WO2012144654A1 - Fluorescent probe for measuring hydrogen sulfide - Google Patents

Abstract

A compound represented by formula (I) (wherein R¹ represents a group represented by formula (A); R² represents a hydrogen atom or a univalent substituent; R³ and R⁴ independently represent a hydrogen atom or a halogen atom; R⁵ and R⁶ independently represent a hydrogen atom, an alkylcarbonyl group or the like; R⁷ represents a hydrogen atom, an alkyl group or an alkoxyalkyl group; and R⁸ and R⁹ independently represent a hydrogen atom, a halogen atom or the like), which is useful as a fluorescent probe that can measure hydrogen sulfide specifically and with high sensitivity without being subjected to the influence of reduced glutathione.

Description

Fluorescent probe for hydrogen sulfide measurement

The present invention relates to a fluorescent probe for measuring hydrogen sulfide.

The toxicity of hydrogen sulfide (H ₂ S) has been known for 300 years, and many reports on toxicity have been made so far. On the other hand, in recent years, it has become clear that H ₂ S functions as a physiological signal transmitter, and is the third gaseous signal transmitter after nitrogen monoxide (NO) and carbon monoxide (CO). It is attracting attention as.

Role H 2 _S play is being studied using NaHS and Na 2 _S is H _{2 S} donor, the roles that are best studied is an operation related to relaxation of smooth muscle. H ₂ S causes relaxation of vascular smooth muscle and intestinal smooth muscle, and causes symptoms such as headache and blood pressure reduction at the individual level. On the other hand, it has been reported that H ₂ S is involved in memory formation in nerve cells, and there is also a report that H ₂ S is involved in O ₂ sensing and control of insulin secretion in the carotid body.

Thus, although it has been suggested that H ₂ S contributes to intracellular signal transduction, there is still room for doubt as to whether physiological H ₂ S really exists. In addition, identification of molecules that are targets of H ₂ S has not progressed, and there are many unresolved points in specific intracellular signal transduction mechanisms. From such a background, development of a means for visualizing H ₂ S in a living body is eagerly desired.

In order to specifically visualize H ₂ S in a living body using a fluorescent probe and perform highly sensitive measurement, it is possible to measure H ₂ S in water, and selectivity between other anion species And a condition such as having selectivity with reduced glutathione (GSH) are essential. Conventionally, a 2,4,6-triarylpyrylium cation (Journal of the American Chemical Society, 125) which is an absorption change type probe having an amino group at the p-position as a fluorescent probe for visualizing and measuring H ₂ S. , 9000, 2003) has been proposed, but there is a problem that competition of reaction with GSH cannot be avoided. A method using 2,4-dinitrobenzenesulfonylfluorescein as a fluorescence-enhanced probe has also been proposed (Analytica Chimica Acta, 631, 91, 2009). However, the sulfonic acid ester is hydrolyzed over time and the fluorescence intensity is increased. Have the problem of changing.

As another fluorescence-enhancing probe, an aminofluorescein compound (DPA-4-AF) having a 2,2′-dipicolylamine moiety coordinated with divalent copper ion (Cu ²⁺ ) has been proposed ( Chem. Commun., Pp. 7390-7392, 2009). In this compound, the fluorescence is quenched by the coordination of Cu ²⁺ , but when the sulfide ion (HS ⁻ ) acts, Cu ²⁺ becomes CuS and is insolubilized, the quenching is canceled and strong fluorescence is emitted. This fluorescent probe can detect sulfide ions in water, and is highly specific and sensitive even in the presence of various anions (such as halogen ions, sulfate ions, acetate ions, nitrate ions, hypochlorite ions). _{2 It} has the characteristic that S can be measured. However, according to a follow-up test by the present inventors, this compound has low selectivity with reduced glutathione, and H ₂ S present in cells or the like in vivo due to glutathione present in large quantities in the living body. There is a problem that it cannot be measured specifically and with high sensitivity. A compound using a cyclic polyamine as a zinc ion capturing group is known (dansylaminoethylcyclene: J. Am. Chem. Soc., 118, 12696, 1996). The coordinated compound is not known, and there is no report on the reactivity with H ₂ S. Further, a compound using a cyclic polyamine as a cadmium ion capturing group is also known, but the reactivity between this compound and H ₂ S is not known (J. Am. Chem. Soc., 124, pp. 3920-3925, 2002).

An object of the present invention is to provide a fluorescent probe capable of specifically visualizing hydrogen sulfide in a living body and performing highly sensitive measurement. In particular, it is an object of the present invention to provide a fluorescent probe capable of measuring hydrogen sulfide with high sensitivity and specificity without being affected by reduced glutathione present in large quantities in a living body.

As a result of intensive studies to solve the above problems, the present inventors have used the compound represented by the following general formula (I) as a fluorescent probe for measuring hydrogen sulfide in the presence of reduced glutathione. It was also found that hydrogen sulfide can be measured with extremely high sensitivity and specificity. The present invention has been completed based on the above findings.

That is, according to the present invention, the following general formula (IA) or (IB):

[Wherein R ¹ represents the following formula (A):

(In the formula, p, q, r, and s each independently represent an integer of 2 or 3, t represents 0 or 1, and R ¹¹ , R ¹² , and R ¹³ each independently represent a hydrogen atom or a carbon atom. R ² represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R ³ and R ⁴ represent Each independently represents a hydrogen atom or a halogen atom; R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; and R ⁷ represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. R ⁸ and R ⁹ are each independently a hydrogen atom, a halogen atom, or — (CH ₂ ) _x —N (R ¹⁴ ) (R ¹⁵ ) (wherein x represents an integer of 1 to 4; R ¹⁴ and ^{R 15} are each The standing ^{_{- (CH 2) y -COOR 16}} ( wherein, y represents an integer of 1 ^{4, R 16} is a hydrogen atom, an alkylcarbonyl group, or an alkyl carbonyl an oxy alkyl group) a group represented by Or a salt thereof is provided.

According to a preferred aspect of the present invention, p, q, r, and s are 2, t is 1, and R ¹¹ , R ¹² , and R ¹³ are hydrogen atoms, A compound represented by (IB) or a salt thereof; p, q, r, and s are 2, t is 1, R ¹¹ , R ¹² , and R ¹³ are hydrogen atoms, and R ² is A compound represented by the above general formula (IA) or (IB) which is a hydrogen atom or a salt thereof; and R ¹ present on the benzene ring is bonded to the para-position relative to the binding site of the xanthene ring, p, q, r, and s are 2, t is 1, R ¹¹ , R ¹² , and R ¹³ are hydrogen atoms, and R ² is a hydrogen atom, or the above general formula (IA) or ( A compound represented by IB) or a salt thereof is provided.

According to a further preferred embodiment, the compound represented by the above general formula (IA) or (IB) or a salt thereof, wherein R ³ and R ⁴ are hydrogen atoms; R ⁵ and R ⁶ are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IA) or (IB), which is an acetyloxymethyl group, or a salt thereof; R ⁷ is a hydrogen atom, an alkyl group, or a methoxymethyl group; And a compound represented by the above general formula (IA) or (IB) or a salt thereof, wherein both R ⁸ and R ⁹ are hydrogen atoms.

Further, according to the present invention, the following general formula (IIA) or (IIB):

[Wherein R ²¹ represents the following formula (B):

(In the formula, d, e, f, and g each independently represent an integer of 2 or 3, h represents 0 or 1, and R ³¹ , R ³² , and R ³³ each independently represent a hydrogen atom or a carbon atom. R ²² represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R ²³ and R ²⁴ represent Each independently represents a hydrogen atom or a halogen atom; R ²⁵ and R ²⁶ each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R ²⁷ represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group; R ²⁸ and R ²⁹ are each independently a hydrogen atom, a halogen atom, or — (CH ₂ ) _m —N (R ³⁴ ) (R ³⁵ ) (wherein m represents an integer of 1 to 4; R ³⁴ as well as ³⁵ are each independently ^{_{- (CH 2) n -COOR 36}} ( wherein, n represents an integer of 1 to ^{4, R 36} is a hydrogen atom, an alkyl group, or an alkylcarbonyloxy group) is represented by Or a salt thereof is provided.

According to a preferred aspect of the present invention, d, e, f, and g are 2, h is 1, and R ³¹ , R ³² , and R ³³ are hydrogen atoms, A compound represented by (IIB) or a salt thereof; d, e, f, and g are 2, h is 1, R ³¹ , R ³² , and R ³³ are hydrogen atoms, and R ²² is A compound represented by the above general formula (IIA) or (IIB) which is a hydrogen atom or a salt thereof; and R ²¹ present on the benzene ring is bonded to the para-position relative to the binding site of the xanthene ring, wherein d, e, f, and g are 2, h is 1, R ³¹ , R ³² , and R ³³ are hydrogen atoms, and R ²² is a hydrogen atom, the above general formula (IIA) or ( A compound represented by IIB) or a salt thereof is provided.

According to a further preferred embodiment, the compound represented by the above general formula (IIA) or (IIB) or a salt thereof, wherein R ²³ and R ²⁴ are hydrogen atoms; R ²⁵ and R ²⁶ are each independently a hydrogen atom, an acetyl group; Or a compound represented by the above general formula (IIA) or (IIB), which is an acetyloxymethyl group, or a salt thereof; R ²⁷ is a hydrogen atom, an alkyl group, or a methoxymethyl group; IIB) or a salt thereof; and a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, in which R ²⁸ and R ²⁹ are both hydrogen atoms.

From another point of view, according to the present invention, a fluorescent probe for measuring hydrogen sulfide containing the compound represented by the above general formula (IA) or (IB) or a salt thereof, and the above general formula (IA) or (IB). Provided is a reagent for measuring hydrogen sulfide containing the compound or a salt thereof. The present invention also provides use of the compound represented by the above general formula (IA) or (IB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide.

In addition, according to the present invention, a fluorescent probe for measuring hydrogen sulfide containing a compound represented by the above general formula (IIA) or (IIB) or a salt thereof, and a compound represented by the above general formula (IIA) or (IIB) or A reagent for measuring hydrogen sulfide containing the salt is provided. The present invention also provides use of the compound represented by the above general formula (IIA) or (IIB) or a salt thereof for the production of a fluorescent probe for measuring hydrogen sulfide or a reagent for measuring hydrogen sulfide. This fluorescent probe and reagent react with divalent copper ions in the measurement system to generate a compound represented by the above general formula (IA) or (IB) or a salt thereof in situ in the measurement system. It is useful as a fluorescent probe or reagent for measurement.

From another aspect, according to the present invention, there is provided a method for measuring hydrogen sulfide, comprising the following steps: (a) contacting a compound represented by the above general formula (IA) or (IB) or a salt thereof with hydrogen sulfide And (b) a method of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) produced in the step (a) or a salt thereof.

According to the present invention, there is also provided a method for measuring hydrogen sulfide, comprising the steps of: (a) reacting a compound represented by the general formula (IIA) or (IIB) or a salt thereof with a divalent copper ion. A step of producing a compound represented by the above general formula (IA) or (IB) or a salt thereof; (b) a compound represented by the above general formula (IA) or (IB) produced in the above step (a); A step of contacting the salt with hydrogen sulfide; and (c) a step of measuring the fluorescence of the compound represented by the general formula (IIA) or (IIB) generated in the step (b) or the salt thereof. Is provided.

The compound represented by the general formula (IA) or (IB) provided by the present invention or a salt thereof has high reactivity to hydrogen sulfide, and is a general formula having strong fluorescence in the presence of hydrogen sulfide. It has the property of producing a compound represented by (IIA) or (IIB) or a salt thereof. In addition, since the compound represented by the general formula (IA) or (IB) or a salt thereof has substantially no reactivity with reduced glutathione, it can be sulfided with high sensitivity even in the presence of reduced glutathione. Hydrogen can be measured. Furthermore, the compound represented by the general formula (IA) or (IB) or a salt thereof is specific even in the presence of various anions (halogen ion, sulfate ion, acetate ion, nitrate ion, hypochlorite ion, etc.). It can react with hydrogen sulfide with high sensitivity. Therefore, the fluorescent probe for measuring hydrogen sulfide containing the compound represented by the general formula (IA) or (IB) or a salt thereof is a fluorescence for measuring a small amount of hydrogen sulfide existing in a living body with high sensitivity and high accuracy. It is useful as a probe and extremely useful as a reagent for imaging hydrogen sulfide in cells and tissues in an in vivo environment.

It is the figure which showed the result of having measured the absorption and fluorescence spectrum of Cyclne-4-AF-Cu (left side) and Cyclen-4-AF (right side). It is the figure which showed the result of having measured the absorption and fluorescence spectrum of TACN-4-AF (left side) and Cyclam-4-AF (right side). It is the figure which showed the result of having measured the absorption and fluorescence spectrum of TMCyclen-AF (left side) and TMCyclen-4-AF-Cu (right side). It is the figure which showed the result of having evaluated the reactivity of Cyclen-4-AF-Cu. The result of adding 10 μM Na ₂ S (red), 10 μM Na ₂ S and 10 mM GSH (green), or 10 mM GSH (yellow) and the result of adding no negative control (blue) are shown. It is the figure which showed the result of having introduced Cyclen-4-AF-Cu in a HeLa cell by microinjection and measuring hydrogen sulfide. The upper row shows before Na ₂ S addition (0 sec), the middle row shows after Na ₂ S addition (360 sec), and the lower row shows changes in fluorescence intensity over time, and regions 5 and 6 show regions where microinjection is not performed. It is the figure which showed the result of having measured the hydrogen sulfide by the uptake | capture to a HeLa cell using Cyclen-4-AF-Cu diacetate. The upper part shows before Na ₂ S addition (0 sec), the middle part shows after Na ₂ S addition (240 sec), and the lower part shows the change in fluorescence intensity over time. TACN-4-AF alone and 2 the absorption spectrum of the equivalent of TACN-4-AF plus ^{Cu 2+} and (upper left) fluorescence spectrum results obtained by measuring the (upper right), and TACN-4-AF of CuSO ₄ In addition, 100 μM Na ₂ S (red), 10 μM Na ₂ S (green), or 10 mM GSH (yellow) was added, and the results of examining the reactivity with the addition-free experiment as a negative control (blue) (lower) are shown. It is a figure. It is the figure which showed the result of having measured the absorption spectrum (left) and the fluorescence spectrum (right) of Cyclam-4-AF which added Cyclam-4-AF independent and 2 equivalent Cu2 ⁺ . The CuSO ₄ was added to Cyclam-4-AF (top), or TMCyclen-4-AF-Cu (lower) 100 [mu] M Na ₂ S (red), 10 [mu] M Na ₂ S (green), or added 10 mM GSH (yellow) FIG. 5 is a diagram showing the results of examining the reactivity of an additive-free experiment as a negative control (blue). It is the figure which showed the result of having detected hydrogen sulfide using the cell lysate of the 3-mercaptopyruvate sulfate transferase (3MST) expression cell. Using glutathione-S-transferase (GST) tag fusion 3-mercaptopyruvate sulfatransferase (3MST) purified protein, hydrogen sulfide produced using 3-mercaptopyruvate (3MP) as a substrate is Cyclen-4-AF- It is the figure which showed the result detected by Cu.

In the general formula (IA) or (IB), R ¹ represents a group represented by the formula (A). In the group represented by the formula (A), p, q, r, and s each independently represent an integer of 2 or 3, preferably p, q, r, and s are all 2. . t represents 0 or 1, but is preferably 1. R ¹¹ , R ¹² , and R ¹³ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and any or all of R ¹¹ , R ¹² , and R ¹³ are hydrogen atoms. It is preferable that R ¹¹ , R ¹² , and R ¹³ are all hydrogen atoms. Of the group represented by the formula (A) can be bonded to any position on the benzene ring, preferably a benzene ring R ¹ and xanthene ring are bonded, the bonding position of R ¹ and xanthene ring para It is preferable to be in a position.

In the present specification, the term “alkyl group” includes an alkyl group composed of linear, branched, cyclic, and combinations thereof. The same applies to the alkyl part of other substituents having an alkyl part (for example, an alkylcarbonyl group or an alkoxyalkyl group).

R ² represents a hydrogen atom or 1 to 3 monovalent substituents substituted on the benzene ring, preferably 1 or 2 monovalent substituents substituted on the hydrogen atom or benzene ring. More preferably, it represents one monovalent substituent substituted on a hydrogen atom or a benzene ring. When R ² is a monovalent substituent, examples of the substituent include a halogen atom (in this specification, the term halogen atom includes a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom), a hydroxyl group, Examples thereof include, but are not limited to, an oxo group, a carboxyl group, an alkoxycarbonyl group, an acyl group, an amino group, an alkyl group, an alkenyl group, an alkynyl group, an alkoxy group, an aryl group, and an aralkyl group. These substituents may be further substituted with another substituent. Examples of such include, but are not limited to, a fluoroalkyl group, a fluoroacetyl group, a methoxybenzyl group, and the like. R ² is particularly preferably a hydrogen atom.

R ³ and R ⁴ each independently represent a hydrogen atom or a halogen atom, but it is preferable that both R ³ and R ⁴ are hydrogen atoms.
R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group. The alkylcarbonyl group is preferably an alkylcarbonyl group having about 2 to 7 carbon atoms, and for example, an acetyl group can be preferably used. As the alkylcarbonyloxyalkyl group, for example, an alkylcarbonyloxyalkyl group in which an alkylcarbonyloxy group having about 2 to 7 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms is preferable. For example, an acetoxymethyl group Etc. can be preferably used.

R ⁷ represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. The alkoxyalkyl group is preferably an alkoxyalkyl group in which an alkoxy group having about 1 to 6 carbon atoms is substituted with an alkyl group having about 1 to 6 carbon atoms. For example, a methoxymethyl group or an ethoxymethyl group is preferably used. Can do.

R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, or a group represented by — (CH ₂ ) _x —N (R ¹⁴ ) (R ¹⁵ ). x represents an integer of 1 to 4, but x is preferably 1 or 2, and x is particularly preferably 1. R ¹⁴ and R ¹⁵ each independently represent a group represented by — (CH ₂ ) _y —COOR ¹⁶ . y represents an integer of 1 to 4, and y is preferably 1 or 2, and y is particularly preferably 1. R ¹⁶ represents a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group, and the alkylcarbonyl group and the alkylcarbonyloxyalkyl group are the same as those described for R ⁵ above. Preferably, a hydrogen atom or an acetoxymethyl group can be used as R ¹⁶ .

Group represented by formula (B) in general formula (IIA) or (IIB), d, e, f, g, h, R ³¹ , R ³² , R ³³ , R ²² , R ²³ , R ²⁴ , R ²⁵ , R ²⁶ , R ²⁷ , R ²⁸ , R ²⁹ , m, R ³⁴ , R ³⁵ , n, and R ³⁶ are groups represented by the corresponding formula (A) in the general formula (IA) or (IB), respectively. , P, q, r, s, t, R ¹¹ , R ¹² , R ¹³ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , x, R ¹⁴ , R ¹⁵ , n, and R ¹⁶ are the same.

The compound represented by general formula (IA) or (IB), or general formula (IIA) or (IIB) may form an acid addition salt or a base addition salt. Examples of acid addition salts include mineral acid salts such as hydrochloride, sulfate, and nitrate, and organic acid salts such as p-toluenesulfonate, oxalate, and malate. There is no limit. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, magnesium salt, or calcium salt, ammonium salts, and organic amine salts such as triethylamine salt or ethanolamine salt. There is no limit. Among these salts, physiologically acceptable salts are preferable when the compound of the present invention is used as a reagent for measuring hydrogen sulfide in a living body, cell or tissue.

Moreover, the compound represented by general formula (IA) or (IB), or general formula (IIA) or (IIB) may have one or two or more asymmetric carbons depending on the type of the substituent. Arbitrary optical isomers based on these asymmetric carbons, arbitrary mixtures of optical isomers, racemates, diastereoisomers based on two or more asymmetric carbons, arbitrary mixtures of diastereoisomers, etc. Are included in the scope of the present invention. Any hydrate or solvate of the free compound or salt form of the compound is also encompassed within the scope of the present invention. Further, when R ⁷ is a hydrogen atom, the carboxy group may form a lactone, but such structural isomers are also included in the scope of the present invention. The compound in which R ⁵ is a hydrogen atom in the general formula (IA) and the compound in which R ⁷ is a hydrogen atom in the general formula (IB) correspond to tautomers, but such tautomers exist. Is easily understood by those skilled in the art, and any tautomer is included in the scope of the present invention.

The production methods of representative compounds of the compounds of the present invention are shown in detail and specifically in the examples of the present specification. Those skilled in the art can appropriately select the reaction raw materials, reaction conditions, reaction reagents, and the like based on the description of the present example, and modify or modify these methods as necessary. Any of the compounds of the present invention can be prepared. A fluorescein derivative such as 4-aminofluorescein that can be used as a raw material compound can be produced according to the method described in, for example, Tetsuji Kameya, Synthetic Organic Chemistry IX, Nanedo, page 215 (1977). In addition, regarding the cyclic polyamine partial structure, J.A. Am. Chem. Soc. , 118, 12696, 1996, can be referred to the synthesis of the partial structure of the zinc probe.

As used herein, the term “measurement” should be interpreted in the broadest sense, including measurement, examination, detection, etc. performed for the purpose of quantitative, qualitative, or diagnostic purposes. The method for measuring hydrogen sulfide according to the present invention generally comprises (a) a step of reacting a compound represented by the general formula (IA) or (IB) or a salt thereof with hydrogen sulfide, and (b) the above step. A step of measuring fluorescence derived from the compound represented by the general formula (IIA) or (IIB) generated in (a) or a salt thereof. The compound represented by the general formula (IA) or (IB) is quenched by the coordination of divalent copper ions, and is itself non-fluorescent or weakly fluorescent. Copper ions react with hydrogen sulfide to form insoluble CuS, which is removed from the cyclic polyamine moiety to produce a strongly fluorescent compound represented by the general formula (IIA) or (IIB) or a salt thereof.

As a reagent for measuring hydrogen sulfide, a compound represented by the general formula (IA) or (IB) in which a divalent copper ion is coordinated or a salt thereof is dissolved in an appropriate aqueous medium such as water. In a measurement system, a compound represented by the general formula (IIA) or (IIB) or a salt thereof and a divalent copper ion are reacted to represent in situ the general formula (IA) or (IB). Or a salt thereof may be produced in a measurement system and the compound represented by the general formula (IA) or (IB) or a salt thereof may be reacted with hydrogen sulfide. It goes without saying that such a measuring method is also included in the scope of the present invention.

The fluorescence measuring means using the hydrogen sulfide measuring reagent of the present invention is not particularly limited, but a method of measuring a fluorescence spectrum in vitro, a method of measuring a fluorescence spectrum in vivo using a bioimaging technique, etc. Can be adopted. For example, when quantification is performed, it is desirable to prepare a calibration curve in advance according to a conventional method. If the reagent of the present invention is incorporated into cells by a microinjection method or the like, hydrogen sulfide localized in individual cells can be measured with high sensitivity in real time by a bioimaging technique, and a cell culture solution or tissue slice The hydrogen sulfide released from cells and living tissues can be measured by using it in a culture solution or perfusate. Therefore, by using the hydrogen sulfide measurement reagent of the present invention, it is possible to measure the behavior of hydrogen sulfide in cells or living tissues in real time. In addition to elucidating the mechanism of signal transmission by hydrogen sulfide, It can be suitably used for investigating the cause of the disease and developing therapeutic agents.

For example, when an ester group that can be hydrolyzed by esterase or the like is introduced into any one or more of R ⁵ , R ⁶ , R ⁷ , R ⁸ , or R ⁹ , the entire molecule becomes fat-soluble. Thus, it easily penetrates the cell membrane and reaches the cytoplasm. However, when a hydrophilic carboxyl group is generated by esterase hydrolysis in the cytoplasm, the cell membrane cannot be easily penetrated. Accordingly, such ester-introduced compounds (for example, compounds into which acetoxymethyl ester or methoxymethyl ester is introduced as an ester) are extremely useful as reagents for measuring the concentration of hydrogen sulfide in the cytoplasm.

The reagent of the present invention may be used as a composition by blending additives usually used in the preparation of the reagent as necessary. For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible. These compositions are provided as a composition in an appropriate form such as a mixture in a powder form, a lyophilized product, a granule, a tablet, or a liquid.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
Cyclen-4-AF and Cyclen-4-AF-Cu were synthesized according to the following synthesis scheme.

(A) Chloroacetylamide-4-AF
4-Aminofluorescein (948.5 mg, 2.73 mmol) was dissolved in 30 mL of dehydrated tetrahydrofuran (THF), and NaHCO ₃ (761.5 mg, 9.06 mmol) was added. ClCH ₂ COCl (436.8 mg, 3.87 mmol) dissolved in 40 mL of dehydrated THF was added dropwise over 10 minutes with stirring. Thereafter, the mixture was stirred overnight at room temperature under argon, the solvent was removed after filtration, and the residue was purified by column chromatography (silica gel, ethyl acetate / hexane = 6/4) to obtain the desired product (756.9 mg, 1 .79 mmol, y.65%).
¹ H NMR (300 MHz, CD ₃ OD): δ 8.34 (d, 1H, J = 1.5 Hz), 7.86 (dd, 1H, J = 8.1, 1.5 Hz), 7.16 ( d, 1H, J = 8.1 Hz), 6.67 (d, 2H, J = 2.9 Hz), 6.61 (d, 2H, J = 8.8 Hz), 6.52 (dd, 2H, J = 8.8, 2.9 Hz), 4.24 (s, 2H)
¹³ C NMR (75 MHz, CD ₃ OD): δ 171.1, 167.8, 161.4, 154.1, 149.5, 141.2, 130.2, 129.1, 128.3, 125. 9,116.4,113.6,111.3,103.5,44.0
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 424.0588, Found, 424.0578 (-1.0mmu)

(B) Cyclen-4-AF
Chloroacetylamido-4-aminofluorescein (106.6 mg, 251.5 μmol) was dissolved in 30 mL of dehydrated acetonitrile, cyclen (368.0 mg, 2.14 mmol) and diisopropylethylamine (DIEA, 300 μL, 1.72 mmol) were added, Stir at 70 ° C. for 8 hours under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF (TFA salt) (103.3 mg, 109.0 μmol, y.43%).
¹ H NMR (300 MHz, D ₂ O): δ 8.34 (d, 1H, J = 2.2 Hz), 7.83 (dd, 1H, J = 8.1, 2.2 Hz), 7.28 ( d, 2H, J = 8.8 Hza), 7.23 (d, 1H, J = 8.1 Hz), 6.92 (dd, 2H, J = 8.8, 2.2H), 6.86 (d , 2H, J = 2.2 Hz), 3.69 (s, 2H), 3.05 to 3.16 (m, 16H)
¹³ C NMR (100 MHz, D ₂ O): δ 170.9, 167.5, 165.9, 155.8, 137.8, 131.7, 130.5, 129.2, 128.1, 124. 1,120.2,116.8,116.3,113.7,113.4,100.9,54.5,48.3,42.9,41.2,40.7
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 560.2509, Found, 560.2506 (−0.3 mmu)

(C) Cyclen-4-AF-Cu
CuSO ₄ pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1M CuSO ₄ aqueous solution. Cyclen-4-AF (73.8 mg, 77.9 μmol) was dissolved in an aqueous CuSO ₄ solution (7792 μL, 7.79 mmol), stirred overnight at room temperature, purified by HPLC, and purified with Cyclen-4-AF-Cu ( 65.2 mg, quant yield).
HRMS (ESI ⁺ ): Calcd for [M−H] ⁺ , 621.1649, Found, 621.1690 (4.1 mmu)

(D) Cyclen-4-AF-Cu diacetate
Cyclen-4-AF-Cu (5.60 μmol) was dissolved in 4 mL of dehydrated acetonitrile, pyridine (4.14 mmol) and acetic anhydride (41.9 mmol) were added, and the mixture was stirred at 60 ° C. for 6 hours under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclen-4-AF-Cu diacetate (3.6 mg, 5.1 μmol, y. 91%).
HRMS (ESI ⁺ ): Calcd for [M−H] ⁺ , 705.1860, Found, 705.1843 (−1.7 mmu)

Example 2
TACN-4-AF was synthesized by the following scheme.

Chloroacetylamide-4-aminofluorescein (53.8 mg, 126.9 μmol) was dissolved in 8 mL of dehydrated acetonitrile, and 1,4,7-triazacyclononane (TACN, 101.4 mg, 784.8 μmol), potassium iodide. (6.8 mg, 41.0 μmol) and K ₂ CO ₃ (111.0 mg, 803.1 μmol) were added and stirred overnight at room temperature under argon. After removing the solvent, the product was purified by HPLC to obtain TACN-4-AF (TFA salt) (47.4 mg, 55.2 μmol, y.44%).
¹ H NMR (300 MHz, D ₂ O): δ 8.35 (d, 1H, J = 2.2 Hz), 7.84 (dd, 1H, J = 8.4, 2.2 Hz), 7.27− 7.30 (m, 3H), 6.98 (d, 2H, J = 1.5 Hz), 6.93 (dd, 2H, J = 9.2, 1.5 Hz), 3.80 (s, 2H) ), 3.71 (s, 4H), 3.36 (t, 4H, J = 5.5 Hz), 3.15 (t, 4H, J = 5.5 Hz)
¹³ C NMR (100 MHz, D ₂ O): δ 171.4, 168.1, 165.4, 155.6, 137.5, 132.6, 130.2, 129.6, 127.8, 123. 9, 119.6, 116.3, 116.2, 113.3, 113.3, 100.9, 55.1, 47.6, 42.8, 41.5
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 517.2087, Found, 517.2106 (1.9 mmu)

Example 3
Cyclam-4-AF was synthesized according to the following scheme.

Chloroacetylamido-4-aminofluorescein (51.5 mg, 121.5 μmol) was dissolved in 8 mL of dehydrated acetonitrile, and 1,4,8,11-tetraazacyclotetradecane (cyclam, 191.2 mg, 954.5 μmol), iodine Potassium chloride (8.7 mg, 52.4 μmol) and K ₂ CO ₃ (136.7 mg, 989.1 μmol) were added, and the mixture was stirred overnight at room temperature under argon. After removing the solvent, the residue was purified by HPLC to obtain Cyclam-4-AF (TFA salt) (22.5 mg, 21.6 μmol, y.18%).
¹ H NMR (300 MHz, D ₂ O): δ 8.53 (d, 1H, J = 2.2 Hz), 7.89 (dd, 1H, J = 8.8, 2.2 Hz), 7.49 ( d, 2H, J = 8.4 Hz), 7.37 (d, 1H, J = 8.8 Hz), 7.18 (d, 2H, J = 2.2 Hz), 7.09 (dd, 2H, J = 8.4, 2.2 Hz), 3.56 (s, 2H), 3.13-3.25 (m, 12H), 2.86-2.91 (m, 4h), 1.94-1 .98 (m, 4H)
¹³ C NMR (100 MHz, D ₂ O): δ 171.1, 167.2, 166.7, 156.4, 138.1, 130.7, 130.5, 128.6, 123.6, 120.4 , 117.3, 116.3, 114.3, 113.4, 100.9, 54.4, 53.6, 52.7, 46.5, 46.0, 45.7, 44.8, 43 .6, 42.0, 22.7, 21.4
HRMS (ESI ⁺ ): Calcd for [M + M] ⁺ , 588.8222, Found, 5888.2794 (-2.8mmu).

Example 4
TMCyclen-4-AF and TMCyclen-4-AF-Cu were synthesized by the following scheme.

(A) 1,4,7,10-tetraazacyclododecane (cyclen, 614.6 mg, 3.57 mmol) was dissolved in 15 mL of dehydrated dichloromethane, and diisopropylethylamine (1.86 g, 14.35 mmol) was added thereto. Was stirred. Then, p-toluenesulfonic acid chloride (699.2 mg, 3.67 mmol) dissolved in 10 mL of dehydrated dichloromethane was added dropwise over 10 minutes. Thereafter, the mixture was stirred at room temperature under argon for 16 hours, basified with 2N NaOH aqueous solution, extracted with dichloromethane and dehydrated to remove the solvent. It was dissolved in 99% formic acid (1.83 g), 37% aqueous formaldehyde solution (3.10 mL, 41.6 mmol) was added, and the mixture was stirred at 110 ° C. for 8.5 hours. Hydrochloric acid was added and the mixture was stirred at 50 ° C. for 3.5 hours. The solution was made basic, extracted with dichloromethane and dehydrated, and the solvent was removed. Further, the product was purified by HPLC to make the liquid basic, followed by extraction with dichloromethane and dehydration. The solvent was removed to obtain the desired product (669.7 mg, 1.82 mmol, y. 51%).
¹ H NMR (300 MHz, CDCl ₃ ) δ: 7.53 (d, 2H, J = 8.1 Hz), 7.14 (d, 2H, J = 8.1 Hz), 3.10 (t, 4H, J = 6.2 Hz), 2.65 (t, 4H, J = 6.2 Hz), 2.32-2.36 (m, 8H), 2.26 (s, 3H), 2.11 (s, 6H) ), 2.07 (s, 3H)
¹³ C NMR (75 MHz, CDCl ₃ ) δ: 142.8, 135.8, 129.4, 126.9, 56.0, 55.3, 55.1, 47.5, 43.5, 43.0 , 21.2
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 369.2324, Found, 369.2298 (−2.6 mmu)

(B) The compound obtained in the above step (a) (669.7 mg, 1.82 mmol) was dissolved in 5 mL of concentrated sulfuric acid and stirred at 110 ° C. for 11 hours under argon. After dilution with water, the solution was made basic with 2N NaOH solution and extracted with dichloromethane. After dehydration, the solvent was removed to obtain the desired product (348.7 mg, 1.63 mmol, y.89%).
¹ H NMR (300 MHz, CDCl ₃ ) δ: 2.80 (t, 4H, J = 5.0 Hz), 2.61 (t, 4H, J = 5.0 Hz), 2.45-2.48 (m , 8H), 2.36 (s, 6H), 2.20 (s, 3H)
¹³ C NMR (75 MHz, CDCl ₃ ) δ: 55.6, 53.6, 53.3, 47.0, 45.2, 21.4
LRMS (ESI ⁺ ): [M + H] ⁺ , 215

(C) TMCyclen-4-AF
Chloroacetylamide-4-aminofluorescein (73.2 mg, 172.7 μmol) was dissolved in 40 mL of dehydrated dimethylformamide, and the compound (117.4 mg, 547.7 μmol) obtained in step (b) and diisopropylethylamine (371 mg, 2.87 mmol) was added and stirred overnight under reflux with heating under argon. After removal of the solvent, purification by HPLC gave TMCyclen-4-AF (TFA salt) (42.2 mg, 42.6 μmol, y.25%).
¹ H NMR (300 MHz, D ₂ O) δ: 8.41 (d, 1H, J = 2.2 Hz), 7.85 (dd, 1H, J = 8.8, 2.2 Hz), 7.27 ( d, 2H, J = 8.4 Hz), 7.19 (d, 1H, J = 8.8 Hz), 6.90 (dd, 2H, J = 8.4, 2.2 Hz), 6.85 (d , 2H, J = 2.2 Hz), 3.75 (s, 2H), 2.90 (s, 6H), 2.62-3.67 (m, 16H), 2.41 (s, 3H)
¹³ C NMR (75 MHz, D ₂ O) δ: 171.8, 167.4, 166.4, 156.4, 137.6, 131.4, 130.7, 129.4, 128.6, 124. 1, 120.6, 117.1, 116.8, 114.1, 112.9, 101.0, 55.6, 52.3, 52.0, 49.0, 48.2, 40.5, 40.4
HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ , 602.2979, Found, 602.2939 (−4.0 mmu)

(D) TMCyclen-4-AF-Cu
CuSO ₄ pentahydrate was dissolved in 30 mM HEPES buffer (pH 7.4) to prepare a 1M CuSO ₄ aqueous solution. TMCyclen-AF (12.1 mg, 12.2 μmol) was dissolved in an aqueous CuSO ₄ solution (1220 μL, 1.22 mmol), stirred at room temperature for 4 hours, purified by HPLC, and purified by TMCyclen-4-AF-Cu. (9.52 mg, y.quant) was obtained.
HRMS (ESI ⁺ ): Calcd for [M−H] ⁺ , 663.2118, Found, 663.2106 (−1.2 mmu)

Example 5
The absorption and fluorescence spectra of Cyclne-4-AF-Cu were measured. Absorption and fluorescence spectra were measured in 30 mM HEPES buffer (pH 7.4). In the measurement of the fluorescence spectrum, Cyclen-4-AF-Cu was excited at 491 nm. In the fluorescence quantum yield measurement, fluorescein (Φ _fl = 0.85) was used as a fluorescence standard in a 0.1N NaOH solution. The results are shown in FIG. 1 (left side) and Table 1. It was shown that the fluorescence quantum yield of Cyclen-4-AF-Cu was lowered. Similarly, Cyclen-4-AF (FIG. 1, right side), TACN-4-AF (left side of FIG. 2), Cyclam-4-AF (FIG. 2, right side), TMCyclen-4-AF (FIG. 3, left side), And the absorption and fluorescence spectrum of TMCyclen-4-AF-Cu (FIG. 3, right side) were measured.

Example 6
The reactivity of Cyclen-4-AF-Cu was evaluated. 10 μM Na ₂ S (red), 10 μM Na ₂ S, 10 mM GSH (green), and 10 mM GSH (yellow) were added to 1 μM Cyclen-4-AF-Cu 300 seconds later, and the experiment without addition was negative control The reactivity was investigated as (blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. The results are shown in FIG. Cyclen-4-AF-Cu showed an increase in fluorescence after reaction with Na ₂ S, indicating that it has selectivity compared to GSH. Cyclen-4-AF-Cu was also found to increase in fluorescence intensity when 10 μM Na ₂ S was added even in the presence of 10 mM GSH.

Example 7
It was evaluated whether or not hydrogen sulfide in vivo can be measured using Cyclen-4-AF-Cu. HeLa cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin in Dulbecco's modified Eagle medium (DMEM) at 37 ° C. in 5% CO ₂ mixed air. Washed twice with Hanks balanced salt solution (HBSS). HBSS (0.1% DMSO) containing 100 μM Cyclen-4-AF-Cu was microinjected (region 1-4) into HeLa cells in HBSS. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured. 5 minutes after the start of measurement, 10 mM Na ₂ S was added to the extracellular fluid. As a result, an increase in fluorescence intensity was observed in the region 1-4 in the microinjected HeLa cells. The upper part of FIG. 5 is before Na ₂ S addition (0 sec), the middle part is after addition of Na ₂ S (360 sec), and the lower part is a change in fluorescence intensity with time, and regions 5 and 6 are regions that are not microinjected. .

Similarly, FIG. 6 shows the results of measurement of hydrogen sulfide by incorporation into HeLa cells using Cyclen-4-AF-Cu diacetate. Hela cells cultured as described above were incubated at 37 ° C. for 2 hours in HBSS containing 100 μM Cyclen-4-AF-Cu diacetate. Excitation was performed at 470-490 nm with a fluorescence microscope, and the fluorescence intensity at 515-550 nm was measured. 210 mM after the start of measurement, 10 mM Na ₂ S was added to the extracellular fluid. Increased fluorescence intensity was observed inside and outside HeLa cells. The upper part of FIG. 6 shows before Na ₂ S addition (0 sec), the middle part after Na ₂ S addition (240 sec), and the lower part shows the change in fluorescence intensity with time.

Example 8
The absorption spectrum and fluorescence spectrum of TACN-4-AF alone and TACN-4-AF added with 2 equivalents of Cu ²⁺ were measured. The measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm in the measurement of the fluorescence spectrum. Addition of Cu ²⁺ significantly reduced the fluorescence intensity (FIG. 7, upper left: absorption spectrum, upper right: fluorescence spectrum). Also, 2 μM CuSO ₄ was added to 1 μM TACN-4-AF, and after 300 seconds, 100 μM Na ₂ S (red), 10 μM Na ₂ S (green), or 10 mM GSH (yellow) was added, and the experiment without addition was negative The reactivity was examined as a control (blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. As a result, TACN-4-AF + Cu ²⁺ showed a large increase in fluorescence after reaction with Na ₂ S, indicating that it has a higher selectivity for hydrogen sulfide than GSH (FIG. 7, lower panel).

Example 9
The absorption spectrum and fluorescence spectrum of Cyclam-4-AF alone and Cyclam-4-AF added with 2 equivalents of Cu ²⁺ were measured. The measurement was performed in 30 mM HEPES buffer (pH 7.4), and excitation was performed at 491 nm in the measurement of the fluorescence spectrum. Addition of Cu ²⁺ significantly decreased the fluorescence intensity (FIG. 8).

Example 10
2 μM CuSO ₄ was added to 1 μM Cyclam-4-AF, and after 300 seconds, 100 μM Na ₂ S (red), 10 μM Na ₂ S (green), or 10 mM GSH (yellow) was added. The reactivity was investigated as blue). Excitation was performed at 491 nm in 30 mM HEPES buffer (pH 7.4), and the fluorescence intensity at 516 nm was measured. As a ^{result, Cyclam-4-AF + Cu} 2+ after the reaction with Na ₂ S, shows the fluorescence increase was shown to have a selectivity compared with GSH (Fig. 9 top). Similarly, 100 μM Na ₂ S (red), 10 μM Na ₂ S (green), or 10 mM GSH (yellow) was added to 1 μM TMCyclen-4-AF-Cu after 300 seconds. When the reactivity was examined as a negative control (blue), TMCyclen-4-AF-Cu also showed an increase in fluorescence after reaction with Na ₂ S, indicating that it has selectivity compared to GSH (lower part of FIG. 9). ).

Example 11: Detection of hydrogen sulfide using cell lysate of 3MST-expressing cells Express 3-mercaptopyruvate sulfate transferase (3MST), an enzyme that produces H ₂ S using 3-mercaptopyruvate (3MP) as a substrate The plasmid was introduced into HEK293 cells by a method described in the literature (Antioxid. Redox Signal, 11, 703, 2009). The cells were suspended in 30 mM HEPES buffer (pH 7.4) containing 100 μM dithiothreitol and 1% protease inhibitor cocktail (Sigma), lysed using an ultrasonic crusher, and used for experiments. Similarly, HEK293 cells into which no plasmid was introduced were also lysed and used for the experiment. Then, 1 μM Cycle-4-AF-Cu was added, and 100 μM 3MP was added after 180 seconds to evaluate the responsiveness. As a result of excitation at 491 nm and measurement of fluorescence intensity at 516 nm, it was shown that when 3MP was added to a lysate containing 3MST, the fluorescence intensity significantly increased (left figure in FIG. 10). Further, after 10 minutes in the presence of 3-mercaptopyruvic acid, a difference was observed in fluorescence intensity with a significant difference (p <0.05) depending on the presence or absence of 3MST (the right diagram in FIG. 10).

Example 12: Detection of hydrogen sulfide using purified 3MST Glutathione-S-transferase (GST) -tagged 3-mercaptopyruvate sulfatransferase (3MST) purified protein and 3-mercaptopyruvate (3MP) as substrate It was evaluated whether the produced H ₂ S was detectable by Cyclen-4-AF-Cu. 1 μM Cyclen-4-AF-Cu was added, and GST tag fusion 3MST (red, green) or GST tag (yellow, blue) was added 60 seconds later. Further, after 120 seconds, 100 μM 3MP or the same amount of HEPES buffer (pH 7.4) was added, and excitation was performed at 491 nm, and fluorescence intensity at 516 nm was measured. As a result, Cyclen-4-AF-Cu showed a significant increase in fluorescence intensity in response to H ₂ S produced by the addition of GST-tag-fused 3MST and 3MP, which are purified proteins (left panel in FIG. 11). In addition, with the aim of high-throughput screening, it was evaluated whether H ₂ S produced by an enzyme reaction could be detected in a 96-well plate. Then, 3MP or the same amount of HEPES buffer (pH 7.4) was added and incubated at 37 ° C. for 60 minutes, and fluorescence was measured. As a result, the same result as in the cuvette was shown, and it was shown that H ₂ S production can be detected even in a 96-well plate by using Cyclen-4-AF-Cu (the right diagram in FIG. 11).

The compound of the present invention can be used as a fluorescent probe capable of measuring hydrogen sulfide with high sensitivity and specificity without being affected by reduced glutathione present in large quantities in the living body.

Claims (10)

1. The following general formula (IA) or (IB):
   

   

   [Wherein R ¹ represents the following formula (A):
   

   

   (In the formula, p, q, r, and s each independently represent an integer of 2 or 3, t represents 0 or 1, and R ¹¹ , R ¹² , and R ¹³ each independently represent a hydrogen atom or a carbon atom. R ² represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R ³ and R ⁴ represent Each independently represents a hydrogen atom or a halogen atom; R ⁵ and R ⁶ each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; and R ⁷ represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group. R ⁸ and R ⁹ are each independently a hydrogen atom, a halogen atom, or — (CH ₂ ) _x —N (R ¹⁴ ) (R ¹⁵ ) (wherein x represents an integer of 1 to 4; R ¹⁴ and ^{R 15} are each The standing ^{_{- (CH 2) y -COOR 16}} ( wherein, y represents an integer of 1 ^{4, R 16} is a hydrogen atom, an alkylcarbonyl group, or an alkyl carbonyl an oxy alkyl group) a group represented by Or a salt thereof.
2. R ¹ present on the benzene ring is bonded to the bonding position of the xanthene ring at the para position, p, q, r, and s are 2, t is 1, and R ¹¹ , R ¹² , And R ¹³ is a hydrogen atom, and R ² is a hydrogen atom, The compound represented by the general formula (IA) or (IB) or a salt thereof according to claim 1.
3. R ³ and R ⁴ are a hydrogen atom, R ⁵ and R ⁶ are each independently a hydrogen atom, an acetyl group, or an acetyloxymethyl group, R ⁷ is a hydrogen atom, an alkyl group, or a methoxymethyl group, The compound represented by general formula (IA) or (IB) or a salt thereof according to claim 1 or 2, wherein R ⁸ and R ⁹ are both hydrogen atoms.
4. The following general formula (IIA) or (IIB):
   

   

   [Wherein R ²¹ represents the following formula (B):
   

   

   (In the formula, d, e, f, and g each independently represent an integer of 2 or 3, h represents 0 or 1, and R ³¹ , R ³² , and R ³³ each independently represent a hydrogen atom or a carbon atom. R ²² represents a hydrogen atom or 1 to 3 monovalent substituents substituted on a benzene ring; R ²³ and R ²⁴ represent Each independently represents a hydrogen atom or a halogen atom; R ²⁵ and R ²⁶ each independently represent a hydrogen atom, an alkylcarbonyl group, or an alkylcarbonyloxyalkyl group; R ²⁷ represents a hydrogen atom, an alkyl group, or an alkoxyalkyl group; R ²⁸ and R ²⁹ are each independently a hydrogen atom, a halogen atom, or — (CH ₂ ) _m —N (R ³⁴ ) (R ³⁵ ) (wherein m represents an integer of 1 to 4; R ³⁴ as well as ³⁵ are each independently ^{_{- (CH 2) n -COOR 36}} ( wherein, n represents an integer of 1 to ^{4, R 36} is a hydrogen atom, an alkyl group, or an alkylcarbonyloxy group) is represented by Or a salt thereof.
5. R ²¹ present on the benzene ring is bonded to the bonding position of the xanthene ring in the para position, d, e, f, and g are 2, h is 1, R ³¹ , R ³² , And R ³³ is a hydrogen atom, and R ²² is a hydrogen atom, The compound represented by formula (IIA) or (IIB) or a salt thereof according to claim 4.
6. R ²³ and R ²⁴ are a hydrogen atom, R ²⁵ and R ²⁶ are each independently a hydrogen atom, an acetyl group, or an acetyloxymethyl group, R ²⁷ is a hydrogen atom, an alkyl group, or a methoxymethyl group, The compound represented by the general formula (IIA) or (IIB) or a salt thereof according to claim 4 or 5, wherein R ²⁸ and R ²⁹ are both hydrogen atoms.
7. A fluorescent probe for measuring hydrogen sulfide comprising the compound represented by the general formula (IA) or (IB) according to any one of claims 1 to 3 or a salt thereof.
8. A fluorescent probe for measuring hydrogen sulfide comprising the compound represented by the general formula (IIA) or (IIB) according to any one of claims 4 to 6 or a salt thereof.
9. A method for measuring hydrogen sulfide, comprising the following steps:
   (A) a step of bringing the compound represented by the general formula (IA) or (IB) or a salt thereof according to any one of claims 1 to 3 into contact with hydrogen sulfide; and (b) the step (a). A method comprising a step of measuring the fluorescence of the compound represented by the above general formula (IIA) or (IIB) or a salt thereof generated in (1).
10. A method for measuring hydrogen sulfide, comprising the following steps:
    (A) A compound represented by the general formula (IIA) or (IIB) according to any one of claims 4 to 6 or a salt thereof and a divalent copper ion are reacted to form the above general formula (IA) or A step of producing a compound represented by (IB) or a salt thereof;
    (B) a step of bringing the compound represented by the general formula (IA) or (IB) or a salt thereof generated in the step (a) into contact with hydrogen sulfide; and (c) the step (b). The method including the process of measuring the fluorescence of the compound or its salt represented by the said general formula (IIA) or (IIB).

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