JP2018145126A - Fluorescent probe for detection of carboxypeptidase activity - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel fluorescent probe for detection of carboxypeptidase activity that can be applied to a wide range of carboxypeptidases.SOLUTION: The present invention provides a compound represented by formula (I) or a salt thereof (NH-L1 and NH-L2 independently represent an amino acid residue, a peptide or a C1-6 aminoalkyl group, where, at least one of NH-L1 and NH-L2 is an amino acid residue or, preferably, the same amino acid residue, or particularly preferably, a phenylalanine residue, a leucine residue or a lysine residue).SELECTED DRAWING: None

Description

  The present invention relates to a novel fluorescent probe for detecting carboxypeptidase activity.

  Carboxypeptidase is a general term for a group of enzymes that recognize and cleave one or more amino acids from the C-terminus of a peptide chain. Many relations with various biological phenomena and diseases in the living body have been reported so far, and inhibitors of angiotensin converting enzyme (ACE) are widely used as antihypertensive drugs, Prostate membrane specific antigen (PSMA) Is a very important group of enzymes not only from biological research but also from the viewpoint of drug discovery and diagnosis, because it is a prostate cancer and Carboxypeptidase A is a biomarker of pancreatic disease.

  Due to its importance, a number of techniques for detecting the enzymatic activity of carboxypeptidases have been developed so far. Among them, some techniques using a fluorescence method with excellent sensitivity have been reported, but most of them have complicated operations such as derivatization or extraction of enzyme reaction products. For example, as a method for detecting the enzyme activity of carboxypeptidase by a fluorescence method, a method for detecting the activity of carboxypeptidase M using Dns-Ala-Arg (Non-patent Document 1), or an enzyme reaction using a peptide substrate. And angiotensin converting enzyme activity detection method using non-patent document 2 using the subsequent derivatization.

In addition, a few types of fluorescent substrates in which fluorescence is increased by the metabolism of the substrate by the enzyme have been reported. For example, as a method for observing the increase in fluorescence by the metabolism of the substrate to the enzyme, carboxy Examples using a fluorescent substrate based on the principle of FRET have been reported, including an example of detecting peptidase A activity (Non-patent Document 3) and an example of detecting angiotensin converting enzyme activity (Non-patent Document 4). However, in these molecular designs, since the enzyme recognition site is used as a fluorescence control site such as a fluorophore or a quencher, there is a problem that the number of enzymes capable of detecting the activity is extremely limited.
Moreover, it can be said that it is a problem from the viewpoint of cytotoxicity that ultraviolet light excitation is necessary for fluorescence detection in all the above-described examples.

  The research group of the present inventors has developed an aminopeptidase fluorescent probe that hydrolyzes the amino terminus of a protein in order to visualize minute cancers. As mentioned above, carboxypeptidase, a hydrolase at the carboxy terminus, is also known to be involved in important biological functions. Until now, fluorescent probes capable of detecting carboxypeptidase activity in living cells or in vivo Not developed.

Journal of Biological Chemistry, 1990, 25, 15083 Journal of neurochemistry, 1972, 10, 2443 Analytical biochemistry, 1972, 1, 56 European Journal of Biochemistry, 1978, 87, 265

  An object of the present invention is to provide a novel fluorescent probe for detecting carboxypeptidase activity that can be applied to a wide range of carboxypeptidases.

  As a result of intensive studies, the present inventors have established a fluorescent probe design method applicable to carboxypeptidases in general by introducing a nucleophilic substituent that exhibits optimal intramolecular spirocyclization properties to the rhodamine skeleton. Successfully completed the present invention.

（式中、
Ｒ^１は、水素原子を示すか、又はベンゼン環上に存在する１ないし４個の同一又は異なる一価の置換基を示し：
Ｒ^２及びＲ^３は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^４及びＲ^５は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^６及びＲ^７は、それぞれ独立に、水素原子、炭素数１〜３の分枝鎖を有してもよい炭素数１〜７のアルキル基、エチル基、フェニルメチル基、４−ヒドロキシフェニルメチル基、１−ハイドロキシエチル基、又は−（ＣＨ₂）_ｎ−Ｚ基を表し、ｎは１〜４の数を表し、Ｚはヒドロキシ基、カルボキシル基、アミノ基、チオール基、メチルチオ基、グアニジノ基、カルバモイル基、又は窒素原子を１〜３個有してもよい５員環若しくは６員環又は５員環と６員環から構成される縮合複素環を示し；
Ｘは、酸素原子、Ｓｉ（Ｒ^ａ）（Ｒ^ｂ）、Ｃ（Ｒ^ａ）（Ｒ^ｂ）、Ｇｅ（Ｒ^ａ）（Ｒ^ｂ）、Ｐ（＝Ｏ）Ｒ^ｃ又はＳｅから選択され、
ここで、Ｒ^ａ及びＲ^ｂは、それぞれ独立に、炭素数１〜６個のアルキル基又は置換されていてもよいアリール基であり、Ｒ^ｃは、炭素数１〜６個のアルキル基又は置換されていてもよいフェニル基であり；
Ｙは、ＮＨ、Ｏ、Ｓ又はＮＲから選択され、
ここで、ＮＲは、置換されていてもよいアミノ基又は置換されていてもよいアミド基を表し；
式（Ｉ）のＮＨ−Ｌ^１およびＮＨ−Ｌ^２は、それぞれ独立に、アミノ酸残基、ペプチド、又は炭素数１〜６のアミノアルキル基を表し、ただし、ＮＨ−Ｌ^１及びＮＨ−Ｌ^２の少なくとも１つはアミノ酸残基である；
ｍは、１〜３の整数を表す；
で表される化合物又はその塩。
［２］ＹはＮＨである、［１］に記載の化合物又はその塩。
［３］ＮＨ−Ｌ^１及びＮＨ−Ｌ^２は、同一又は異なるアミノ酸残基である、［１］又は［２］に記載の化合物又はその塩。
［４］ＮＨ−Ｌ^１及びＮＨ−Ｌ^２は、同一のアミノ酸残基である、［３］に記載の化合物又はその塩。
［５］ＮＨ−Ｌ^１及びＮＨ−Ｌ^２の両方が、フェニルアラニン残基、ロイシン残基又はリジン残基である、［４］に記載の化合物又はその塩。
［６］Ｘは酸素原子である、［１］〜［５］のいずれか１項に記載の化合物又はその塩。
［７］［１］〜［６］のいずれか１項に記載の化合物又はその塩を含むカルボキシペプチダーゼ活性検出用蛍光プローブ。
［８］細胞内のカルボキシペプチダーゼを検出する方法であって、（ａ）［７］に記載の蛍光プローブを細胞内に導入する工程、及び（ｂ）当該蛍光プローブが細胞内で発する蛍光を測定することを含む方法。
［９］以下の一般式（ＩＩＩ）：

（式中、
Ｒ^１は、水素原子を示すか、又はベンゼン環上に存在する１ないし４個の同一又は異なる一価の置換基を示し：
Ｒ^２及びＲ^３は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^４及びＲ^５は、それぞれ独立に、水素原子、炭素数１〜６個のアルキル基又はハロゲン原子を示し；
Ｒ^６及びＲ^７は、それぞれ独立に、水素原子、炭素数１〜３の分枝鎖を有してもよい炭素数１〜７のアルキル基、エチル基、フェニルメチル基、４−ヒドロキシフェニルメチル基、１−ハイドロキシエチル基、又は−（ＣＨ₂）_ｎ−Ｚ基を表し、ｎは１〜４の数を表し、Ｚはヒドロキシ基、カルボキシル基、アミノ基、チオール基、メチルチオ基、グアニジノ基、カルバモイル基、又は窒素原子を１〜３個有してもよい５員環若しくは６員環又は５員環と６員環から構成される縮合複素環を示し；
Ｘは、酸素原子、Ｓｉ（Ｒ^ａ）（Ｒ^ｂ）、Ｃ（Ｒ^ａ）（Ｒ^ｂ）、Ｇｅ（Ｒ^ａ）（Ｒ^ｂ）、Ｐ（＝Ｏ）Ｒ^ｃ又はＳｅから選択され、
ここで、Ｒ^ａ及びＲ^ｂは、それぞれ独立に、炭素数１〜６個のアルキル基又は置換されていてもよいアリール基であり、Ｒ^ｃは、炭素数１〜６個のアルキル基又は置換されていてもよいフェニル基であり；
Ｙは、ＮＨ、Ｏ、Ｓ又はＮＲから選択され、
ここで、ＮＲは、置換されていてもよいアミノ基又は置換されていてもよいアミド基を表し；
ｍは、１〜３の整数を表す；
で表される化合物又はその塩。
を提供するものである。 That is, the present invention
[1] The following general formula (I):

(Where
R ¹ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on the benzene ring:
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms, an ethyl group, a phenylmethyl group, or 4-hydroxyphenylmethyl. Represents a group, 1-hydroxyethyl group, or — (CH ₂ ) _n —Z group, n represents a number of 1 to 4, and Z represents a hydroxy group, a carboxyl group, an amino group, a thiol group, a methylthio group, a guanidino group. , A carbamoyl group, or a 5- or 6-membered ring optionally having 1 to 3 nitrogen atoms, or a condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring;
X is selected from an oxygen atom, Si (R ^a ) (R ^b ), C (R ^a ) (R ^b ), Ge (R ^a ) (R ^b ), P (═O) R ^c or Se;
Here, R ^a and R ^b are each independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, and R ^c is an alkyl group having 1 to 6 carbon atoms or a substituted group. An optionally substituted phenyl group;
Y is selected from NH, O, S or NR;
Here, NR represents an amino group which may be substituted or an amide group which may be substituted;
NH-L ¹ and NH-L ^{2 in} formula (I) each independently represent an amino acid residue, a peptide, or an aminoalkyl group having 1 to 6 carbon atoms, provided that NH-L ¹ and NH-L ² At least one of the is an amino acid residue;
m represents an integer of 1 to 3;
Or a salt thereof.
[2] The compound or salt thereof according to [1], wherein Y is NH.
[3] The compound or a salt thereof according to [1] or [2], wherein NH-L ¹ and NH-L ² are the same or different amino acid residues.
[4] The compound or a salt thereof according to [3], wherein NH-L ¹ and NH-L ² are the same amino acid residue.
[5] The compound or salt thereof according to [4], wherein both NH-L ¹ and NH-L ² are a phenylalanine residue, a leucine residue, or a lysine residue.
[6] The compound or a salt thereof according to any one of [1] to [5], wherein X is an oxygen atom.
[7] A fluorescent probe for detecting carboxypeptidase activity comprising the compound or salt thereof according to any one of [1] to [6].
[8] A method for detecting intracellular carboxypeptidase, wherein (a) the step of introducing the fluorescent probe according to [7] into the cell, and (b) measurement of fluorescence emitted from the fluorescent probe in the cell. A method comprising:
[9] The following general formula (III):

(Where
R ¹ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on the benzene ring:
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms, an ethyl group, a phenylmethyl group, or 4-hydroxyphenylmethyl. Represents a group, 1-hydroxyethyl group, or — (CH ₂ ) _n —Z group, n represents a number of 1 to 4, and Z represents a hydroxy group, a carboxyl group, an amino group, a thiol group, a methylthio group, a guanidino group. , A carbamoyl group, or a 5- or 6-membered ring optionally having 1 to 3 nitrogen atoms, or a condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring;
X is selected from an oxygen atom, Si (R ^a ) (R ^b ), C (R ^a ) (R ^b ), Ge (R ^a ) (R ^b ), P (═O) R ^c or Se;
Here, R ^a and R ^b are each independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, and R ^c is an alkyl group having 1 to 6 carbon atoms or a substituted group. An optionally substituted phenyl group;
Y is selected from NH, O, S or NR;
Here, NR represents an amino group which may be substituted or an amide group which may be substituted;
m represents an integer of 1 to 3;
Or a salt thereof.
Is to provide.

  According to the present invention, a fluorescent probe for detecting carboxypeptidase activity applicable to a wide range of carboxypeptidases can be provided.

The fluorescence spectrum of the probe before and after enzyme addition is shown. (A) is a measurement result about AMRBC-Phe and carboxypeptidase A, (b) is a measurement result about AMRBC-Lys and carboxypeptidase B. The result of the UPLC analysis of the probe after reaction with an enzyme is shown. (A) is a measurement result about AMRBC-Phe and carboxypeptidase A, (b) is a measurement result about AMRBC-Lys and carboxypeptidase B.

  In the present specification, the “alkyl group” or the alkyl part of a substituent containing an alkyl part (for example, an alkoxy group or the like) unless otherwise specified, for example, has 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, More preferably, it means an alkyl group composed of a linear, branched, cyclic, or combination thereof having about 1 to 3 carbon atoms. More specifically, as the alkyl group, for example, methyl group, ethyl group, n-propyl group, isopropyl group, cyclopropyl group, n-butyl group, sec-butyl group, isobutyl group, tert-butyl group, cyclopropyl A methyl group, n-pentyl group, n-hexyl group, etc. can be mentioned.

  In the present specification, the term “halogen atom” may be any of a fluorine atom, a chlorine atom, a bromine atom or an iodine atom, preferably a fluorine atom, a chlorine atom or a bromine atom.

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

In the general formula (I), R ¹ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on the benzene ring.
R ¹ may be the same or different.

Although the kind of monovalent substituent represented by R ¹ is not particularly limited, for example, an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, an alkynyl group having 1 to 6 carbon atoms, carbon It is preferably selected from the group consisting of several to six alkoxy groups, hydroxyl groups, carboxy groups, sulfonyl groups, alkoxycarbonyl groups, halogen atoms, amino groups, amide groups, and alkylamide groups.
These monovalent substituents may further have one or more arbitrary substituents. For example, the alkyl group represented by R ¹ may have one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, and the like. For example, the alkyl group represented by R ¹ is a halogen atom. An alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or an aminoalkyl group may be used. Further, for example, the amino group represented by R ¹ may be present one or two alkyl groups, an amino group represented by R ¹ may be a monoalkylamino group or a dialkylamino group. Furthermore, examples of the case where the alkoxy group represented by R ¹ has a substituent include a carboxy-substituted alkoxy group or an alkoxycarbonyl-substituted alkoxy group, and more specifically, a 4-carboxybutoxy group or 4-acetoxymethyloxy. A carbonyl butoxy group etc. can be mentioned.

In a preferred aspect of the present invention, each R ¹ is a hydrogen atom.

In general formula (I), R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.
When R ² or R ³ represents an alkyl group, the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, For example, the alkyl group represented by R ² or R ³ may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like. R ² and R ³ are preferably each independently a hydrogen atom or a halogen atom. When R ² and R ³ are both hydrogen atoms, or R ² and R ³ are both fluorine atoms or chlorine atoms. More preferred.

In the general formula (I), R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom, and are the same as those described for R ⁴ and R ^5. . R ⁴ and R ⁵ are preferably both hydrogen atoms, both chlorine atoms, or both fluorine atoms.

X is selected from an oxygen atom, Si (R ^a ) (R ^b ), C (R ^a ) (R ^b ), Ge (R ^a ) (R ^b ), P (═O) R ^c or Se.
In a preferred embodiment of the present invention, X is an oxygen atom.

R ^a and R ^b are each independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group. R ^a and R ^b are each independently preferably an alkyl group having 1 to 3 carbon atoms, and more preferably both R ^a and R ^b are methyl groups. The alkyl group represented by R ^a and R ^b may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, and the like, for example, R ^a and R ^b represent The alkyl group may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like. When R ^a and R ^b represent an aryl group, the aryl group may be either a monocyclic aromatic group or a condensed aromatic group, and the aryl ring has one or more ring-constituting heteroatoms. (For example, a nitrogen atom, an oxygen atom, or a sulfur atom) may be contained. The aryl group is preferably a phenyl group. One or more substituents may be present on the aryl ring. As the substituent, for example, one or two or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups and the like may be present.

R ^c is an alkyl group having 1 to 6 carbon atoms or an optionally substituted phenyl group. Examples of the substituent for the phenyl group include a methyl group, a hydroxy group, and a methoxy group.
From the viewpoint of ease of introduction in the synthesis, R ^c is preferably a methyl group or a phenyl group. In addition, it is more preferable that R ^c is a methyl group because of high water solubility.

  One feature of the compound of the general formula (I) is that a spiro ring containing Y is formed between the xanthene skeleton and the benzene ring bonded thereto. One characteristic required of a fluorescent probe for detecting carboxypeptidase activity is that fluorescence increases before and after the enzyme reaction. From the fluorophore side before and after the enzyme reaction, carboxypeptidase is amide → carboxyl. The rate change caused a structural change, but with this structural change, the electron withdrawing change was small, and it was expected that it was difficult to obtain a dynamic fluorescence increase. Therefore, by introducing an intramolecular spiro ring into the rhodamine skeleton, the change in the electron-withdrawing property of amide → carboxylate into a change in the spiro cyclization equilibrium makes the rhodamine skeleton ring-closed (non-fluorescent) → ring-opened. It is possible to cause structural changes in the body (fluorescence) and increase fluorescence before and after the enzyme reaction.

In general formula (I), Y is selected from NH, O, S or NR. Here, NR represents an amino group which may be substituted or an amide group which may be substituted.
In one preferred embodiment of the invention, Y is NH. Although not intended to be bound by theory, when Y is NH, the amino group functions as an intramolecular nucleophilic species having a higher nucleophilicity in the ring-opened form of the compound of the general formula (I) described later. Therefore, the ratio of the ring-closed body under physiological conditions (pH around 7.5) is significantly increased, and can be used effectively as a probe.

  In general formula (I), m represents an integer of 1 to 3. Preferably, m is 1.

In the general formula (I), —NH—CH (R ⁶ ) C (═O) NH—L ¹ and —NH—CH (R ⁷ ) C (═O) NH—L ² are an enzyme (carboxypeptidase). ) Represents a portion recognized as a substrate. Here, at least one of NH-L ¹ and NH-L ² is an amino acid residue.
In the present invention, by introducing the above site having a structure similar to hippuryl amino acid into the amino group at the 3rd and 6th positions of the rhodamine skeleton, the carboxypeptidase has a function of recognizing as a substrate. It is.

R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms, an ethyl group, a phenylmethyl group, 4-hydroxy Represents a phenylmethyl group, a 1-hydroxyethyl group, or a — (CH ₂ ) _n —Z group, n represents a number of 1 to 4, and Z represents a hydroxy group, a carboxyl group, an amino group, a thiol group, a methylthio group, A fused heterocycle composed of a guanidino group, a carbamoyl group, or a 5-membered ring or 6-membered ring which may have 1 to 3 nitrogen atoms, or a 5-membered ring and a 6-membered ring.
In one preferred embodiment of the present invention, R ⁶ and R ⁷ are both hydrogen atoms.

In General Formula (I), NH-L ¹ and NH-L ² in Formula (I) each independently represent an amino acid residue, a peptide, or an aminoalkyl group having 1 to 6 carbon atoms. However, at least one of NH-L ¹ and NH-L ² is an amino acid residue.
The amino acid residue can be selected from any amino acid residue, and is preferably a phenylalanine residue, a leucine residue or a lysine residue.

In a preferred embodiment of the invention, NH-L ¹ and NH-L ² are the same or different amino acid residues.
In a more preferred embodiment of the present invention, NH-L ¹ and NH-L ² are the same amino acid residue.

Another embodiment of the present invention is a compound represented by the following general formula (II) or a salt thereof.

In the general formula (II), R ^{1 to} R ⁷ , X, Y, NH—L ¹ , NH—L ² and m are the same as described in the general formula (I).

  The compound of general formula (II) corresponds to a ring-opened product of the compound of general formula (I). The compounds of the general formulas (I) and (II) take an equilibrium state, and these ratios change depending on pH, particularly in an aqueous solution.

Preferred non-limiting examples of compounds of general formula (II) are:

Another embodiment of the present invention is a compound represented by the following general formula (III) or a salt thereof.

R ^{1 to} R ⁷ , X, Y, and m in the general formula (II) are the same as those described in the general formula (I).

  The compound represented by the general formula (III) or a salt thereof corresponds to a reaction product of the compound of the general formula (I) and carboxypeptidase.

  The compounds of general formulas (I), (II) and (III) of the present invention can exist as acid addition salts or base addition salts. Examples of the acid addition salt include mineral acid salts such as hydrochloride, sulfate, and nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, and tartrate. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, and magnesium salt, organic amine salts such as ammonium salt, and triethylamine salt. In addition to these, a salt with an amino acid such as glycine may be formed. The compounds of general formulas (I), (II) and (III) or salts thereof of the present invention may exist as hydrates or solvates, and these substances are also within the scope of the present invention.

  The compounds of the general formulas (I), (II) and (III) of the present invention may have one or more asymmetric carbons depending on the type of substituent, but one or two or more asymmetric carbons may be present. In addition to stereoisomers such as optically active substances based on asymmetric carbon and diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates, etc. are all within the scope of the present invention. Is included.

  The production methods of representative compounds of the compounds of the general formulas (I) and (II) of the present invention are specifically shown in the examples of this specification. Accordingly, those skilled in the art can appropriately select reaction raw materials, reaction conditions, reaction reagents, and the like based on these explanations, and modify or modify these methods as necessary, so that the general formula (I ) And (II) can be produced.

  Another embodiment of the present invention is a fluorescent probe for detecting carboxypeptidase activity comprising a compound of general formula (I) or a salt thereof.

  The fluorescent probe for detecting carboxypeptidase activity of the present invention can be applied to detection of a wide range of carboxypeptidases. For example, it can be applied to carboxypeptidase A, carboxypeptidase B, carboxypeptidase M and the like.

  Another embodiment of the present invention is a method for detecting carboxypeptidase in a cell, wherein (a) the step of introducing the fluorescent probe of the present invention into the cell, and (b) the fluorescent probe is intracellular. Measuring the emitted fluorescence.

  The method of using the fluorescent probe of the present invention is not particularly limited, and can be used in the same manner as conventionally known fluorescent probes. In general, an aqueous medium such as physiological saline or buffer, or a mixture of an aqueous medium such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, dimethylformamide and an aqueous medium and the like represented by the general formula (I) or ( The compound represented by II) or a salt thereof is dissolved, this solution is added to an appropriate buffer containing cells and tissues, and the fluorescence spectrum is measured. The fluorescent probe of the present invention may be used in the form of a composition in combination with appropriate additives. For example, it can be combined with additives such as a buffer, a solubilizing agent and a pH adjuster.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to this.

[Synthesis Example 1]
According to the following scheme 1, compound 5 (AMBC-Phe) and compound 6 (AMRBC-Lys), which are compounds of the present invention, were synthesized.

(a) 1) MeOH, H₂SO₄, reflux, 2) LAH, THF, y. 56 % in 2 steps (b) TBS-Cl, Imidazole, DMF, y. 94 % (c) 1) glyoxylic acid, AcOH, EtOH, 2) 2-picoline borane, 3) MeOH, H₂SO₄, y. 20 % in 3 steps (d) 1) DPPA, DBU, THF, 2) PPh3, THF, H₂O, 3) Chloranil, DCM, 4) NaOH, MeOH, H₂O, y. 19 % in 4 steps (e) 1) H-Phe-OtBu-HCl, HATU, DIEA, DMF 2) TFA, MeCN, y. 68 % in 2 steps (f) H-Lys(Boc)-OtBu-HCl, HATU, DIEA, DMF 2) TFA, MeCN, y. 73 % in 2 steps
Scheme 1. Synthesis of AMRBC-Phe (5) and AMRBC-Lys (6)

(a) 1) MeOH, H ₂ SO ₄ , reflux, 2) LAH, THF, y. 56% in 2 steps (b) TBS-Cl, Imidazole, DMF, y. 94% (c) 1) glyoxylic acid, AcOH, EtOH, 2) 2-picoline borane, 3) MeOH, H ₂ SO ₄ , y. 20% in 3 steps (d) 1) DPPA, DBU, THF, 2) PPh3, THF, H ₂ O, 3) Chloranil, DCM, 4) NaOH, MeOH, H ₂ O, y. 19% in 4 steps (e) 1) H-Phe-OtBu-HCl, HATU, DIEA, DMF 2) TFA, MeCN, y. 68% in 2 steps (f) H-Lys (Boc) -OtBu-HCl, HATU, DIEA, DMF 2) TFA, MeCN, y. 73% in 2 steps

Synthesis of Compound 1 Rhodamine 110 Chloride 1127 mg (3.12 mmol, 1 eq) was dissolved in 180 mL of methanol, 9 mL of sulfuric acid was added, and the mixture was stirred overnight at 80 ° C. under an argon atmosphere. After cooling to room temperature, the reaction solvent was removed under reduced pressure, and the residue was neutralized with a saturated aqueous sodium hydrogen carbonate solution and then filtered. The solid obtained by filtration was dissolved in methanol and collected, and the methanol was removed under reduced pressure. The residue was dissolved in 200 mL of tetrahydrofuran, 1545 mg (40.7 mmol, 13 eq) of lithium aluminum hydride was added while stirring in an ice bath, and the mixture was stirred overnight at room temperature while being protected from light with aluminum foil under an argon atmosphere. While stirring the reaction solution in an ice bath, 30 mL of methanol was added, and then the reaction solvent was removed under reduced pressure. 200 mL of saturated Rochelle salt aqueous solution and 100 mL of ethyl acetate were added to the residue, and the mixture was stirred overnight under an argon atmosphere while being shielded from light with aluminum foil. The ethyl acetate layer was extracted from the reaction solution by liquid separation, and then the ethyl acetate was removed under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane / methanol = 90/10) to obtain the target compound (557 mg, 56%). .
¹ H NMR (300 MHz, CD ₃ OD): δ 4.63 (s, 2H), 5.38 (s, 1H), 6.30 (dd, 2H, J = 2.1, 9.0 Hz), 6.41 (d, 2H, J = 2.1 Hz), 6.64 (d, 2H, J = 9.0 Hz), 7.04-7.08 (m, 1H), 7.14-7.16 (m, 2H), 7.39-7.42 (m, 1H)
¹³ C NMR (100 MHz, CD ₃ OD): δ 40.2, 63.0, 103.4, 112.2, 115.4, 127.3, 128.7, 128.9, 131.2, 131.9, 145.7, 148.5, 152.8
HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 319.14018; found, 319.14465 (-4.48 mmu)

Synthesis of Compound 2 Compound 557 mg (1.75 mmol, 1 eq), t-butyldimethylsilyl chloride 402 mg (2.66 mmol, 1.5 eq), imidazole 242 mg (3.55 mmol, 2 eq) The resultant was dissolved in 25 mL of N′-dimethylformamide, and stirred for 4 hours while being shielded from light with aluminum foil under an argon atmosphere. After adding 100 mL of water, ethyl acetate was added and the ethyl acetate layer was extracted by liquid separation operation. Ethyl acetate was removed under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate / hexane = 2/3) to obtain the desired compound (713 mg, 94%).
¹ H NMR (400 MHz, aceton-d ₆ ): δ 0.11 (s, 6H), 0.98 (s, 9H), 4.72 (s, 4H), 4.79 (s, 2H), 5.44 (s, 1H), 6.35 (dd, 2H, J = 2.9, 10.7 Hz), 6.46 (d, 2H, J = 2.9 Hz), 6.71 (d, 2H, J = 10.7 Hz), 7.22-7.29 (m, 3H), 7.53-7.57 ( m, 1H)
¹³ C NMR (75 MHz, CDCl ₃ ): δ-5.47, 18.3, 25.9, 39.1, 62.9, 102.0, 110.6, 114.1, 126.4, 127.2, 127.4, 130.4, 130.7, 138.5, 143.9, 146.0, 151.3
HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 433.23113; found, 433.23114 (0.01 mmu)

Compound 3 Synthesis Compound 2 432 mg (1.00 mmol, 1 eq) and 9 M glyoxylic acid aqueous solution 300 μL (3.00 mmol, 3 eq) were dissolved in a mixed solvent of ethanol 4.95 mL and acetic acid 50 μL, and light-shielded with aluminum foil under an argon atmosphere. And stirred for 1 hour. To the reaction solution, 641 mg (6.00 mmol, 6 eq) of 2-picoline borane was added, and the mixture was further stirred overnight under light shielding with aluminum foil under an argon atmosphere. After adding ethyl acetate and a saturated aqueous citric acid solution, the ethyl acetate layer was extracted by a liquid separation operation. The extracted organic layer was dried over sodium sulfate, and then the solvent was removed by removing under reduced pressure. The residue was dissolved in 50 mL of methanol and 2.5 mL of sulfuric acid, heated under reflux in an argon atmosphere, and stirred overnight. The reaction solution was cooled to room temperature, the solvent was removed under reduced pressure, ethyl acetate was added, the solution was neutralized with saturated aqueous sodium hydrogen carbonate solution, and the ethyl acetate layer was extracted by liquid separation operation. The extracted organic layer was dried over sodium sulfate, and then the solvent was removed by removing under reduced pressure. The residue was partially purified by amino silica gel column chromatography (ethyl acetate / hexane = 2/1 → ethyl acetate). The fractions containing the target compound were collected, the solvent was removed by removal under reduced pressure, and the residue was purified using a GPC recycling column to obtain the target compound (88 mg, 19%).
¹ H NMR (300 MHz, CDCl ₃ ): δ 3.76 (s, 6H), 3.86 (s, 4H), 4.56 (s, 2H), 5.33 (s, 1H), 6.19 (dd, 2H, J = 2.6, 8.4 Hz), 6.26 (d, 2H, J = 2.2 Hz), 6.65 (d, 2H, J = 8.1 Hz), 7.17-7.20 (m, 3H), 7.34-7.39 (m, 1H)
¹³ C NMR (75 MHz, CDCl ₃ ): δ39.6, 45.6, 52.2, 62.9, 99.7, 109.1, 114.0, 126.8, 128.1, 129.2, 130.1, 131.2, 138.2, 144.6, 146.8, 151.3, 171.4
HRMS (ESI ⁺ ): calcd for [M + Na] ⁺ , 485.16886; found, 485.16633 (-2.52 mmu)

Synthesis of Compound 4 Compound 88 88 mg (0.190 mmol, 1 eq), diphenyl phosphate azide 163 μL (0.758 mmol, 4 eq), diazabicycloundecene 114 μL (0.816 mmol, 4.3 eq) were dissolved in tetrahydrofuran 15 mL. And stirred at 65 ° C. overnight under an argon atmosphere. Ethyl acetate and a saturated aqueous ammonium chloride solution were added to the reaction solution, and then the ethyl acetate layer was extracted by a liquid separation operation. The extracted organic layer was dried over sodium sulfate, and then the solvent was removed by removing under reduced pressure. After partial purification by amino silica gel column chromatography (ethyl acetate / hexane = 2/1), fractions containing the target compound were collected and the solvent was removed by removal under reduced pressure. To the residue was added a mixed solvent of 10 mL of tetrahydrofuran and 1 mL of water, 100 mg (0.381 mmol, 2 eq) of triphenylphosphine was further added, and the mixture was heated to reflux under an argon atmosphere and stirred overnight. The reaction solution is cooled to room temperature, the solvent is removed under reduced pressure, the residue is partially purified by amino silica gel column chromatography (dichloromethane → dichloromethane / methanol = 9/1), fractions containing the target compound are collected, and the solvent is removed by removing under reduced pressure. Was excluded. The residue was dissolved in 5 mL of dichloromethane, 47 mg (0.191 mmol, 1 eq) of chloranil was added, and the mixture was stirred for 30 minutes. After removing the solvent under reduced pressure, the residue was partially purified by silica gel column chromatography (dichloromethane / methanol = 95/5), fractions containing the target compound were collected, and the solvent was removed by removing under reduced pressure. The residue was dissolved in 5 mL of methanol, 0.5 M aqueous sodium hydroxide solution was added thereto, and the mixture was stirred for 30 minutes. After neutralization with 2M hydrochloric acid, the solvent was removed under reduced pressure, and HPLC (eluent A (H ₂ O 0.1% TFA) and eluent B (CH ₃ CN 80%, H ₂ O 20%, 0.1% TFA) (A / B = 80/20 for 6 min, then 0/100 in 36 min)) to obtain the target compound (26 mg, 25%).
¹ H NMR (400 MHz, CD ₃ OD + NaOD ): δ 3.54 (s, 4H), 4.16 (s, 2H), 6.20 (d, 1H, J = 2.4 Hz), 6.23 (t, 3H, J = 2.4 Hz), 6.46 (d, 2H, J = 8.8 Hz), 6.74 (d, 1H, J = 7.3 Hz), 7.14 (t, 1H, J = 6.8 Hz), 7.22 (td, 1H, J = 1.0, 7.3 Hz), 7.29 (d, 1H, J = 7.3 Hz)
¹³ C NMR (100 MHz, CD ₃ OD + NaOD): δ30.7, 51.2, 67.6, 99.7, 110.6, 116.5, 123.0, 125.8, 128.6, 128.8, 130.0, 142.6, 149.0, 150.5, 153.4, 178.1
HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 432.15595; found, 432.15567 (-0.27 mmu)

Compound 5 Synthesis Compound 4 8.5 mg (0.016 mmol, 1 eq), H-Phe-OtBu hydrochloride 9.4 mg (0.036 mmol, 2.3 eq), N, N′-diisopropylethylamine 14.2 μL (0. 080 mmol, 5 eq) was dissolved in 1 mL of N, N′-dimethylformamide, and 15.4 mg (0.041 mmol, 2.6 eq) of HATU was added thereto, followed by stirring for 3 hours. Ethyl acetate and saturated aqueous sodium hydrogen carbonate solution were added to the reaction solution, and the ethyl acetate layer was extracted by a liquid separation operation. The extracted organic layer was dried over sodium sulfate, and then the solvent was removed by removing under reduced pressure. The residue was dissolved in 1 mL of acetonitrile and 2 mL of trifluoroacetic acid and stirred for 1 hour. After removing the solvent by removal under reduced pressure, HPLC (eluent A (H ₂ O 0.1% TFA) and eluent B (CH ₃ CN 80%, H ₂ O 20%, 0.1% TFA) (A / B = 80/20 for 10 min, then 0/100 in 35 min)) to obtain the target compound (8.9 mg, 68%).
¹ H-NMR (400 MHz, CD ₃ OD + NaOD) δ: 2.84-2.89 (2H, m), 3.01 (t, 2H, J = 4.1 Hz), 3.04 (t, 2H, J = 4.1 Hz), 3.51 (dd, 2H, J = 17.1, 11.2 Hz), 3.66 (dd, 2H, J = 17.6, 4.9 Hz), 4.22 (s, 2H), 4.43-4.47 (m, 2H), 6.05 (dd, 1H, J = 8.8, 2.4 Hz), 6.13-6.16 (m, 1H), 6.22 (d, 1H, J = 2.4 Hz), 6.50 (dd, 2H, J = 8.3, 4.4 Hz), 6.78 (d, 1H, J = 7.3 Hz), 6.86-6.88 (m, 2H), 6.92-6.94 (m, 8H), 7.16 (t, 1H, J = 7.1 Hz), 7.26 (td, 1H, J = 7.3, 1.0 Hz), 7.34 ( d, 1H, J = 7.8 Hz), ¹³ C-NMR (100 MHz, CD ₃ OD + NaOD) δ: 39.4, 51.4, 56.9, 67.5, 99.9, 100.6, 110.1, 110.9, 117.5, 117.7, 123.2, 125.8, 127.3, 128.7, 129.0, 129.2, 130.4, 130.6, 149.0, 149.9, 150.0, 153.1, 153.1, 171.8, 177.9 (diastereomer mixture), HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 726.29277; found, 726.29199 (-0.89 mmu)

Compound 6 Synthesis Compound 4 4.2 mg (0.0077 mmol, 1 eq), H-Lys (Boc) -OtBu hydrochloride 6.4 mg (0.019 mmol, 2.5 eq), N, N′-diisopropylethylamine 6.5 μL (0.037 mmol, 4.8 eq) was dissolved in 1 mL of N, N′-dimethylformamide, and 7.2 mg (0.019 mmol, 2.5 eq) of HATU was added thereto, followed by stirring for 90 minutes. Ethyl acetate and saturated aqueous sodium hydrogen carbonate solution were added to the reaction solution, and the ethyl acetate layer was extracted by a liquid separation operation. The extracted organic layer was dried over sodium sulfate, and then the solvent was removed by removing under reduced pressure. The residue was dissolved in 1 mL of acetonitrile and 2 mL of trifluoroacetic acid and stirred for 2 hours. After removing the solvent by removal under reduced pressure, HPLC (eluent A (H ₂ O 0.1% TFA) and eluent B (CH ₃ CN 80%, H ₂ O 20%, 0.1% TFA) (A / B = 90/10 for 10 min, then 0/100 in 35 min)) to obtain the target compound (4.8 mg, 73%).
¹ H-NMR (400 MHz, CD ₃ OD + NaOD) δ: 1.22-1.24 (4H, m), 1.32-1.40 (4H, m), 1.59-1.69 (2H, m), 1.78-1.87 (2H, m ), 2.47 (4H, q, J = 7.1 Hz), 3.68 (2H, d, J = 17.6 Hz), 3.82 (2H, d, J = 17.2, Hz), 4.27-4.31 (4H, m), 6.30- 6.34 (4H, m), 6.60 (2H, t, J = 8.8 Hz), 6.82 (1H, d, J = 7.3 Hz), 7.23 (1H, t, J = 7.0 Hz), 7.31 (1H, t, J = 7.0 Hz), 7.38 (1H, d, J = 7.3 Hz).
¹³ C-NMR (100 MHz, CD ₃ OD + NaOD) δ: 23.8, 23.9, 33.8, 42.5, 42.6, 51.3, 55.9, 67.4, 99.8, 100.0, 110.7, 111.0, 116.8, 117.7, 117.8, 119.7, 123.2, 125.7, 128.8, 128.9, 130.3, 130.4, 142.5, 148.7, 150.0, 153.1, 153.2, 163.0, 163.3, 172.7, 178.7 (diastereomer mixture)
HRMS (ESI ⁺ ): calcd for [M + H] ⁺ , 688.34587; found, 688.34498 (-0.89 mmu)

Functional mechanism of carboxypeptidase activity detection By cutting out amino acids from a fluorescent probe (AMRBC-Phe or AMRBC-Lys) in which amino acids used as substrates by each carboxypeptidase are condensed with AMRBC-COOH by a two-step enzymatic reaction , AMRBC-COOH is produced. Pre-reaction fluorescent probes are mostly colorless and non-fluorescent ring-closed structures under physiological conditions, while the reaction product AMRBC-COOH contains approximately 50% of the molecules as strong fluorescent ring-opened bodies Therefore, an increase in fluorescence can be observed by the enzyme reaction.
In the following scheme 2, AMRBC-Phe is shown as a probe for carboxypeptidase A using a bulky amino acid as a substrate, and AMRBC-Lys is shown as a probe for carboxypeptidase B using a basic amino acid as a substrate.

Scheme 2: Enzymatic reaction of AMRBC derivatives with carboxypeptidase A or B

[Example 1]
Absorption / fluorescence spectrum measurement of AMRBC-COOH (4), AMRBC-Phe (5), AMRBC-Lys (6) Table 1 shows the results of absorption / fluorescence spectrum measurement of AMRBC-COOH, AMRBC-Phe, and AMRBC-Lys. From the results in the table, the fluorescent probes AMRBC-Phe (5) and AMRBC-Lys (6) are mostly colorless and non-fluorescent ring-closed under physiological conditions, and AMRBC-COOH (4) is about 50 % Were taken to have a strong fluorescent ring-opening. The absorption spectrum was measured using UV-1800 (SIMADZU), and the fluorescence spectrum was measured using F-7000 (HITACHI).

（ａ）０．１Ｍリン酸ナトリウム緩衝液（ｐＨ７．０）中で測定した。
（ｂ）０．１Ｍリン酸ナトリウム緩衝液（ｐＨ７．５）中で測定した。
（ｃ）０．１Ｍリン酸ナトリウム緩衝液（ｐＨ５．５）中で測定した。
（ｄ）フルオレセイン（Φ_ｆｌ＝０．８５）を参照として用いて決定した相対量子収率

(A) Measured in 0.1 M sodium phosphate buffer (pH 7.0).
(B) Measured in 0.1 M sodium phosphate buffer (pH 7.5).
(C) Measured in 0.1 M sodium phosphate buffer (pH 5.5).
(D) Relative quantum yield determined using fluorescein (Φ _fl = 0.85) as a reference.

[Example 2]
When carboxypeptidase A was applied to the enzyme assay AMRBC-Phe (5) and carboxypeptidase B was applied to the AMRBC-Lys (6), an increase in fluorescence due to the enzyme reaction was observed. The fluorescence spectra before and after the enzyme reaction were measured as follows.

(1) 260 μL of 5 μM AMRBC-Phe and AMRBC-Lys in PBS (−) were prepared.
(2) 80 μL of (1) was diluted to 800 μL with PBS (−) and measured as a fluorescence spectrum before the enzyme reaction.
(3) In 180 μL of (1), 8.8 μg of enzyme (carboxypeptidase A for AMRBC-Phe solution, carboxypeptidase B for AMRBC-Lys solution) was added and incubated at 37 ° C. (AMRBC-Phe is 2.5 hours, 4 hours for AMRBC-Lys).
(4) 80 μL of the solution after incubation in (3) was diluted to 800 μL with PBS (−) and measured as a fluorescence spectrum after the enzyme reaction.

Moreover, it confirmed that the enzyme reaction product was AMRBC-COOH ( 4 ) by the analysis by LC-MS, and the apparent enzyme-reaction rate constant by Lineweaver-Burk Plot was also computed.

The fluorescence spectra of the probe before and after the enzyme addition are shown in FIG. (A) is a measurement result about AMRBC-Phe and carboxypeptidase A, (b) is a measurement result about AMRBC-Lys and carboxypeptidase B. The excitation wavelength is 530 nm.
FIG. 2 shows the results of UPLC analysis of the probe after reaction with the enzyme. (A) is a measurement result about AMRBC-Phe and carboxypeptidase A, (b) is a measurement result about AMRBC-Lys and carboxypeptidase B.
Table 2 shows the enzyme reaction rate constant of each probe.

  The fluorescent probe of the present invention has an amino acid that is recognized by the target carboxypeptidase and is colorless and non-fluorescent before the reaction with the target carboxypeptidase. However, the reaction with the enzyme causes absorption and fluorescence in the visible light region. Recovered. In addition, as a result of applying the fluorescent probe of the present invention to an in vitro enzyme system, it was shown that the target carboxypeptidase activity can be rapidly detected by fluorescence.

(Industrial applicability)
The fluorescent probe of the present invention is the world's first carboxypeptidase fluorescent probe, and has the potential to detect carboxypeptidase activity of interest on clinical specimens as well as various diseased cells. This reveals disease-specific enzyme activity that could not be clarified by conventional methods. As a result, in addition to establishing a visualization method using this target, it is extremely effective as a drug discovery target. There is expected.

Claims (9)

The following general formula (I):

(Where
R ¹ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on the benzene ring:
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms, an ethyl group, a phenylmethyl group, or 4-hydroxyphenylmethyl. Represents a group, 1-hydroxyethyl group, or — (CH ₂ ) _n —Z group, n represents a number of 1 to 4, and Z represents a hydroxy group, a carboxyl group, an amino group, a thiol group, a methylthio group, a guanidino group. , A carbamoyl group, or a 5- or 6-membered ring optionally having 1 to 3 nitrogen atoms, or a condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring;
X is selected from an oxygen atom, Si (R ^a ) (R ^b ), C (R ^a ) (R ^b ), Ge (R ^a ) (R ^b ), P (═O) R ^c or Se;
Here, R ^a and R ^b are each independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, and R ^c is an alkyl group having 1 to 6 carbon atoms or a substituted group. An optionally substituted phenyl group;
Y is selected from NH, O, S or NR;
Here, NR represents an amino group which may be substituted or an amide group which may be substituted;
NH-L ¹ and NH-L ^{2 in} formula (I) each independently represent an amino acid residue, a peptide, or an aminoalkyl group having 1 to 6 carbon atoms, provided that NH-L ¹ and NH-L ² At least one of the is an amino acid residue;
m represents an integer of 1 to 3;
Or a salt thereof.   The compound or a salt thereof according to claim 1, wherein Y is NH. ^{NH-L 1} and ^{NH-L 2} are the same or different amino acid residue, or a salt thereof according to claim 1 or 2. The compound according to claim 3 or a salt thereof, wherein NH-L ¹ and NH-L ² are the same amino acid residue. Both ^{NH-L 1} and ^{NH-L 2} is a phenylalanine residue, a leucine residue or a lysine residue, a compound or a salt thereof according to claim 4.   X is an oxygen atom, The compound or its salt of any one of Claims 1-5.   A fluorescent probe for detecting carboxypeptidase activity, comprising the compound according to any one of claims 1 to 6 or a salt thereof.   A method for detecting intracellular carboxypeptidase, comprising: (a) introducing the fluorescent probe according to claim 7 into a cell; and (b) measuring fluorescence emitted from the fluorescent probe in the cell. Including methods. The following general formula (III):

(Where
R ¹ represents a hydrogen atom or 1 to 4 identical or different monovalent substituents present on the benzene ring:
R ² and R ³ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R ⁶ and R ⁷ are each independently a hydrogen atom, an alkyl group having 1 to 7 carbon atoms which may have a branched chain having 1 to 3 carbon atoms, an ethyl group, a phenylmethyl group, or 4-hydroxyphenylmethyl. Represents a group, 1-hydroxyethyl group, or — (CH ₂ ) _n —Z group, n represents a number of 1 to 4, and Z represents a hydroxy group, a carboxyl group, an amino group, a thiol group, a methylthio group, a guanidino group. , A carbamoyl group, or a 5- or 6-membered ring optionally having 1 to 3 nitrogen atoms, or a condensed heterocyclic ring composed of a 5-membered ring and a 6-membered ring;
X is selected from an oxygen atom, Si (R ^a ) (R ^b ), C (R ^a ) (R ^b ), Ge (R ^a ) (R ^b ), P (═O) R ^c or Se;
Here, R ^a and R ^b are each independently an alkyl group having 1 to 6 carbon atoms or an optionally substituted aryl group, and R ^c is an alkyl group having 1 to 6 carbon atoms or a substituted group. An optionally substituted phenyl group;
Y is selected from NH, O, S or NR;
Here, NR represents an amino group which may be substituted or an amide group which may be substituted;
m represents an integer of 1 to 3;
Or a salt thereof.

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