (selectivity) for mercapto compounds.

  Compounds 5a, 7, 8b and 8c were also tested in the same manner, and similar results were confirmed.

[3. Acetylcholinesterase assay]
As shown in Scheme 9 below, an enzymatic reaction using acetylthiocholine as a substrate and acetylcholinesterase as an enzyme produces thiocholine, which is a kind of mercapto compound (thiol). If this thiocholine is detected by the sulfonate compound of the present invention as shown in the following scheme 10, for example, it can be expected that the activity of acetylcholinesterase (AChE) can be measured in a short time. Scheme 10 is merely an example of a possible mechanism and does not limit the present invention.

  The present inventors have confirmed that the above-mentioned compounds can actually be used for measuring the activity of acetylcholinesterase. Specific operations for Compound 8a are shown below. That is, first, an EtOH solution (10 mM) of compound 8a was prepared, which was further diluted 500-fold with a HEPES buffer of pH 7.4 to prepare a probe solution (20 μM) containing compound 8a. On the other hand, a solution (1 mM) in which commercially available acetylthiocholine was dissolved in HEPES buffer at pH 7.4 was prepared. Furthermore, various concentrations (activity amounts) of acetylcholinesterase (AChE) solutions (solutions dissolved in pH 7.4 HEPES buffer) were prepared.

Next, 200 μL of the probe solution, 10 μL of acetylthiocholine solution, and 10 μL of acetylcholinesterase (AChE) solution of various concentrations (activity) are added to each well of a 96-well microplate and incubated at 37 ° C. for 10 minutes. Enzymatic reaction was performed. Immediately thereafter, the fluorescence response was measured at λ _ex (excitation wavelength) = 483 nm and λ _em (radiation wavelength) = 515 nm. FIG. 6 shows the measurement result of the fluorescence response. In the figure, the horizontal axis represents the amount of AChE activity (μU), and the vertical axis represents the fluorescence intensity (au). As shown in the figure, the fluorescence response obtained for each well showed a good dependence on the amount of AChE activity. Furthermore, in the range of 0.1 μU to 0.5 μU, a good linear relationship was observed between AChE activity and the fluorescence response (slope = 28.8 au / μU, r = 0.9998). This AChE assay was characterized not only by the sensitivity due to the small background response, but also by a very short reaction time of 10 minutes.

Furthermore, it was confirmed by using the well-known anticholinergic drugs neostigmine and pyridostigmine that the above assay method can also be used for measuring the activity of AChE inhibitors. The specific operation is as follows. That is, first, a probe solution containing 8a, an acetylthiocholine solution, and an acetylcholinesterase (AChE) solution were prepared in the same manner as described above. However, the concentration (activity) of the AChE solution was fixed at 5 U / mL. Meanwhile, various concentrations of neostigmine solution and pyridostigmine solution (both dissolved in pH 7.4 HEPES buffer) were prepared. The probe solution (25 μM, 150 μL), acetylthiocholine solution (1 mM, 10 μL), AChE solution (5 U / mL, 10 μL) and various concentrations of neostigmine solution or pyridostigmine solution in 96 wells In addition to each well of the microplate, an enzyme reaction was performed at 37 ° C for 10 minutes, and then the fluorescence response was measured for each well. Measurements were performed at λ _ex (excitation wavelength) = 483 nm and λ _em (radiation wavelength) = 515 nm. Then, the inhibition% value was calculated from the fluorescence intensity, and an inhibition activity curve was prepared. FIG. 7 shows the inhibitory activity curve. The horizontal axis represents the concentration (μmol) of neostigmine or pyridostigmine, and the vertical axis represents the% inhibition value calculated from the fluorescence intensity.

When the IC ₅₀ values of neostigmine and pyridostigmine were estimated from the inhibition curve of FIG. 7, they were 0.18 μM and 0.29 μM. These values do not agree with literature values known as IC ₅₀ values for neostigmine and pyridostigmine because the measurement conditions and the like are different. However, the relative degree of difference between the IC ₅₀ values of these anticholinergic drugs correlated well with the literature values. That is, it was found that compound 8a can be used as a highly accurate activity measuring agent for AChE inhibitors.

  The measurement of AChE assay and AChE inhibitory activity is very important for diagnosis of diseases involving AChE, such as Alzheimer's disease, and screening of new drug candidate compounds. In particular, AChE inhibitors have recently attracted attention as therapeutic agents for Alzheimer's disease, and the sulfonate compounds of the present invention can be expected to be very useful for screening AChE inhibitors according to the above test results.

[4. Utilization as a thiophosphate group-responsive probe]
Furthermore, the possibility of the sulfonate compound of the present invention as a fluorescent probe for mercapto compounds other than thiol (compound in which —SH group is bonded to a carbon atom) was examined. More specifically, compounds 5a, 6a, 7, and 8a-8c are converted to adenosine 5 '-[γ-thio] triphosphate (γ-S-ATP) or adenosine. It was clarified that it responds also to the thiol group in the so-called thiophosphate group which adenosine monothiophosphate (S-AMP) has. The test results using compounds 8a to 8c and γ-S-ATP are described below.

Add 8a solution (20 μM, 200 μL) and various concentrations of γ-S-ATP solution (both in imidazole buffer at pH 7.4) to each well of a 96-well microplate and add 37 ° After a 180 minute reaction at C, the fluorescence response was measured for each well. Measurements were performed at λ _ex (excitation wavelength) = 483 nm and λ _em (radiation wavelength) = 515 nm. FIG. 8 shows the result. As shown, the response observed after the reaction showed a good dependence on the γ-S-ATP concentration. The detection limit was 5 pmol and 8a showed high sensitivity to γ-S-ATP. In addition, when the same test was performed on 8b and 8c, it was revealed that γ-S-ATP could be detected with higher sensitivity than 8a.

  Thus, since 8a-8c showed a response to the thiophosphate group, a kinase assay was constructed based on the concept shown in the following schemes 11 and 12 by using a mercapto compound-responsive fluorescent probe for these compounds. It is expected to be possible. Actually, the fluorescence response to thiophosphorylated proteins (provided by Sysmex Corporation) with different degrees of introduction of thiophosphate groups was measured using 8a, and fluorescence responses corresponding to the degree of introduction were obtained.

  The schemes 11 and 12 will be described more specifically as follows. Kinase is an enzyme that phosphorylates hydroxyl groups such as tyrosine residues of proteins in the presence of ATP. However, when γ-S-ATP is used instead of ATP, a thiophosphorylated protein is produced. If this thiophosphorylated protein is detected with a thiol-responsive fluorescent probe, a kinase assay can be performed. For example, the following two methods (1) and (2) are conceivable as actual methods when this kinase assay is used for clinical diagnosis.

(1) A method using a microplate on which a specific substrate for kinase is immobilized can be performed, for example, by sequentially performing the following operations (i) to (v).
(i) separation of the target kinase from the sample (blood or tissue) with the antibody, ie immunoprecipitation
(ii) Thiophosphorylation of immobilized substrate by isolated kinase and γ-S-ATP, ie, enzyme reaction with kinase
(iii) Additive wash-out
(iv) Fluorescing process by reaction with thiophosphate group-responsive probe
(v) Detection with a fluorescence plate reader
(2) A method for blotting a specific substrate of a kinase can be carried out, for example, by sequentially performing the following operations (i) to (v).
(i) separation of the target kinase from the sample (blood or tissue) with the antibody, ie immunoprecipitation
(ii) Thiophosphorylation of substrate by isolated kinase and γ-S-ATP, ie enzyme reaction with kinase
(iii) Separation of thiophosphorylated substrate and its blotting on a membrane
(iv) Fluorescing process by reaction with thiophosphate group-responsive probe
(v) Detection with a fluorescence image analyzer
According to the method of (1) or (2) above, by using specific antibodies and substrates, among various kinases relating to various life phenomena (cell differentiation, division and proliferation, and cycle), It is thought that a specific kinase can be specifically detected. Therefore, the thiophosphate group-responsive probe is expected not only as a research reagent for measuring the amount of kinase activity but also in clinical applications such as cancer diagnosis.

  As described above, from the results of this Example, it was found that the acetyl AChE assay and kinase assay of the present invention can perform simple measurements without using radioisotope labels that affect the environment. Specifically, in the AChE assay, the enzyme reaction and the fluorescence reaction can be performed simultaneously, and in the kinase assay, the measurement can be performed by a simple operation of performing the fluorescence reaction after the enzyme reaction. It was also possible to measure using only the substrate and probe. Furthermore, the detection sensitivity of the measuring method using the fluorescent probe for detecting a mercapto compound according to the present invention is considered to be comparable to that of chemiluminescence from the results of the above-mentioned examples.

  Furthermore, other enzyme activity measurement methods using the mercapto compound detection probe of the present invention can be easily constructed by molecular design so that thiols are generated from these enzyme substrates by enzymatic reaction. For example, as shown in Scheme 13 below. Considering such advantages, the mercapto compound-responsive fluorescent probe can be expected as a reagent having various functions suitable for construction of various enzyme assay kits.

  As described above, the sulfonic acid ester compound of the present invention is suitable for the use of a fluorescent probe that responds with high selectivity to superoxide. Fluorescent probes are important reagents in current cell physiology research. Furthermore, considering the recent hard and soft developments in image analysis systems and flow cytometry (cell sorters) using fluorescent microscopes, fluorescent probes will be increasingly used in the future. The market is expected to expand. The present invention is suitable for, for example, bioimaging and clinical analysis, and can be used favorably for developing new therapeutic methods and drug discovery seeds for diseases associated with reactive oxygen species and acetylcholinesterase. In particular, a probe for detecting a mercapto compound can be expected to be useful for screening therapeutic agents such as Alzheimer's disease and cancer through detection of acetylcholinesterase activity and use in a kinase assay. Furthermore, the use of the sulfonic acid ester compound of the present invention is not limited to a fluorescent probe, and can be used for any application.

2 is a graph showing the superoxide detection performance of compound 1d. 2 is a graph showing test results of applying Compound 1d to a cell line. 4 is another graph showing the test results of applying Compound 1d to a cell line. It is a graph which shows the specificity with respect to a superoxide or glutathione about each of compound 5a, 7, 6a, 8a, and 1d. It is a graph which shows the fluorescence response with respect to glutathione and cysteine, FIG. 5 (a) shows the measurement result about the compound 6a, FIG.5 (b) shows the measurement result about the compound 8a. 3 is a graph showing the relationship between acetylcholinesterase activity and the fluorescence response of compound 8a. 3 is a graph showing measurement results of acetylcholinesterase inhibitory activity of neostigmine and pyridostigmine using compound 8a. 4 is a graph showing a fluorescence response of compound 8a to γ-S-ATP.

Claims (14)

The fluorescent probe for superoxide detection containing the sulfonate compound represented by either of following formula 1 and 3-6.

In Formula 1, X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a methyl group or a halogen, and may be the same or different,
In Formula 4, Formula 5 and Formula 6, R ¹ is a hydrogen atom, nitro group, methyl group, chloro group or methoxy group, R ² is a hydrogen atom, nitro group, trifluoromethyl group, methyl group, isopropyl group, It is a chloro group or a methoxy group, and at least one of R ¹ and R ² is a nitro group. The compound represented by the formula 1 is a compound represented by the following formulas 1a to 1d,
The compound represented by the formula 4 is a compound represented by the following formulas 4a to 4e,
The compound represented by the formula 5 is a compound represented by the following formulas 5a to 5b,
The fluorescent probe for superoxide detection according to claim 1, wherein the compound represented by formula 6 is a compound represented by the following formulas 6a to 6b.

  The fluorescent probe for detecting superoxide according to claim 1 or 2 used for bioimaging.   Used in at least one method selected from the group consisting of an assay method using blotting (transcription), a superoxide dismutase quantification method, a superoxide elimination ability measurement method of a functional food or pharmaceutical, a clinical analysis method and a pharmaceutical screening method The fluorescent probe for detecting superoxide according to claim 1 or 2. A kit for measuring a superoxide dismutase concentration in a human or animal body fluid, comprising the fluorescent probe for detecting superoxide according to claim 1 or 2,
In the measurement of the superoxide dismutase concentration, the fluorescence response after reacting the fluorescent probe for detecting superoxide according to any one of claims 1 and 2 with superoxide is used as the body fluid of human or animal or the body fluid thereof. A kit for quantifying superoxide dismutase by comparing the presence and absence of the sample extracted from the sample. A reagent for clinical analysis for superoxide detection, which comprises a sulfonate compound represented by any one of the following formulas 1 and 3-6.

In Formula 1, X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a methyl group or a halogen, and may be the same or different,
In Formula 4, Formula 5 and Formula 6, R ¹ is a hydrogen atom, nitro group, methyl group, chloro group or methoxy group, R ² is a hydrogen atom, nitro group, trifluoromethyl group, methyl group, isopropyl group, It is a chloro group or a methoxy group, and at least one of R ¹ and R ² is a nitro group. A fluorescent probe for detecting a mercapto compound comprising a sulfonate compound represented by any of the following formulas (i) to (iv).

In formulas (i) to (iv),
X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or halogen, which may be the same or different,
X ³ is a linear or branched alkyl group having 1 to 6 carbon atoms, or a trifluoromethyl group,
R ¹ and R ³ are each a hydrogen atom, a nitro group, a methyl group, a chloro group or a methoxy group, and may be the same or different;
R ² and R ⁴ are each a hydrogen atom, a nitro group, a trifluoromethyl group, a methyl group, an isopropyl group, a chloro group or a methoxy group, and may be the same or different;
And at least one of R ¹ and R ² is a nitro group, and at least one of R ³ and R ⁴ is a nitro group, and in formula (iv), X ³ is a straight chain having 1 to 6 carbon atoms or In the case of a branched alkyl group, R ¹ and R ² are both groups other than hydrogen atoms. The compound represented by the formula (i) is represented by the following formula 1 or 3,
The compound represented by the formula (ii) is represented by the following formula 8,
The compound represented by the formula (iii) is represented by the following formula 5,
The fluorescent probe according to claim 7, wherein the compound represented by the formula (iv) is represented by the following formula 6 or 7.

In formulas 1 and 8, X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a methyl group or a halogen, which may be the same or different,
In formulas 5 and 6, R ¹ is a hydrogen atom, nitro group, methyl group, chloro group or methoxy group, and R ² is a hydrogen atom, nitro group, trifluoromethyl group, methyl group, isopropyl group, chloro group or methoxy group And at least one of R ¹ and R ² is a nitro group. The compound represented by the formula 1 is a compound represented by the following formulas 1a to 1d,
The compound represented by the formula 5 is a compound represented by the following formulas 5a to 5b,
The compound represented by the formula 6 is a compound represented by the following formulas 6a to 6b,

The fluorescent probe for detecting a mercapto compound according to claim 8, wherein the compound represented by the formula 8 is a compound represented by the following formulas 8a to 8c.

  The fluorescent probe for detecting a mercapto compound according to any one of claims 7 to 9, which is used for bioimaging.   The method according to any one of claims 7 to 9, which is used in at least one method selected from the group consisting of a kinase assay method, an acetylcholinesterase assay method, an assay method using blotting (transcription), a clinical analysis method, and a pharmaceutical screening method. A fluorescent probe for detecting a mercapto compound according to 1. A reagent for clinical analysis for detecting a mercapto compound comprising a sulfonate compound represented by any of the following formulas (i) to (iv).

In formulas (i) to (iv),
X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a linear or branched alkyl group having 1 to 6 carbon atoms, or halogen, which may be the same or different,
X ³ is a linear or branched alkyl group having 1 to 6 carbon atoms, or a trifluoromethyl group,
R ¹ and R ³ are each a hydrogen atom, a nitro group, a methyl group, a chloro group or a methoxy group, and may be the same or different;
R ² and R ⁴ are each a hydrogen atom, a nitro group, a trifluoromethyl group, a methyl group, an isopropyl group, a chloro group or a methoxy group, and may be the same or different;
And at least one of R ¹ and R ² is a nitro group, and at least one of R ³ and R ⁴ is a nitro group, and in formula (iv), X ³ is a straight chain having 1 to 6 carbon atoms or In the case of a branched alkyl group, R ¹ and R ² are both groups other than hydrogen atoms. A sulfonic acid ester compound represented by any one of the following formulas 1, 3 to 5 and 8.

In formulas 1 and 8, X ¹ , X ² , Y ¹ and Y ² are each a hydrogen atom, a methyl group or a halogen, which may be the same or different,
In Formula 4 and Formula 5, R ¹ is a hydrogen atom, nitro group, methyl group, chloro group or methoxy group, and R ² is a hydrogen atom, nitro group, trifluoromethyl group, methyl group, isopropyl group, chloro group or It is a methoxy group, and at least one of R ¹ and R ² is a nitro group. The compound represented by the formula 1 is a compound represented by the following formulas 1a to 1d,
The compound represented by the formula 4 is a compound represented by the following formulas 4a to 4e,
The compound represented by the formula 5 is a compound represented by the following formulas 5a to 5b,
The sulfonic acid ester compound according to claim 13, wherein the compound represented by the formula 8 is a compound represented by the following formulas 8a to 8c.

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