Fluorescent probe for detecting peptide bond hydrolase

Abstract

The purpose of the present invention is to provide a fluorescent probe that can be used in an enzymatic activity detection method. The present invention provides a fluorescent probe for detecting a hydrolase, the fluorescent probe containing a compound represented by general formula (II) or a salt thereof, the compound having at least one water-soluble substituent selected from the group consisting of-CO2H,-PO3H2, and-SO3H. The fluorescent probe of the present invention can be used in an enzyme activity detection method using a micro device. [Chemical Formula 1] # imgabs0 #

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority from prior Japanese Patent Application No. 2022-79082 (filing date: May 12, 2022), the entire disclosure of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to a fluorescent probe for detecting peptide bond hydrolase. Background Art

[0004] Enzyme activity detection methods at the single enzyme level using microdevices have been widely used for purposes such as clarifying a single biochemical parameter of an enzyme and using a single biochemical parameter as a reporter protein to detect the presence of a specific protein with high sensitivity.

[0005] The inventor's research group has so far used a group of microdevice-compatible enzyme fluorescent probes for activity detection, performed analysis based on the detection and activity of enzymes in blood at the single-molecule level, and discovered new correlations with diseases.

[0006] On the other hand, in the method of detecting enzyme activity at a single enzyme level using a microdevice, by sufficiently diluting a biological sample and adding the biological sample to the microdevice, a state in which one molecule or less of the enzyme is present in one well can be formed, and so-called multicolor analysis, in which fluorescent probes of multiple colors having different reactivities between enzymes react simultaneously, the increase in fluorescence of multiple wavelengths in each well is measured, and the enzyme in the well is specified from the combination thereof, is theoretically possible.

[0007] However, as fluorescent probes that can be used in enzyme activity detection methods using microdevices, practically usable probes that emit red fluorescence (red fluorescent probes) or green fluorescence (green fluorescent probes) have not yet been found. Summary of the invention

[0008] Technical issues

[0009] The object of the present invention is to provide a fluorescent probe which can be used in a method for detecting enzyme activity.

[0010] Solutions to technical problems

[0011] The inventors have conducted in-depth research to solve the above-mentioned technical problems, and found that by introducing specific water-soluble substituents into a compound having a rhodamine skeleton and further setting the LogP value of the compound within a specific range, a fluorescent probe can be provided that can also be used in an enzyme activity detection method using a microdevice, thereby completing the present invention.

[0012] According to the present invention, the following inventions are provided. [1]

[0014] A fluorescent probe for detecting a hydrolase, the fluorescent probe comprising a compound having at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H and represented by the following general formula (II) or a salt thereof:

[0015] [Chemical formula 1]

[0016]

[0017] wherein Ring A is an aromatic ring, which is phenyl or a 5- or 6-membered heteroaryl;

[0018] Ring Q1 is a ring structure of the following formula (i) or (ii),

[0019] [Chemical formula 2]

[0020]

[0021] Where R 2 , R 4 , R 6 and R0 is defined as follows;

[0022] Ring Q2 is a ring structure of the following formula (iii), (iv), (v) or (vi),

[0023] [Chemical formula 3]

[0024]

[0025] Where R 3 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 The following definitions are given;

[0026] In a ring structure containing an X atom, the bond represented by 1 and the key represented by 2 each represents a single bond or a double bond, provided that both bonds cannot simultaneously represent a double bond;

[0027] R is selected from the group consisting of -CO2H, -PO3H2, -SO3H, -L-CO2H, -L-PO3H2 and -L-SO3H, wherein L is a linker;

[0028] n is an integer from 0 to 5;

[0029] R 1 represents a monovalent substituent optionally present on the aromatic ring, wherein when two or more monovalent substituents are present, the substituents may be the same or different,

[0030] Where R 1 With R 1 The carbon atoms on the bonded ring A, the carbon atoms on the ring structure containing the X atom to which the ring A is bonded, and a part of the ring A may form a 5- or 6-membered carbocyclic or heterocyclic ring;

[0031] R 2 and R 3 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0032] R 4 and R 5 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0033] R 6 and R 7 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0034] R 8 and R 9 each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent (the alkyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent), or an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent (the alkenyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent),

[0035] Where R 8 and R 9 can form a 4- to 7-membered heterocyclic group together, the heterocyclic group may contain a nitrogen atom, R 8 and R 9 is connected to the nitrogen atom and may have a substituent,

[0036] R 8 or R 9 , or R 8 and R 9 Both, with R 5 or R 7 Together, they can each form a 5- to 7-membered heterocyclic or heteroaryl group containing a nitrogen atom, R 8 or R 9is connected to the nitrogen atom, and further, the heterocyclic group or heteroaryl group may have a substituent selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, an arylalkyl group having 6 to 10 carbon atoms, and an alkyl-substituted alkenyl group having 6 to 10 carbon atoms,

[0037] The water-soluble substituent may be bonded to these heterocyclic groups and heteroaryl groups directly or through a substituent;

[0038] R 11 is a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent (the alkyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent), or an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent (the alkenyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent);

[0039] X is selected from oxygen atoms, Si(R a )(R b )、C(R a )(R b )、P(=O)R c 、S(R d )2、Ge(R a )(R b ), tellurium atoms or bismuth atoms,

[0040] Where R a and R b are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted aryl group,

[0041] R c is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted phenyl group,

[0042] R d are the same or different substituted or unsubstituted aryl groups,

[0043] R a , R b , R c and R d The alkyl, aryl and phenyl groups may be substituted with water-soluble substituents; and

[0044] R0 and R 10 Each is independently selected from a partial structure of an amino acid, a partial structure of a carboxylic acid, TO-, or a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group,

[0045] The partial structure of the amino acid is similar to R0 or R 10 The bonded C=O together constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0046] The partial structure of the carboxylic acid is similar to R0 or R 10 The bonded C=O together form part of the carboxylic acid,

[0047] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0048] The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar. [2]

[0050] The fluorescent probe according to [1], wherein Q2 represents a ring structure of formula (iii). [3]

[0052] The fluorescent probe according to [1], wherein Q2 represents a ring structure of formula (iv), (v) or (vi). [4]

[0054] The fluorescent probe according to any one of [1] to [3], wherein n is 1 to 3. [5]

[0056] The fluorescent probe according to any one of [1] to [4], wherein R 2 To R 9 and R a , R b , R c and R d At least one of the following is a water-soluble substituent, an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms, an aryl group having a water-soluble substituent, and a phenyl group having a water-soluble substituent. [6]

[0058] The fluorescent probe according to any one of [1] to [5], wherein X is an oxygen atom or Si(R a )(R b ). [7]

[0060] The fluorescent probe according to any one of [1] to [6], wherein R is -SO3H or -L-SO3H. [8]

[0062] The fluorescent probe according to any one of [1] to [7], wherein the hydrolase is a peptide bond hydrolase selected from aminopeptidase, peptidase, protease or amidase. [9]

[0064] The fluorescent probe according to any one of [1] to [8], wherein the MiLogP value of the compound or the salt thereof is 2.56 or less.

[10]

[0066] The fluorescent probe according to any one of [1] to [8], wherein the MiLogP value of the fluorescent core of the compound or the salt thereof is less than 2.47.

[11]

[0068] The fluorescent probe according to any one of [1] to [8], wherein the MiLogP value of the compound or the salt thereof is 2.56 or less, and the MiLogP value of the fluorescent core of the compound or the salt thereof is less than 2.47.

[12]

[0070] The fluorescent probe according to any one of [1] to

[11] , which is a fluorescent probe for a microdevice.

[13]

[0072] The fluorescent probe according to any one of [1] to

[12] , wherein the compound represented by general formula (II) or a salt thereof is a compound represented by any one of the following general formulas (Ia) to (Ie) or a salt thereof:

[0073] [Chemical formula 4]

[0074]

[0075]

[0076] Where R 4 To R 9 , R a , R b and R0 are as defined in the general formula (II).

[14]

[0078] The fluorescent probe according to any one of [1] to

[13] , wherein the compound represented by the general formula (II) or a salt thereof is a compound represented by the following general formula (If) or a salt thereof:

[0079] [Chemical formula 5]

[0080]

[0081] Where R 4 To R 9 and R0 are as defined in the general formula (II).

[15]

[0083] The fluorescent probe according to any one of [1] to

[14] , wherein the compound or the salt thereof is a compound or the salt thereof selected from the following compounds:

[0084] [Chemical formula 6]

[0085]

[16]

[0087] A method for diagnosing pancreatic cancer or a method for predicting the possibility that a test subject from which a biological sample is derived has pancreatic cancer, the method comprising using the fluorescent probe according to any one of [1] to

[15] to detect the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (II).

[17]

[0089] A method for diagnosing pancreatic cancer or a method for predicting the possibility that a test subject from which a biological sample is derived has pancreatic cancer, the method comprising using the fluorescent probe according to any one of [1] to

[15] to detect the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 It is -Pro-Xaa in the general formula (II) (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamic acid).

[18]

[0091] A method for diagnosing pancreatic cancer or a method for predicting the likelihood that a test subject from which a biological sample is derived has pancreatic cancer, the method comprising:

[0092] Using the fluorescent probe according to any one of [1] to

[15] , detecting the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (II); and

[0093] Using the fluorescent probe according to any one of [1] to

[15] , detecting the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 It is -Pro-Xaa in the general formula (II) (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamine).

[19]

[0095] The method according to any one of

[16] to

[18] , wherein the biological sample is a biological sample of a pancreatic cancer patient, a patient suspected of having pancreatic cancer, or a healthy subject.

[20]

[0097] A compound represented by any one of the following formulae (1) to (5) or a salt thereof:

[0098] [Chemical formula 7]

[0099]

[0100] in

[0101] R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-,

[0102] The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0103] The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid.

[0104] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0105] The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar. [twenty one]

[0107] A compound represented by the following formula (6) or a salt thereof:

[0108] [Chemical formula 8]

[0109]

[0110] in

[0111] R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-,

[0112] The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0113] The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid.

[0114] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0115] The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar. [twenty two]

[0117] The fluorescent probe according to any one of [1] to

[15] , wherein the solubility of the compound or a salt thereof in hexadecane is 0.3 μM or less in a solubility measurement test (25° C.) in sealing oil. [twenty three]

[0119] The fluorescent probe according to any one of [1] to

[15] and

[22] , wherein in a fluorescence intensity measurement test in a microdevice, the ratio of the fluorescence intensity of the pore edge region to the fluorescence intensity of the pore center region of the enzymatic hydrolysate of the compound or its salt is 0.6 or less. [twenty four]

[0121] A fluorescent probe according to any one of [1] to

[15] ,

[22] and

[23] , wherein in an enzymatic hydrolysis test of the fluorescent probe in a microdevice and a cuvette, the ratio of the signal-to-noise ratio (first ratio) of the enzymatic hydrolysate of the compound or its salt in the microdevice to the signal-to-noise ratio (second ratio) of the enzymatic hydrolysate in the cuvette is 0.4 or greater.

[101]

[0123] Use of a compound represented by the following general formula (III) or a salt thereof for producing a compound or a salt thereof according to any one of [1] to

[15] :

[0124] [Chemical formula 9]

[0125]

[0126] Wherein ring A, ring Q2, X, n, R, R 1 , R 2 , R 4 and R 6 As defined in general formula (II), the bond Represents a single bond or a double bond.

[102]

[0128] Use of a compound represented by the following general formula (IV) or a salt thereof for producing a compound or a salt thereof according to any one of [1] to

[15] :

[0129] [Chemical formula 10]

[0130]

[0131] Among them, ring A, X, n, R and R 1 To R 9 As defined in general formula (II).

[103]

[0133] A compound represented by the following general formula (V) or a salt thereof:

[0134] [Chemical formula 11]

[0135]

[0136] in

[0137] Ring A, R, R 8 and R 9 As defined in general formula (II),

[0138] n is 1 or 2,

[0139] R 1 is a hydrogen atom, an unsubstituted alkyl group having 1 to 4 carbon atoms, or an alkyl group having 1 to 4 carbon atoms substituted with a hydroxyl group,

[0140] R 2 To R 7 is a hydrogen atom,

[0141] R 8 and R 9 are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and

[0142] X is an oxygen atom or Si(R a )(R b )(R a and R b As defined in formula (II)).

[104]

[0144] The compound or salt thereof according to

[103] , which is a compound or salt thereof selected from the following compounds:

[0145] [Chemical formula 12]

[0146]

[105]

[0148] Use of the compound according to

[103] or

[104] or a salt thereof for producing the compound according to any one of [1] to

[15] or a salt thereof.

[201]

[0150] A method for selecting a fluorescent probe, the method comprising:

[0151] preparing the fluorescent probe according to any one of [1] to

[15] ; and

[0152] Select a fluorescent probe that meets the following conditions: (A) in a solubility measurement test in sealing oil (25°C), the solubility of the fluorescent probe and its hydrolysate in hexadecane is 0.3 μM or less; (B) in a fluorescence intensity measurement test in a microdevice, the ratio of the fluorescence intensity of the pore edge area to the fluorescence intensity of the pore center area of ​​the hydrolysate of the fluorescent probe is 0.6 or less, and (C) in a fluorescent probe enzymatic hydrolysis test in a microdevice and a cuvette, the ratio of the signal-to-noise ratio (first ratio) of the hydrolysate in the microdevice to the signal-to-noise ratio (second ratio) of the hydrolysate in the cuvette is 0.4 or greater.

[0153] According to the present invention, the following inventions are also provided.

[301]

[0155] A fluorescent probe for detecting a hydrolase in a microdevice, the fluorescent probe comprising a compound having at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H and represented by the following general formula (I) or a salt thereof:

[0156] [Chemical formula 13]

[0157]

[0158] in

[0159] [Chemical formula 14]

[0160]

[0161] is an aromatic ring, which is a phenyl group or a 5- or 6-membered heteroaryl group;

[0162] R is selected from the group consisting of -CO2H, -PO3H2, -SO3H, -L-CO2H, -L-PO3H2 and -L-SO3H, wherein L is a linker;

[0163] n is an integer from 0 to 5;

[0164] R 1 represents a monovalent substituent optionally present on the aromatic ring, wherein when two or more monovalent substituents are present, the substituents may be the same or different,

[0165] R 2 and R 3 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0166] R 4 and R 5Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0167] R 6 and R 7 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent;

[0168] R 8 and R 9 each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms, or an alkenyl group having a water-soluble substituent and having 1 to 6 carbon atoms,

[0169] Where R 8 and R 9 can form a 4- to 7-membered heterocyclic group together, the heterocyclic group may contain a nitrogen atom, R 8 and R 9 is connected to the nitrogen atom and may have a substituent,

[0170] R 8 or R 9 , or R 8 and R 9 Both, with R 5 or R 6 Together, they can each form a 5- to 7-membered heterocyclic or heteroaryl group containing a nitrogen atom, R 8 or R 9 is connected to the nitrogen atom, and further, the heterocyclic group or heteroaryl group may have a substituent selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, an arylalkyl group having 6 to 10 carbon atoms, and an alkyl-substituted alkenyl group having 6 to 10 carbon atoms,

[0171] The water-soluble substituent may be bonded to these heterocyclic groups and heteroaryl groups directly or through a substituent;

[0172] X is selected from oxygen atoms, Si(R a )(R b )、C(R a )(R b )、P(=O)R c 、S(R d )2、Ge(R a )(R b ), tellurium atoms or bismuth atoms,

[0173] Where R a and R b are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted aryl group,

[0174] R c is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted phenyl group,

[0175] R d are the same or different substituted or unsubstituted aryl groups,

[0176] R a , R b , R c and R d The alkyl, aryl and phenyl groups may be substituted with water-soluble substituents; and

[0177] R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-,

[0178] The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0179] The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid.

[0180] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0181] The sugar part of T together with the O to which T is bonded constitutes a sugar or a part of a sugar.

[0182] The compound or a salt thereof has a LogP value of 1.09 or less.

[302]

[0184] The fluorescent probe according to

[301] , wherein n is 1 to 3.

[303]

[0186] The fluorescent probe according to any one of

[301] or

[302] , wherein R 2 To R 9 and R a , R b , R c and R d At least one of the following is a water-soluble substituent, an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms, an aryl group having a water-soluble substituent, and a phenyl group having a water-soluble substituent.

[304]

[0188] The fluorescent probe according to any one of

[301] to

[303] , wherein X is an oxygen atom or Si(R a )(R b ).

[305]

[0190] The fluorescent probe according to

[302] , wherein R is -SO3H or -L-SO3H.

[306]

[0192] The fluorescent probe according to any one of

[301] to

[305] , wherein the hydrolase is a peptide bond hydrolase selected from aminopeptidase, peptidase, protease or amidase.

[307]

[0194] The fluorescent probe according to any one of

[301] to

[306] , wherein the compound represented by general formula (I) or a salt thereof is a compound represented by any one of the following general formulas (Ia) to (Ie) or a salt thereof:

[0195] [Chemical formula 15]

[0196]

[0197] Where R 4 To R 9 , R a , R b and R0 are as defined in the general formula (I).

[308]

[0199] The fluorescent probe according to any one of

[301] to

[307] , wherein the compound represented by general formula (I) or a salt thereof is a compound represented by the following general formula (If) or a salt thereof:

[0200] [Chemical formula 16]

[0201]

[0202] Where R 4 To R 9 and R0 are as defined in the general formula (I).

[309]

[0204] A method for diagnosing pancreatic cancer, the method comprising using a fluorescent probe according to any one of

[301] to

[308] to detect the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 is an alanine, lysine, arginine or methionine residue in the general formula (I).

[310]

[0206] A method for diagnosing pancreatic cancer, the method comprising using a fluorescent probe according to any one of

[301] to

[308] to detect the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 is -Pro-Xaa in the general formula (I) (Pro represents a proline residue, and Xaa represents an amino acid residue, such as glycine, serine or glutamic acid).

[311]

[0208] A method for diagnosing pancreatic cancer, the method comprising:

[0209] Using the fluorescent probe according to any one of

[301] to

[308] , detecting the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 is an alanine, lysine, arginine or methionine residue in the general formula (I); and

[0210] Using a fluorescent probe according to any one of

[301] to

[308] , the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject is detected by a microdevice, wherein -CO-R0 is -Pro-Xaa in the general formula (I) (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamine).

[312]

[0212] A compound represented by any one of the following formulae (1) to (5) or a salt thereof:

[0213] [Chemical formula 17]

[0214]

[0215]

[0216] in

[0217] R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-,

[0218] The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0219] The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid.

[0220] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0221] The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar.

[313]

[0223] A compound represented by the following formula (6) or a salt thereof:

[0224] [Chemical formula 18]

[0225]

[0226] in

[0227] R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-,

[0228] The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid,

[0229] The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid.

[0230] T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and

[0231] The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar.

[0232] Technical Effects of the Invention

[0233] According to the present invention, a red fluorescent probe and a green fluorescent probe that can be used in a method for detecting enzyme activity can be provided. The present invention is particularly advantageous in that these fluorescent probes can be used in a method for detecting enzyme activity using a microdevice.

[0234] The fluorescent probe of the present invention can be effectively used for detecting pancreatic cancer and the like.

[0235] For example, according to the present invention, by using the fluorescent probe of the present invention, a method for diagnosing pancreatic cancer by detecting the single-molecule enzyme activity of DPP4 in blood and detecting the single-molecule enzyme activity of CD13 in blood can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0236] Figure 1 is a perspective view schematically illustrating a microdevice using the fluorescent probe of the present invention.

[0237] Figure 2 Fluorescence microscopy images of measurements performed using sHMRG- and sSiR-based peptidase and protease probes in Examples 1 to 15 are shown.

[0238] Figure 3 The results of the pancreatic disease (pancreatic cancer) diagnosis experiment using the single-molecule enzyme activity of CD13 in Example 17 are shown.

[0239] Figure 4 The results of the pancreatic disease (pancreatic cancer) diagnosis experiment using the single-molecule enzyme activity of DPP4 in Example 18 are shown.

[0240] Figure 5 The figure shows the calibration curve used for calculating the solubility in the sealing oil in Example 19. The horizontal axis represents the concentration (μm) of the fluorescent probe, and the vertical axis represents the peak area of ​​LC.

[0241] Figure 6 The results of evaluating the adsorption degree of the test compound on the pore wall surface based on the fluorescence intensity (AU) of the center area (Center) and the fluorescence intensity (AU) of the edge area (Edge) in Example 20 are shown. The images in the middle row of the center part of the figure represent the center area for analyzing the fluorescence intensity, and the images in the right row represent the edge area for analyzing the fluorescence intensity.

[0242] Figure 7 The results of measuring fluorescence spectra before and after enzyme reaction using a cuvette in Example 21 are shown. A is the result of Glu-sSiR, and B is the result of GP-sHMRG.

[0243] Figure 8 The results of measuring fluorescence spectra before and after enzyme reactions using a microdevice in Example 21 are shown. A is the result of Glu-siR, and B is the result of GP-sHMRG. In A and B, the images in the left row show the results of using only the assay buffer, the images in the middle row show the results in the presence of the test compound, and the images in the right row show the results in the presence of the test compound and the enzyme. DETAILED DESCRIPTION

[0244] In the present specification, the "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

[0245] In the present specification, "alkyl" may be any aliphatic hydrocarbon group that is linear, branched, cyclic, or a combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but is, for example, 1 to 4 (C 1-4 ), 1 to 6 (C 1-6 ), 1 to 10 (C 1-10 ), 1 to 15 (C 1-15 ) or 1 to 20 (C 1-20 ). When the number of carbon atoms is specified, it refers to an "alkyl group" with a carbon number within that numerical range. For example, C 1-8Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, n-octyl etc. In this specification, alkyl may have one or more optional substituents. Examples of such substituents include, but are not limited to, alkoxy, halogen atoms, amino, monosubstituted or disubstituted amino, substituted silicon and acyl. When alkyl has two or more substituents, they may be the same or different from each other. The same is true for the alkyl moieties of other substituents (e.g., alkoxy, arylalkyl etc.) including the alkyl moiety.

[0246] In this specification, when certain functional groups are defined as "optionally substituted", the type, position and number of substituents are not particularly limited, and when there are two or more substituents, they may be the same or different from each other. Examples of substituents include, but are not limited to, alkyl, alkoxy, hydroxy, carboxyl, halogen atoms, sulfo, amino, alkoxycarbonyl and oxo groups. Other substituents may also exist in these substituents. Examples of this include, but are not limited to, alkyl halide groups and dialkylamino groups.

[0247] In this specification, "aryl" can be either a monocyclic aromatic hydrocarbon group or a condensed polycyclic aromatic hydrocarbon group, or can be an aromatic heterocycle containing one or more heteroatoms (such as oxygen atoms, nitrogen atoms or sulfur atoms) as ring atoms. In this case, the aromatic heterocycle can also be referred to as "heteroaryl" or "heteroaromatic". The aryl group can be connected to all possible positions, whether it is a monocyclic ring or a condensed ring. The non-limiting examples of monocyclic aromatic groups include phenyl (Ph), thienyl (2- or 3-thienyl), pyridyl, furyl, thiazolyl, oxazolyl, pyrazolyl, 2-pyrazinyl, pyrimidinyl, pyrrolyl, imidazolyl, pyridazinyl, 3-isothiazolyl, 3-isoxazolyl, 1,2,4-oxadiazole-5-yl and 1,2,4-oxadiazole-3-yl. Non-limiting examples of condensed polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthracenyl, indazolyl, quinolyl, isoquinolyl, 1,2-dihydroisoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2,3-dihydrobenzofuran-1-yl, 2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzothiophene-1-yl, 2,3-dihydrobenzothiophene-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl and thioxanthenyl. In the present specification, aryl may have one or more optional substituents on its ring. Examples of substituents include, but are not limited to, alkoxy, halogen atoms, amino, monosubstituted or disubstituted amino, substituted silicon, and acyl. When the aryl group has two or more substituents, they may be the same or different from each other. The same is true for the aryl moiety of other substituents (e.g., aryloxy, arylalkyl, etc.) including the aryl moiety.

[0248] In the present specification, "alkoxy" refers to a structure in which the above-mentioned alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups of linear, branched, cyclic or combinations thereof. Preferred examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropyloxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclobutyloxy, cyclopropylmethoxy, n-pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropoxy, cyclobutylethyloxy and cyclopentylmethyloxy.

[0249] In the present specification, the "alkylene group" is a divalent group composed of a straight-chain or branched saturated hydrocarbon, and examples thereof include methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1,1-dimethyltrimethylene, methyl, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2-diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene and 2,2-di-n-propyltrimethylene.

[0250] The "amide" used in the present specification includes RNR'CO- (in the case of R = alkyl, alkylaminocarbonyl-) and RCONR'- (in the case of R = alkyl, alkylcarbonylamino-).

[0251] The "ester" used in the present specification includes ROCO- (in the case where R = alkyl, alkoxycarbonyl-) and RCOO- (in the case where R = alkyl, alkylcarbonyloxy-).

[0252] 1. Fluorescent probe of the present invention

[0253] According to the present invention, there is provided a fluorescent probe for detecting a hydrolase, the fluorescent probe comprising a compound represented by the following general formula (II) or a salt thereof.

[0254] [Chemical formula 19]

[0255]

[0256] One mode of the present invention is a fluorescent probe for detecting a hydrolase, the fluorescent probe comprising a compound represented by the following general formula (I) or a salt thereof.

[0257] [Chemical formula 20]

[0258]

[0259] In this specification, a compound or a salt thereof satisfying the above requirements is also referred to as "the compound of the present invention", and a fluorescent probe containing the compound of the present invention is also referred to as "the fluorescent probe of the present invention".

[0260] The compounds of the present invention have at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H.

[0261] As described below, the compounds of the present invention can be specified by MiLogP value or LogP value, which is a lipophilicity index. For example, the MiLogP value of the compounds of the present invention can be set to 2.56 or less, or less than 2.47, preferably 2.40 or less, 2.30 or less, 2.20 or less, 2.10 or less, 2.00 or less, 1.90 or less, 1.80 or less, 1.70 or less, or 1.64 or less, more preferably 1.60 or less, 1.50 or less, 1.40 or less, 1.34 or less, 1.30 or less, 1.22 or less, 1.20 or less, 1.10 or less, or 1.00 or less (especially preferably 0 or less). The lower limit of the MiLogP value of the compounds of the present invention is not particularly specified, but is, for example, about -3.1 or more. For compounds of the present invention wherein X is an oxygen atom in formula (I) and (II), the MiLogP value may be set to 2.40 or less, 2.30 or less, 2.20 or less, 2.10 or less, 2.00 or less, 1.90 or less, 1.80 or less, or 1.70 or less, preferably 1.60 or less, 1.50 or less, 1.40 or less, 1.30 or less, 1.20 or less, 1.10 or less, or 1.00 or less (more preferably 0 or less), and the lower limit of the MiLogP value may be set to, for example, about -3.1 or more. For compounds of the present invention wherein X is Si(R) in formula (I) and (II), the MiLogP value may be set to 2.40 or less, 2.30 or less, 2.20 or less, 2.10 or less, 2.00 or less, 1.90 or less, 1.80 or less, or 1.70 or less, preferably 1.60 or less, 1.50 or less, 1.40 or less, 1.30 or less, 1.20 or less, 1.10 or less, or 1.00 or less (more preferably 0 or less), and the lower limit of the MiLogP value may be set to, for example, about -3.1 or more. a )(R b ), the MiLogP value can be set to 2.56 or lower, 2.50 or lower, 2.40 or lower, 2.30 or lower, 2.20 or lower, 2.10 or lower, 2.00 or lower, 1.90 or lower, 1.80 or lower, 1.70 or lower, or 1.64 or lower, preferably 1.60 or lower, 1.50 or lower, 1.40 or lower, 1.34 or lower, 1.30 or lower, 1.22 or lower, 1.20 or lower, or 1.10 or lower (more preferably 1.00 or lower), and the lower limit of the MiLogP value can be set to, for example, about -3.1 or higher, or 0 or higher. In a preferred mode of the present invention, the MiLogP value of the enzymolyte of the compound of the present invention can also be specified in the same manner as above. The enzymolyte of the compound of the present invention can have, for example, a structure of formula (III), and the structure of formula (III) includes the structure of the fluorescent mother core.

[0262] The LogP value of the compound of the present invention can be set to less than 1.17, 1.16 or lower, 1.15 or lower, 1.14 or lower, 1.13 or lower, 1.12 or lower, 1.11 or lower, or 1.10 or lower, preferably 1.09 or lower, 1.08 or lower, 1.07 or lower, 1.06 or lower, 1.05 or lower, 1.04 or lower, 1.03 or lower, or 1.02 or lower, more preferably 1.01 or lower, 1.0 or lower, 0.9 or lower, 0.8 or lower, 0.7 or lower, 0.6 or lower, or 0.5 or lower (particularly preferably 0.46 or lower, or 0.42 or lower). The lower limit of the LogP value of the compound of the present invention is not particularly specified, and the lower limit value is higher in water solubility, but for example, it is about -3 or higher. In a preferred mode of the present invention, the LogP value of the enzymatic hydrolyzate of the compound of the present invention can also be specified in the same manner as above. The enzymatic hydrolysate of the compound of the present invention may have, for example, the structure of formula (III), which includes the structure of a fluorescent core.

[0263] As described below, the fluorescent probe of the present invention can be preferably used as a fluorescent probe for a microdevice.

[0264] In the compounds of the present invention, it is important to essentially have at least one water-soluble substituent selected from the group consisting of a carboxyl group (-CO2H), a phosphate group (-PO3H2) and a sulfonic acid group (-SO3H).

[0265] Among the compound structures used in microdevices that have been studied so far, the mother core is limited to the xanthene skeleton, and the rhodamine skeleton, which has a higher lipophilicity than the xanthene skeleton, has not been studied. Since rhodamine and Si-rhodamine have high lipophilicity, when fluorescent probes using these compounds (e.g., OFF-ON type fluorescent probes that emit fluorescence by reacting with a target enzyme) are used in a method for detecting enzyme activity using a microdevice (such as a porous chamber type microdevice), when the device is in a closed state, the fluorescent probe is more likely to leak from the aqueous solution in the device hole to the organic solvent used for sealing outside the device, so rhodamine and Si-rhodamine are considered unsuitable for measurements requiring high quantitativeness and sensitivity.

[0266] As for the enzyme recognition site, in the fluorescent probes used in microdevices that have been studied so far, the water solubility of the enzyme recognition site is relatively high (such as phosphate), but in many fluorescent probes used to detect enzymes that hydrolyze peptide bonds, glycosidic bonds, etc. targeted by the present invention, the enzyme recognition site is relatively lipophilic (such as peptides), so the problem of fluorescent probe leakage into organic solvents is relatively large.

[0267] As a result of in-depth research on the above problems, the inventors found that in the fluorescent probe of the present invention, by introducing a specific water-soluble substituent into the molecular skeleton of rhodamine, silyl rhodamine, etc., the above problems can be solved. The inventors also found that by introducing a specific water-soluble substituent into the molecular skeleton of rhodamine and setting the MiLogP value and / or LogP value as the lipophilicity index of the compound molecule within a specific range, excellent applicability to microdevices can be achieved. Therefore, as a fluorescent probe with a rhodamine skeleton confirmed for the first time, it is possible to provide a fluorescent probe that can be used in an enzyme activity detection method using a microdevice.

[0268] According to one mode of the present invention, a fluorescent probe for detecting a hydrolase in a microdevice is provided, the fluorescent probe containing a compound or a salt thereof having at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H and represented by the general formula (II), wherein the MiLogP value is less than 2.47 and / or the LogP value is less than 1.09. According to one mode of the present invention, a fluorescent probe for detecting a hydrolase in a microdevice is also provided, the fluorescent probe containing a compound or a salt thereof having at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H and represented by the general formula (I), wherein the MiLogP value is less than 2.47 and / or the LogP value is less than 1.09. Each component will be described in detail below.

[0269] (1) Compounds of the present invention

[0270] In the general formulae (I) and (II), ring A:

[0271] [Chemical formula 21]

[0272]

[0273] is an aromatic ring, which is a phenyl group (benzene ring) or a 5- or 6-membered heteroaryl group.

[0274] A 5- or 6-membered heteroaryl group is an aromatic heterocycle containing one to three heteroatoms such as oxygen, nitrogen and sulfur, examples of which include, but are not limited to, pyrrole, furan, thiophene, imidazole, pyrazole, triazole, pyridine, pyran, thiopyran, pyrazine, pyrimidine, pyridazine, 1,2-dioxin, 1,4-dioxin, 1,2-dithiine, 1,4-dithiine and triazine.

[0275] The aromatic ring is preferably phenyl (benzene ring), pyrrole, thiophene, furan or pyridine.

[0276] In the general formula (II), when Q2 represents a ring structure of formula (iii), it represents a bond of 1 Represents a single bond, expressed as a bond of 2 represents a double bond, and Q1 represents a ring structure of formula (i).

[0277] In the general formula (II), when Q2 represents a ring structure of formula (iv), (v) or (vi),

[0278] R 1 The bond to the carbon atom of the ring structure containing the X atom that is directly bonded to ring A to form a spiro ring is represented by 1 and the key represented by 2 represents a single bond, Q1 represents a ring structure of formula (i), or

[0279] Keys represented as 1 Represents a double bond, which is represented by 2 bonds represents a single bond, and Q1 represents a ring structure of formula (ii).

[0280] In the present invention, when X is an oxygen atom, the "ring structure containing an X atom" is a xanthene ring, but in the present invention, the "xanthene skeleton" is used to mean that X includes a ring structure other than an oxygen atom.

[0281] In the general formulae (I) and (II), R is selected from the group consisting of -CO2H, -PO3H2, -SO3H, -L-CO2H, -L-PO3H2 and -L-SO3H. L represents a linker. That is, in the compounds represented by the general formulae (I) and (II) or their salts, the water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H may be bonded to the aromatic ring directly or through a linker.

[0282] The linker of L is selected from the group consisting of: an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which may have a substituent (provided that one or more of the -CH2- of the alkyl group may be substituted by -O-, -S-, -NH- or -CO-); an aryl group (including a heteroaryl group); a cycloalkyl group; an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms; an alkoxy group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms; an alkoxycarbonyl group (-CO-OR'); an alkylamino group (-NHR', -NR'2); an ester group (-O-CO-R'); an amide group (-NHCOR', -CONHR', -CONR'2); a polyethylene glycol chain; and a group formed by optionally bonding two or more groups selected from these groups. R' is a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group, and when there are two or more R's, they may be the same or different from each other.

[0283] As the linker of L, from the perspective of ease of synthesis, amide can be appropriately used, but from the perspective of the physical properties of the fluorescent probe, any of the above can be used, and preferably an alkyl group having 1 to 6 carbon atoms (the alkyl group may have a substituent), an amide group (more preferably -CONHR' or -CONR'2) or a phenyl group can be used.

[0284] In the present invention, R is preferably -CO2H, -SO3H, -L-CO2H or -L-SO3H, and R is more preferably -SO3H or -L-SO3H.

[0285] n is an integer from 0 to 5. In the compounds represented by the general formula (I) and (II) or their salts, the water-soluble substituents can be introduced into the aromatic ring directly or through a linker. In the compounds represented by the general formula (I) and (II) or their salts, the water-soluble substituents can be introduced into the xanthene skeleton directly or through a linker, but not into the aromatic ring (i.e., n=0). In addition to or in lieu of introducing the water-soluble substituents into the xanthene skeleton directly or through a linker, the water-soluble substituents can also be introduced into the cyclohexyl ring or cycloaryl ring formed by bonding to the xanthene skeleton directly or through another substituent, as described below.

[0286] In a preferred mode of the present invention, n is 1-5, more preferably 1-3.

[0287] In another preferred mode of the present invention, the water-soluble substituent is introduced only into the aromatic ring directly or via a linker, and n is 1-3 or 1-2.

[0288] When n is 2 or more, R may be the same or different.

[0289] In the general formulae (I) and (II), R 1 represents a monovalent substituent optionally present on the aromatic ring. When two or more monovalent substituents are present, they may be the same or different. 1 The fact that it is optionally present on an aromatic ring means that R 1 The presence of R on the aromatic ring 1 This does not occur on aromatic rings.

[0290] R 1 The type of the representative monovalent substituent is not particularly limited, and for example, is preferably selected from the group consisting of 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, an alkoxy group having 1 to 6 carbon atoms, a hydroxyl group, an alkoxycarbonyl group, a halogen atom and an amino group.

[0291] These monovalent substituents may further have one or two or more arbitrary substituents. For example, R 1The alkyl group represented by R may contain one or two or more halogen atoms, hydroxyl groups, amino groups, alkoxy groups, etc. For example, R 1 Representative alkyl groups may be haloalkyl, hydroxyalkyl, aminoalkyl and the like.

[0292] R 1 The monovalent substituent represented by is introduced into the aromatic ring separately from the above R (selected from the group consisting of -CO2H, -PO3H2, -SO3H, -L-CO2H, -L-PO3H2 and -L-SO3H), and when n is 0, R 1 It may exist or not exist. When n is 1 or more, R 1 It can exist or it can not exist.

[0293] For example, in the case where the aromatic ring is a phenyl group (benzene ring), when n is 1, a monovalent substituent may be present on 1 to 4 benzene rings, and when n is 2, a monovalent substituent may be present on 1 to 3 benzene rings.

[0294] R 1 With R 1 The carbon atom on the bonded ring A, the carbon atom on the ring structure containing the X atom to which the ring A is bonded (spiro atom), and a part of the ring A may form a 5- or 6-membered carbocyclic ring or heterocyclic ring (spirocyclic ring).

[0295] In a preferred mode of the present invention, R 1 It is absent or is an alkyl group having 1 to 6 carbon atoms, preferably a methyl group or an ethyl group.

[0296] In another preferred mode of the present invention, as R 1 , there is an alkyl group having 1 to 6 carbon atoms on the aromatic ring, preferably a methyl group.

[0297] In this case, an alkyl group (preferably methyl group) having 1 to 6 carbon atoms is preferably bonded to the 2-position (para position when the aromatic ring is a benzene ring) of the aromatic ring relative to the position at which the alkyl group is bonded to the xanthene ring.

[0298] In the general formulae (I) and (II), R 2 and R 3 Each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent.

[0299] When R 2 or R 3 When R represents an alkyl group, there may be one or two or more halogen atoms, hydroxyl groups, amino groups, alkoxy groups, etc. in the alkyl group. For example, R 2 or R 3Representative alkyl groups may be haloalkyl, hydroxyalkyl, and the like.

[0300] R 2 and R 3 At least one of the may be a water-soluble substituent selected from -CO2H, -PO3H2 or -SO3H, or an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms.

[0301] In one aspect of the present invention, R 2 and R 3 are each independently a hydrogen atom or a halogen atom, more preferably, R 2 and R 3 All are hydrogen atoms.

[0302] In another aspect of the present invention, R 2 and R 3 Each is independently a hydrogen atom, a water-soluble substituent selected from -CO2H, -PO3H2 or -SO3H, or an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms.

[0303] In the general formulae (I) and (II), R 4 and R 5 Each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent, and the details are the same as for R 2 and R 3 Same as the description.

[0304] In one aspect of the present invention, R 4 and R 5 are each independently a hydrogen atom or a halogen atom, more preferably, R 4 and R 5 All are hydrogen atoms.

[0305] In another aspect of the present invention, R 4 and R 5 Each is independently a hydrogen atom, a water-soluble substituent selected from -CO2H, -PO3H2 or -SO3H, or an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms.

[0306] In the general formulae (I) and (II), R 6 and R 7 Each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent, the details of which are the same as those for R 2 and R 3 Same as the description.

[0307] In one aspect of the present invention, R 6 and R 7 are each independently a hydrogen atom or a halogen atom, more preferably, R 6 and R 7 All are hydrogen atoms.

[0308] In another aspect of the present invention, R 6 and R 7 Each is independently a hydrogen atom, a water-soluble substituent selected from -CO2H, -PO3H2 or -SO3H, or an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms.

[0309] In the general formulae (I) and (II), R 8 and R 9 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, or an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, having a water-soluble substituent (the alkyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent), or an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, having a water-soluble substituent (the alkenyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent).

[0310] R 8 and R 9 can form a 4- to 7-membered heterocyclic group containing a nitrogen atom, R 8 and R 9 Attached to the nitrogen atom.

[0311] R 8 or R 9 , or R 8 and R 9 Both, with R 5 or R 7 Together, they can each form a 5- to 7-membered heterocyclic or heteroaryl group containing a nitrogen atom, R 8 or R 9 The heterocyclic group or heteroaryl group is connected to the nitrogen atom, and further, the heterocyclic group or heteroaryl group may have a substituent selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, an arylalkyl group having 6 to 10 carbon atoms, and an alkyl-substituted alkenyl group having 6 to 10 carbon atoms.

[0312] As ring members of the heterocyclic group or heteroaryl group, one to three additional hetero atoms selected from the group consisting of an oxygen atom, a nitrogen atom and a sulfur atom may be contained.

[0313] The water-soluble substituent may be bonded to these heterocyclic groups and heteroaryl groups directly or through a substituent.

[0314] In a preferred mode of the present invention, R 8 and R 9 All are hydrogen atoms.

[0315] In the general formulae (I) and (II), R 11 is a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, having a water-soluble substituent (the alkyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent), or an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, having a water-soluble substituent (the alkenyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent).

[0316] In the general formulae (I) and (II), X is selected from oxygen atoms, Si(R a )(R b ), C(Ra)(Rb), P(=O)Rc, S(R d )2、Ge(R a )(R b ), tellurium atoms or bismuth atoms.

[0317] R a and R b Each is independently a substituted or unsubstituted alkyl group or a substituted or unsubstituted aryl group having 1 to 6 carbon atoms.

[0318] R a and R b Each is independently preferably an alkyl group having 1 to 3 carbon atoms, more preferably, R a and R b All are methyl.

[0319] R a and R b The alkyl group represented by may contain one or two or more halogen atoms, hydroxyl groups, amino groups, alkoxy groups, etc. For example, the alkyl group may be a halogenated alkyl group, a hydroxyalkyl group, etc. a and / or R b When it is an aryl group, the aryl group may be a monocyclic aromatic group or a condensed aromatic group, and the aryl ring may contain one or two or more heteroatoms constituting the ring (e.g., nitrogen atom, oxygen atom, sulfur atom, etc.). The aryl group is preferably a phenyl group. One or two or more substituents may exist on the aryl ring. For example, as a substituent, one or two or more halogen atoms, hydroxyl, amino, alkoxy, etc. may exist.

[0320] Defined as R a and R bThe alkyl and aryl groups may be substituted with water-soluble substituents selected from -CO2H, -PO3H2 or -SO3H.

[0321] R c It is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted phenyl group.

[0322] Defined as R c The alkyl group or phenyl group may be substituted with a water-soluble substituent.

[0323] R d are the same or different substituted or unsubstituted aryl groups.

[0324] Defined as R d The aryl group may be substituted with a water-soluble substituent.

[0325] In a preferred mode of the present invention, X is an oxygen atom or Si(R a )(R b ).

[0326] In the general formulas (I) and (II), R0 and R 10 Each is independently selected from a partial structure of an amino acid, a partial structure of a carboxylic acid, TO-, or a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group.

[0327] The partial structure of an amino acid refers to its relationship with R0 or R 10 The bonded C=O together constitute an amino acid, an amino acid residue, a peptide or a part of an amino acid.

[0328] T is a sugar, a partial structure of a sugar, or a sugar having a self-cleavable linker, and the partial structure of the sugar of T together with O to which T is bonded constitutes a sugar or a part of the sugar.

[0329] The example of saturated or unsaturated 5 or 6 yuan carbocyclic or heterocyclic group comprises aryl and contains 5 or 6 yuan saturated heterocyclic groups of one or two heteroatoms as ring member atoms.Heteroatoms used herein can be selected from nitrogen-atoms, oxygen atoms and sulphur atoms, and when there are two heteroatoms, heteroatoms can be the same or different.Non-limiting examples of saturated heterocycles include morpholine ring.

[0330] In this specification, for "amino acid", any compound can be used as long as it has both an amino group and a carboxyl group, and amino acids include natural and non-natural compounds. The amino acid can be any of a neutral amino acid, a basic amino acid, and an acidic amino acid, and amino acids that function as transmitters such as neurotransmitters can be used, as well as amino acids that are components of polypeptide compounds such as physiologically active peptides (including dipeptides, tripeptides, tetrapeptides, and oligopeptides) or proteins, and the amino acid can be, for example, α amino acids, β amino acids, γ amino acids, etc. It is preferred to use optically active amino acids as amino acids. For example, as α amino acids, D-amino acids or L-amino acids can be used, and optically active amino acids that function in vivo may be preferably selected.

[0331] In the present specification, the "amino acid residue" refers to a structure corresponding to the remaining partial structure obtained by removing a hydroxyl group from a carboxyl group of an amino acid.

[0332] The amino acid residue includes an α-amino acid residue, a β-amino acid residue or a γ-amino acid residue.

[0333] In the present specification, "peptide" refers to a structure in which two or more amino acids are linked by a peptide bond.

[0334] R0 or R 10 With R0 or R 10 Examples of the case where the bonded C═O together constitutes a part of an amino acid include a structure in which a carboxyl group of an amino acid side chain is bonded to —NH 2 to form a carbonyl group, thereby constituting a part of an amino acid, such as the above-mentioned γ-glutamyl group.

[0335] In one mode of the present invention, R0 and R 10 The partial structure of the amino acid is represented by the following formula (A):

[0336] [Chemical formula 22]

[0337]

[0338] In formula (A), R 1a Selected from the group consisting of: groups constituting the side chains of natural amino acids (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine or proline), groups constituting the side chains of unnatural amino acids (such as citrulline or norvaline), and substituted or unsubstituted alkyl groups.

[0339] In formula (A), R 2a It is a hydrogen atom or an N-terminal protecting group, such as acetyl (-COCH3) or succinamide (-CO-C2H4-COOH).

[0340] * represents the bonding site to C=O.

[0341] When R0 or R 10 When represented by formula (A), R0 or R 10 With R0 or R 10 The bonded C=O together constitute an amino acid residue.

[0342] When R is represented by formula (A), the compounds represented by general formulae (I) and (II) or salts thereof can be used as fluorescent probes for detecting aminopeptidase activity.

[0343] In one mode of the present invention, R0 and R 10 The partial structure of the amino acid is represented by the following formula (B):

[0344] [Chemical formula 23]

[0345]

[0346] In formula (B), R 3a Selected from the group consisting of: groups constituting the side chains of natural amino acids (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine or proline), groups constituting the side chains of unnatural amino acids (such as citrulline or norvaline), and substituted or unsubstituted alkyl groups.

[0347] In formula (B), R 4a represents a group constituting a side chain of a natural amino acid (glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine or proline), such as hydrogen or methyl. 4a Also included are groups that form side chains of unnatural amino acids such as citrulline or norvaline. 4a It also includes groups other than the groups constituting the side chains of natural amino acids or non-natural amino acids, such as alkyl groups having various substituents.

[0348] In formula (B)

[0349] [Chemical formula 24]

[0350]

[0351] Representative examples include a structure in which the amino group of formula (B) is bonded to an amino group, an N-acetylated amino group, etc., a structure in which an amino group and an amino acid (the N-terminus may be acetylated, etc.) are bonded to each other, or a peptide in which an amino group and multiple amino acids are bonded via a peptide bond (the N-terminus may be acetylated, etc.).

[0352] * represents the bonding site to C=O.

[0353] When R0 or R 10 When represented by formula (B), R0 or R 10 With R0 or R 10 The bonded C=0 together form a peptide in which two or more amino acids are linked by peptide bonds.

[0354] When R0 or R 10 When represented by formula (B), the compound represented by general formula (I) or (II) or a salt thereof can be used as a fluorescent probe for detecting the activity of peptidase or protease.

[0355] In one mode of the present invention, R0 and R 10 The partial structure of the amino acid is represented by the following formula (C):

[0356] [Chemical formula 25]

[0357]

[0358] * represents the bonding site to C=O.

[0359] When R0 or R 10 When represented by formula (C), R0 or R 10 With R0 or R 10 The bonded C=O together forms a γ-glutamyl bond. In this case, the compounds represented by the general formulae (I) and (II) or salts thereof can be used as fluorescent probes for detecting the activity of γ-glutamyl transpeptidase (GGT).

[0360] In a preferred aspect of the present invention, NH—CO—R0 and -NH—CO—R0 bonded to the benzene ring of the tricyclic structure (especially the xanthene ring) of the general formula (I) and (II) 10 The part is represented by any of the following structures:

[0361] [Chemical formula 26]

[0362]

[0363]

[0364] In the above formula, ** represents the site of bonding to the benzene ring.

[0365] Defined as R0 or R 10The carboxylic acid portion of the structure is R0 or R 10 The bonded C=O together constitutes a part of the carboxylic acid. 5a The carboxyl group of the carboxylic acid of -COOH allows the amino group bonded to the xanthene skeleton to bond with an amide bond, thereby forming R 5a -CO-NH-*** complex group. 5a represents a hydrocarbon group which may have a carboxyl group at its terminal end, and *** represents the site of bonding to the benzene ring of the xanthene skeleton.

[0366] The carboxylic acid is preferably a saturated fatty acid, an unsaturated fatty acid or a dicarboxylic acid.

[0367] The saturated fatty acids are preferably acetic acid (CH3CO2H), butyric acid (CH3CH2CO2H), palmitic acid (CH3(CH2) 14 CO2H) etc.

[0368] The unsaturated fatty acid is preferably oleic acid.

[0369] The dicarboxylic acid is preferably succinic acid (HO2C-CH2-CH2-CO2H).

[0370] When R0 or R 10 When it is a partial structure of a carboxylic acid, the compound represented by the general formula (I) or (II) or a salt thereof can be used as a fluorescent probe for detecting the activity of amidase.

[0371] In another aspect of the present invention, for a 10 Partial structure of carboxylic acid, R 5a -CO-NH-*** in the complex group R 5a It may be a hydrocarbon group having a site reduced by an oxidoreductase (*** represents a site bonded to the benzene ring of the xanthene skeleton).

[0372] Examples of sites reduced by oxidoreductases include quinone moieties, which, when reduced by oxidoreductases to form phenol, attack the amide moiety, cleave the amide, and release the fluorescent dye, thereby enabling detection of enzyme activity. 10 Examples of the partial structure of carboxylic acid are shown below.

[0373] [Chemical formula 27]

[0374]

[0375] The fluorescent probe containing the above compound can detect the activity of oxidoreductase.

[0376] Defined as R0 or R 10In TO-, T is a sugar, a partial structure of a sugar, or a sugar having a self-cleavable linker, and the partial structure of the sugar of T together with O to which T is bonded constitutes a sugar or a part of a sugar.

[0377] Examples of sugars include β-D-glucose, β-D-galactose, β-L-galactose, β-D-xylose, α-D-mannose, β-D-fucose, α-L-fucose, β-L-fucose, β-D-arabinose, β-L-arabinose, β-DN-acetylglucosamine and β-DN-acetylgalactosamine, with β-D-galactose and β-DN-acetylglucosamine being preferred.

[0378] Self-cleaving linkers refer to linkers that can spontaneously cleave and decompose, and examples thereof include carbamate, urea, p-aminobenzyloxy and ester groups. Self-cleaving linkers are well known and can be used in the present invention. Non-limiting examples of self-cleaving linkers include sugar hydrolase probes (J.Am.Chem.Soc.2013,135(1),409-414).

[0379] As described above, the compound of the present invention basically has at least one water-soluble substituent selected from the group consisting of a carboxyl group (—CO2H), a phosphoric acid group (—PO3H2) and a sulfonic acid group (—SO3H). As a mode of introducing a water-soluble substituent, a water-soluble substituent may be introduced only into the aromatic ring of A directly or through a linker (mode A), or a water-soluble substituent may be introduced into the xanthene skeleton directly or through a linker without being introduced into the aromatic ring (i.e., n=0) (mode B). A water-soluble substituent may be introduced into the aromatic ring of A directly or through a linker, and a water-soluble substituent may be introduced into the xanthene skeleton directly or through a linker (mode C).

[0380] In addition to each of the above modes, a water-soluble substituent may be introduced directly or via another substituent into a cyclohexyl ring or a cycloaryl ring formed by bonding a nitrogen atom to a xanthene skeleton (mode D), and only a water-soluble substituent may be introduced directly or via another substituent into a cyclohexyl ring or a cycloaryl ring formed by bonding a nitrogen atom to a xanthene skeleton (mode E).

[0381] In any case of Modes A to E, the compound of the present invention has a MiLogP value of less than 2.47 or a LogP value of 1.09 or less, which is a lipophilicity index. When the MiLogP value is less than 2.47 or the LogP value is 1.09 or less, the microdevice applicability of the compound of the present invention is good.

[0382] The LogP value is the partition coefficient of octanol / water, which indicates the ratio of the equilibrium concentration of a substance dissolved between octanol and water phases, and is an indicator of whether a component is hydrophobic (lipid soluble) or hydrophilic (water soluble). In the present invention, the LogP value is a value measured at room temperature (25° C.), and the details of the measurement method will be described in the Examples.

[0383] LogP value can also be estimated according to chemical structure. Those skilled in the art can use software and program for estimating LogP value, for example, in the present invention, program provided by Molinspiration Cheminformatics can be used to estimate LogP value. The LogP value thus obtained is referred to as MiLogP value in the present invention.

[0384] One preferred mode of the fluorescent probe of the present invention is a fluorescent probe containing at least one compound among the compounds represented by the following general formulae (Ia) to (Ie) or a salt thereof.

[0385] [Chemical formula 28]

[0386]

[0387]

[0388] In formulae (Ia) to (Ie), R 4 To R 9 , R a , R b and R0 are as defined in formulae (I) and (II).

[0389] Another preferred embodiment of the fluorescent probe of the present invention is a fluorescent probe comprising a compound represented by the following general formula (If) or a salt thereof.

[0390] [Chemical formula 29]

[0391]

[0392] In formula (If), R 4 To R 9 and R0 are as defined in formulae (I) and (II).

[0393] Unless otherwise specifically stated, the compounds of the present invention also include stereoisomers thereof, such as tautomers, geometric isomers (e.g., E-form, Z-form, etc.) and enantiomers. That is, when the compounds of the present invention contain one or two or more asymmetric carbons, they can each independently present (R)-form or (S)-form, depending on the stereochemistry of the asymmetric carbons, and sometimes exist as enantiomers or diastereomers of derivatives or other such stereoisomers. Therefore, pure stereoisomers, any mixtures of stereoisomers, racemates, etc. can be used as active ingredients of the fluorescent probes of the present invention, and are all included in the scope of the present invention.

[0394] (2) Fluorescent probes for detecting hydrolases

[0395] Suitably, the fluorescent probes of the invention are used to detect hydrolases.

[0396] Examples of hydrolases include peptide bond hydrolases and O-glycosyl compound hydrolases.

[0397] Peptide bond hydrolases include amidases, aminopeptidases, peptidases and proteases. Examples of peptide bond hydrolases detectable by the fluorescent probe of the present invention include but are not limited to aminopeptidase N, γ-glutamyl transpeptidase, dipeptidyl peptidase, elastase, fatty acid amide hydrolase, caspase, acylamino acid releasing enzyme, fibroblast activation protein and SUMO specific protease.

[0398] Examples of O-glycosyl compound hydrolases detectable by the fluorescent probe of the present invention include, but are not limited to, β-galactosidase and β-GlcNAcase.

[0399] Some enzymes exhibit various enzyme activities depending on the reaction conditions, and even such enzymes are treated as hydrolases in the present invention as long as they have hydrolase activity. For example, transferases such as γ-glutamyl transferase are hydrolases because they exhibit hydrolase activity in the reverse reaction.

[0400] The method for using the fluorescent probe of the present invention is not particularly limited, and the fluorescent probe can be used in the same manner as conventional known fluorescent probes. Typically, the compound of the present invention or its salt can be dissolved in an aqueous medium such as physiological saline or a buffer solution, an aqueous medium and a water-miscible organic solvent such as a mixture of ethanol, acetone, ethylene glycol, dimethyl sulfoxide or dimethylformamide, etc., and the solution is added to a suitable buffer solution containing cells and tissues, and the fluorescence spectrum is measured. The fluorescent probe of the present invention can be used in combination with a suitable additive in the form of a composition. For example, the fluorescent probe of the present invention can be combined with an additive such as a buffer, a solubilizing agent or a pH adjusting agent.

[0401] (3) Fluorescent probes for microdevices

[0402] Suitably, the fluorescent probe of the present invention can be used in a method for detecting enzyme activity using a microdevice.Thus, by providing the fluorescent probe of the present invention in a well of a microdevice, the fluorescent probe can be used in a method for detecting the enzyme activity of a target hydrolase in a biological sample.

[0403] Microdevices include microchamber devices, liposomes, droplets, etc.

[0404] Hereinafter, the micro device will be described by taking a micro chamber type device as an example.

[0405] The material of the microdevice is not particularly limited, and examples thereof include glass materials, silicon, and plastics containing dendritic polymers or copolymers. Examples of glass materials include soda lime glass, Pyrex (registered trademark) glass, Vycor (registered trademark) glass, and quartz glass. Examples of resin polymers include poly (vinyl chloride), poly (vinyl alcohol), poly (methyl methacrylate), poly (vinyl acetate-to-maleic anhydride), poly (dimethylsiloxane) monomethacrylate, cycloolefin polymers, fluorocarbon polymers, polystyrene, polypropylene, and polyethyleneimine. Examples of copolymers include poly (vinyl acetate-to-maleic anhydride), poly (styrene-to-maleic anhydride) and poly (ethylene-to-acrylic acid) or derivatives thereof.

[0406] Examples of microdevice shapes include multiwell plates, such as Figure 1 As shown, any number of holes (eg, microwells) are arranged in the multi-well plate. The number of holes is, for example, 1 or more and 10,000,000 or less, such as 10 or more and 500,000 or less, such as about 100,000, etc. per plate.

[0407] The pore size of the pores of the microdevice may be, for example, 10 nm or more and 10 μm or less, may be, for example, 100 nm or more and 10 μm or less, and may be, for example, 1 μm or more and 10 μm or less.

[0408] The depth of the pores of the microdevice may be, for example, 10 nm or more and 100 μm or less, may be, for example, 100 nm or more and 80 μm or less, and may be, for example, 200 nm or more and 70 μm or less.

[0409] When the pore diameter and depth are within the above ranges, one molecule of the target enzyme can be captured in the pore, and the enzyme activity of each molecule of the enzyme in the biological sample can be detected.

[0410] The microdevice may include one of the fluorescent probes of the present invention described above in one well.

[0411] In this way, the fluorescence intensity of a fluorescent probe of the present invention relative to a molecule of the target enzyme in the biological sample can be detected, and the enzyme activities of the molecule of the target enzyme can be compared with each other.

[0412] The amount of the fluorescent probe of the present invention contained in one well of a microdevice may be, for example, 100 nM or more and 1000 μM or less, may be, for example, 1 μM or more and 1000 μM or less, and may be, for example, 10 μM or more and 1000 μM or less.

[0413] As a method of using the microdevice, first, a solution containing a biological sample is added to the microdevice, and then a sealing oil is added dropwise to seal the target enzyme in the biological sample in the pores of the microdevice.

[0414] The fluorescent probe of the present invention can be added to a solution containing a biological sample.

[0415] 2. Methods for detecting enzyme activity in biological samples

[0416] Another embodiment of the present invention is a method for detecting the activities of multiple enzymes in a biological sample using one or more compounds of the present invention (hereinafter also referred to as "detection method 1 of the present invention").

[0417] In the detection method 1 of the present invention, a microdevice can be used as appropriate.

[0418] The microdevice used in the detection method 1 of the present invention comprises the above-mentioned compound of the present invention.

[0419] The materials and shapes of the microdevices that can be used in the detection method 1 of the present invention are as described in detail with respect to the fluorescent probe of the present invention.

[0420] The microdevice may include one compound of the invention in one well.

[0421] In this way, one fluorescent probe of the present invention can be detected against one molecule of an enzyme in a biological sample, and the enzyme activity of one molecule of each enzyme in a plurality of enzymes and the compound of the present invention can be compared.

[0422] The amount of the fluorescent probe of the present invention contained in one well of a microdevice may be, for example, 100 nM or more and 1000 μM or less, may be, for example, 1 μM or more and 1000 μM or less, and may be, for example, 10 μM or more and 1000 μM or less.

[0423] As a method of using the microdevice, first, a solution containing a biological sample is added to the microdevice. Then, a sealing oil is added dropwise to seal the enzyme in the biological sample in the pores of the microdevice. The fluorescent probe of the present invention can be added to a solution containing a biological sample.

[0424] According to the present invention, a method for detecting the activity of multiple enzymes in a biological sample is provided, the method comprising contacting a biological sample (e.g., a biological sample, a biopsy sample, a body fluid sample, and an aqueous solution, which are separated from a subject) with a compound of formula (I) or a compound of formula (II). In a certain mode, the biological sample can be a blood sample (e.g., a serum sample or a plasma sample). The method may further include measuring the fluorescence of the compound after the compound is contacted with the aqueous solution. When the compound emits fluorescence, the presence or absence of fluorescence indicates the presence of an enzyme in the aqueous solution, and the intensity of the fluorescence indicates the activity intensity of the enzyme in the aqueous solution.

[0425] One mode of the detection method 1 of the present invention is a method for detecting peptide bond hydrolase activity in a biological sample, which comprises contacting the compound of the present invention with a plurality of enzymes.

[0426] In a preferred aspect of the detection method 1 of the present invention, the contacting is performed in the presence of serum.

[0427] According to the detection method 1 of the present invention, the enzyme activities of various enzymes in biological samples can be detected.

[0428] The detection method 1 of the present invention will be described in detail below.

[0429] [Step 1]

[0430] First, a solution containing a biological sample is added to a microdevice comprising the compounds of this invention or a group (library) of the compounds of this invention. Examples of biological samples include biological samples, biopsy samples, body fluid samples and aqueous solutions separated from a subject. The biological sample can be a blood sample (e.g., a serum sample or a plasma sample).

[0431] As the pH of the solution containing the biological sample, any value can be selected. For example, the pH of the solution can be set to a value close to the pH in the living body, in which case the pH can be, for example, 6.0 or higher and 8.0 or lower. In order to narrow the target to a specific enzyme group, the pH of the solution can also be adjusted to acidity to achieve an optimal pH.

[0432] A solution containing a biological sample and a solution containing the fluorescent probe of the present invention can be prepared separately, the ratio of the biological sample and the fluorescent probe can be appropriately adjusted and mixed, and then the mixed solution is added to the microdevice.

[0433] The protein concentration of a biological sample may generally be, for example, 1 pM or more and 100 pM or less, and may be, for example, 10 pM or more and 100 pM or less as the concentration of a "target protein".

[0434] For example, the upper limit of total protein concentration in a sample may be about 10 μM.

[0435] As a method for measuring the protein concentration in a biological sample, for example, a method using an antibody-antigen reaction (e.g., ELISA method, etc.) can be used as a method for measuring the concentration of a "target protein." Examples of methods for measuring the total protein concentration in a sample include colorimetric methods using a reaction between a protein and a reagent (e.g., bicinchoninic acid (BCA) method, Bradford method, Lowry method, and biuret method).

[0436] The biological sample can be diluted to achieve the above concentration using various aqueous solvents, etc. Examples of aqueous solvents include water, physiological saline, phosphate buffered saline (PBS), tris buffered saline (TBS), and HEPES buffered saline, but are not limited thereto.

[0437] [Step 2]

[0438] Then drip sealing oil, the enzyme in the biological sample is sealed in the hole of the microdevice. The sealing oil can be any known sealing oil, which is usually used to seal the sample in the microdevice, and its example includes fluorine-based oil (such as FC-40) and aliphatic hydrocarbon (such as hexadecane).

[0439] [Step 3]

[0440] Next, the fluorescence in the wells of the microdevice is detected using a fluorescence scanner, a fluorescence microscope, etc. The enzyme activity can be evaluated based on the intensity of the detected fluorescence.

[0441] For example, it is possible to determine what enzyme is contained in the wells of a microdevice where enzymatic activity is detected by comparison with the enzymatic activity pattern of a target protein prepared separately.

[0442] The detection method 1 of the present invention may further include observing the fluorescence response using a fluorescence imaging device. As a device for observing the fluorescence response, a fluorimeter with a wide measurement wavelength can be used, but a fluorescence imaging device that can display the fluorescence response as a two-dimensional image can also be used to visualize the fluorescence response. By using a fluorescence imaging device, the fluorescence response can be visualized in a two-dimensional form, so that the target enzyme can be immediately and intuitively identified. As a fluorescence imaging device, a device known in the art can be used. In some cases, the reaction between the sample to be tested and the fluorescent probe can also be detected by changes in the ultraviolet-visible absorption spectrum (for example, changes in absorbance under a specific absorption wavelength).

[0443] In one mode of the detection method 1 of the present invention, by contacting a fluorescent probe of the present invention with multiple enzymes in a biological sample, the fluorescence intensity of the fluorescent probe of the present invention relative to one molecule of the multiple enzymes in the biological sample can be detected, and the enzyme activity of one molecule of the multiple enzymes can be compared. In step 1, the enzyme activity of one molecule of the enzyme can be measured by preparing a well (e.g., a plurality of wells) containing one molecule of the enzyme and multiple fluorescent probes.

[0444] 3. Detection of biomarkers

[0445] The fluorescent probe of the present invention can be based on the introduced functional group R0 or R 10 The types are used as fluorescent probes for detecting the activity of one or more hydrolases. The fluorescent probes of the present invention can be further used to detect specific biomarkers of predetermined diseases to diagnose the diseases.

[0446] That is, another embodiment of the present invention is a method for diagnosing a disease state by detecting single-molecule hydrolase activity using the fluorescent probe of the present invention (hereinafter also referred to as "the diagnostic method of the present invention").

[0447] Non-limiting examples of diagnostic methods of the present invention are shown below.

[0448] That is, another embodiment of the present invention is a method for detecting CD13 activity in a biological sample, the method comprising contacting a compound of the present invention (hereinafter also referred to as "compound A of the present invention") with a biological sample in an aqueous solution, wherein -CO-R0 or -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (I) or (II), and an increase in fluorescence intensity in an aqueous solution indicates the presence of CD13 activity.

[0449] Another embodiment of the present invention is a method for diagnosing pancreatic cancer (hereinafter also referred to as "diagnostic method 1 of the present invention"), which comprises using a fluorescent probe containing the compound of the present invention to detect the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 or -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (I) or (II).

[0450] Another mode of the present invention is a method for diagnosing pancreatic cancer or a method for predicting the possibility that a subject from which a biological sample is derived has pancreatic cancer (hereinafter also referred to as "diagnosis or prediction method 1 of the present invention"), the method comprising (a) contacting a biological sample obtained from a subject with a fluorescent probe containing a compound of the present invention by a microdevice, wherein -CO-R0 or -CO-R 10is an alanine, lysine, arginine or methionine residue in the general formula (I) or (II), and (b) measuring the fluorescence intensity of the biological sample contacted with the fluorescent probe.

[0451] The diagnostic method 1 of the present invention and the diagnostic or prognostic method 1 of the present invention are based on the detection of the enzymatic activity of the compound A of the present invention on one molecule of CD13.

[0452] Another embodiment of the present invention is a method for detecting DPP4 activity in a biological sample, the method comprising contacting a compound of the present invention (hereinafter also referred to as "compound B of the present invention") with a biological sample in an aqueous solution, wherein -CO-R0 or -CO-R 10 It is -Pro-Xaa (Pro represents a proline residue, and Xaa represents an amino acid residue, such as glycine, serine or glutamic acid) in the general formula (I) or (II). The increase in fluorescence intensity in the aqueous solution indicates the presence of DPP4 activity.

[0453] Another embodiment of the present invention is a method for diagnosing pancreatic cancer (hereinafter also referred to as "diagnostic method 2 of the present invention"), which comprises using a fluorescent probe containing the compound of the present invention to detect the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 or -CO-R 10 It is -Pro-Xaa in the general formula (I) or (II) (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamic acid).

[0454] Another mode of the present invention is a method for diagnosing pancreatic cancer or a method for predicting the possibility that a subject from which a biological sample is derived has pancreatic cancer (hereinafter also referred to as "diagnosis or prediction method 2 of the present invention"), the method comprising (a) contacting a biological sample obtained from a subject with a fluorescent probe containing a compound of the present invention by a microdevice, wherein -CO-R0 or -CO-R 10 is -Pro-Xaa (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamic acid) in the general formula (I) or (II), and (b) measuring the fluorescence intensity of the biological sample contacted with the fluorescent probe.

[0455] The diagnostic method 2 of the present invention and the diagnostic or predictive method 2 of the present invention are based on the detection of the enzymatic activity of the compound B of the present invention on one molecule of DPP4.

[0456] Another mode of the present invention is a method for diagnosing pancreatic cancer, the method comprising: using a fluorescent probe containing the compound of the present invention to detect the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 or -CO-R 10is an alanine, lysine, arginine or methionine residue in formula (I) or (II); and

[0457] Using a fluorescent probe containing the compound of the present invention, the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject is detected by a microdevice, wherein -CO-R0 or -CO-R 10 It is -Pro-Xaa in the general formula (I) or (II) (Pro represents a proline residue, and Xaa represents an amino acid residue such as glycine, serine or glutamic acid).

[0458] The diagnostic method 1 (or 2) of the present invention and the diagnostic or predictive method 1 (or 2) of the present invention can be carried out by preparing a solution containing a biological sample and the compound A (or B) of the present invention, adding the solution to a microdevice, sealing the added solution in each well, incubating the microdevice with the sealed solution, and then measuring the fluorescence intensity of each well using a fluorescence microscope.

[0459] Specifically, the diagnostic method 1 of the present invention and the diagnostic or prognostic method 1 of the present invention are performed, for example, as follows, but are not limited thereto.

[0460] The compound A of the present invention is diluted to a predetermined concentration (eg, 100 μM) with an assay buffer. The composition of the assay buffer can be appropriately determined, and may be, for example, 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, and 3 mM TritonX-100.

[0461] Next, a biological sample collected from a subject (including pancreatic cancer patients and healthy subjects), preferably a blood sample (such as a serum sample or a plasma sample), is diluted (e.g., about 10,000 times) with the above-mentioned assay buffer and mixed with the diluted solution of Compound A of the present invention at a predetermined ratio (e.g., equal amounts).

[0462] The mixed solution of the two was added into the microdevice, and then sealing oil was added to seal the solution in each well.

[0463] The microdevice with the blocking solution is incubated under predetermined conditions (eg, 37° C. for 2 hours), and then the fluorescence intensity of each well is measured using a fluorescence microscope.

[0464] The number of wells in which the fluorescence intensity detected was equal to or higher than a predetermined intensity (eg, 2,500 AU or higher) was measured, that is, the number of wells in which CD13 was considered to be blocked.

[0465] In order to diagnose whether the subject from which the biological sample is derived has pancreatic cancer and predict the possibility of pancreatic cancer based on the number of pores in which CD13 is considered to be blocked as measured as above, a ROC curve for the assay is plotted based on the results of CD13 activity measurement using Compound A of the present invention, and the possibility of pancreatic cancer is determined based on the ROC curve.

[0466] Specifically, the diagnostic method 2 of the present invention and the diagnostic or prognostic method 2 of the present invention are performed, for example, as follows, but are not limited thereto.

[0467] The compound B of the present invention is diluted to a predetermined concentration (eg, 100 μM) with an assay buffer. The composition of the assay buffer can be appropriately determined, and may be 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, and 3 mM Triton X-100.

[0468] Next, a biological sample collected from a subject (including pancreatic cancer patients and healthy subjects), preferably a blood sample (such as a serum sample or a plasma sample), is diluted (e.g., about 2,500 times) with the above-mentioned assay buffer and mixed with the diluted solution of the compound B of the present invention at a predetermined ratio (e.g., equal amounts).

[0469] The mixed solution of the two was added into the microdevice, and then sealing oil was added to seal the solution in each well.

[0470] The microdevice in the blocking solution is incubated under predetermined conditions (eg, 25° C. for 2 hours), and then the fluorescence intensity of each well is measured using a fluorescence microscope.

[0471] The histogram plotted based on the fluorescence intensity of each well approximated a curve including two normal distributions. Wells with fluorescence intensities higher than the minimum value of the approximate curve were defined as a high-activity group, wells with fluorescence intensities lower than the minimum value of the approximate curve were defined as a low-activity group, and the ratio of the high-activity group to the sum of these groups was measured.

[0472] In order to diagnose whether the subject from which the biological sample is derived has pancreatic cancer, and to predict the possibility of pancreatic cancer based on the number of pores in which DPP4 is considered to be blocked as measured as above, a ROC curve for the assay is plotted based on the results of DPP4 activity measurement using compound B of the present invention, and the possibility of pancreatic cancer is determined based on the ROC curve.

[0473] Non-limiting examples of Compound A of the present invention include the following compounds.

[0474] [Chemical formula 30]

[0475]

[0476] Non-limiting examples of compound B of the present invention include the following compounds.

[0477] [Chemical formula 31]

[0478]

[0479] 4. Compounds 2 and 3 of the present invention

[0480] Another embodiment of the present invention is a compound represented by any one of the following formulae (1) to (5) or a salt thereof (hereinafter also referred to as "compound 2 of the present invention").

[0481] [Chemical formula 32]

[0482]

[0483]

[0484] In formula (1) to formula (5), R0 is selected from a partial structure of an amino acid, a partial structure of a carboxylic acid, or TO-.

[0485] The partial structure of an amino acid, together with C═O to which R0 is bonded, constitutes an amino acid, an amino acid residue, a peptide, or a part of an amino acid.

[0486] The partial structure of the carboxylic acid constitutes a part of the carboxylic acid together with C═O to which R0 is bonded.

[0487] T is a sugar, a partial structure of a sugar, or a sugar having a self-cleavable linker, and the partial structure of the sugar of T together with O to which T is bonded constitutes a sugar or a part of a sugar.

[0488] The detailed description of the partial structure of the amino acid, the partial structure of the carboxylic acid and TO- are the same as those described in detail for the general formulae (I) and (II).

[0489] Another embodiment of the present invention is a compound represented by the following formula (6) or a salt thereof (hereinafter also referred to as "compound 3 of the present invention").

[0490] [Chemical formula 33]

[0491]

[0492] In formula (6), R0 is selected from a partial structure of an amino acid, a partial structure of a carboxylic acid, or TO-.

[0493] The partial structure of an amino acid, together with C═O to which R0 is bonded, constitutes an amino acid, an amino acid residue, a peptide, or a part of an amino acid.

[0494] The partial structure of the carboxylic acid constitutes a part of the carboxylic acid together with C═O to which R0 is bonded.

[0495] T is a sugar, a partial structure of a sugar, or a sugar having a self-cleavable linker, and the partial structure of the sugar of T together with O to which T is bonded constitutes a sugar or a part of the sugar.

[0496] The detailed description of the partial structure of the amino acid, the partial structure of the carboxylic acid and TO- are the same as those described in detail for the general formulae (I) and (II).

[0497] Specific examples of Compound 2 of the present invention are given below, but are not limited thereto.

[0498] [Chemical formula 34]

[0499]

[0500] Specific examples of Compound 3 of the present invention are given below, but are not limited thereto.

[0501] [Chemical formula 35]

[0502]

[0503] Unless otherwise specified, the compounds represented by formulae (1) to (6) also include stereoisomers thereof, such as isomers, geometric isomers (e.g., E-form, Z-form, etc.) and enantiomers. That is, when the compounds represented by formulae (1) to (6) contain one or two or more asymmetric carbons, they may each independently have an (R)-form or an (S)-form depending on the stereochemistry of the asymmetric carbons, and may sometimes exist as enantiomers or diastereomers of derivatives or other such stereoisomers.

[0504] The method for producing the compounds represented by formula (1) to (6) is not particularly limited, but the synthesis method of typical compounds in the compounds included in formula (1) to (6) is specifically described in the examples of this specification. Those skilled in the art can refer to the examples of this specification and the following schemes, and appropriately change or modify the starting materials, reaction reagents, reaction conditions, etc. as needed to produce the compounds included in formula (1) to (6).

[0505] 5. Intermediates

[0506] The compound represented by the general formula (III) or a salt thereof can be used as an intermediate for producing the compound of the present invention, for example. That is, according to the present invention, there is provided the use of the compound represented by the general formula (III) or a salt thereof for producing the compound of the present invention.

[0507] The compound represented by the general formula (IV) or a salt thereof can be used, for example, as an intermediate for producing the compound of the present invention. That is, according to the present invention, there is provided the use of the compound represented by the general formula (IV) or a salt thereof for producing the compound of the present invention.

[0508] According to the present invention, there is also provided a compound represented by general formula (V) or a salt thereof. For example, the compound can be used as an intermediate for producing the compounds of the present invention. That is, according to the present invention, there is provided a compound represented by general formula (V) or a salt thereof for use in producing the compounds of the present invention.

[0509] In the general formula (V), A preferably represents a phenyl group or a thienyl group. R preferably represents -COOH or -SO3H. When A is a phenyl group, R preferably represents -SO3H. When A is a thienyl group, R preferably represents -COOH.

[0510] In the general formula (V), n is preferably 1. R8 and R9 preferably represent a hydrogen atom. X preferably represents an oxygen atom or Si(-CH3)2.

[0511] More preferred examples of the compound represented by the general formula (V) or a salt thereof include the following compounds, wherein A represents a phenyl group or a thienyl group, R represents -COOH or -SO3H, n is 1, R 8 and R 9 represents a hydrogen atom, X represents an oxygen atom or Si(-CH3)2, or a salt thereof. Particularly preferred examples of the compound represented by general formula (V) or a salt thereof include the following compounds, wherein A represents a phenyl group or a thienyl group, R represents -COOH or -SO3H, n is 1, R 8 and R 9 represents a hydrogen atom, X represents an oxygen atom or Si(-CH3)2, when A is a phenyl group, R represents -SO3H, when A is a thienyl group, R represents -COOH, or a salt thereof.

[0512] 6. Methods for selecting fluorescent probes

[0513] According to the present invention, a method for selecting a fluorescent probe with excellent suitability from the fluorescent probe of the present invention is provided. Examples of indicators for evaluating the suitability of the fluorescent probe include (A), (B) and (C) above. That is, the selection method of the present invention at least includes preparing one or more fluorescent probes of the present invention, and selecting a fluorescent probe that satisfies conditions (A), (B) and (C). Since (A), (B) and (C) are indicators for evaluating suitability in a microdevice, the fluorescent probe selected by the selection method of the present invention can be particularly preferably used as a fluorescent probe for a microdevice.

[0514] In (A), a solubility measurement test in sealing oil (25°C) is performed, and if the solubility of the test compound in hexadecane is 0.4 μM or less, or 0.3 μM or less, preferably 0.2 μM or less, it can be determined that (A) is satisfied. The solubility measurement test in sealing oil can be performed by the following method: measuring the concentration of the test compound transferred to hexadecane, that is, adding 1 μL of a 10 mM DMSO solution of the test compound to 100 μL of hexadecane as a sealing oil, and then ultrasonically treating the mixture at room temperature (25°C) and suspending it for 1 minute. The solubility measurement test in the sealing oil of (A) can be specifically performed according to Example 19.

[0515] In (B), a fluorescence intensity measurement test in a microdevice is performed, and if the ratio of the fluorescence intensity of the edge area of ​​the hole to the fluorescence intensity of the center area of ​​the hole is 0.7 or less, or 0.6 or less, preferably 0.5 or less, it can be determined that (B) is satisfied. The fluorescence intensity measurement in the microdevice can be performed by the following method: adding a test compound to the microdevice, taking an image of the hole encapsulated with the compound with a microscope, and measuring the ratio of the fluorescence intensity of the edge area to the fluorescence intensity of the center area. When the radius of the hole is regarded as 1, the center area and the edge area can be specified according to the following proportions. Central area: the area from the center to a radius of 0.48 to 0.63 (area 1), preferably the area from the center to a radius of 0.53 to 0.58 (area 2). Edge area: the area in the entire hole that does not include area 1, preferably the area in the entire hole that does not include area 2. The fluorescence intensity measurement test in the microdevice of (B) can be specifically performed according to Example 20.

[0516] In (C), the test compound is subjected to an enzymatic hydrolysis test in a microdevice and a cuvette to calculate a signal-to-noise ratio. When the ratio of the signal-to-noise ratio (first ratio) of the hydrolyzate in the microdevice to the signal-to-noise ratio (second ratio) of the hydrolyzate in the cuvette is 0.2 or higher, 0.3 or higher, or 0.4 or higher, preferably 0.5 or higher, it can be determined that (C) is satisfied. When calculating the signal-to-noise ratio, the fluorescence spectra before and after the enzyme reaction are compared, and the fluorescence intensity ratio at the wavelength with the maximum fluorescence intensity can be calculated as the signal-to-noise ratio of the hydrolyzate. The measurement and calculation of the ratio of the signal-to-noise ratio in the microdevice and the cuvette of (C) can be specifically performed according to Example 21.

[0517] In the present invention, among the compounds of the present invention and the compounds of the general formula (III) and salts thereof, compounds satisfying at least one, at least two or all of (A), (B) and (C) may be preferably provided.

[0518] Example

[0519] Hereinafter, the present invention will be described with reference to Examples; however, the present invention is not limited thereto.

[0520] [Sample and measurement method]

[0521] Reagents and solvents used in organic syntheses were provided by Tokyo Chemical Industry Co., Ltd. (TCI), WakoPure Chemical Industries, Ltd. or Aldrich Chemical Company and used without further purification.

[0522] Proton NMR was recorded using a JEOL JMN-LA400 instrument ( 1 H NMR) spectroscopy.

[0523] Using JEOL JMS-T100LP AccuTOF TM Mass spectra were measured by LC-plus4G.

[0524] [Synthesis example 1]

[0525] Synthesis example of sHMRG

[0526] [Chemical formula 36]

[0527]

[0528] (1) Synthesis of Compound 1

[0529] [Chemical formula 37]

[0530]

[0531] In a dry flask, 2.88 mL (54.1 mmol) of concentrated sulfuric acid was added, and 15.7 mL (113.6 mmol) of trifluoroacetic anhydride was slowly added thereto at 0°C under an argon atmosphere. After that, the temperature was returned to room temperature, and the mixture was stirred for 3 hours. When the solution became uniform, 6.25 mL (54.1 mmol) of 2-bromo-benzaldehyde was added in portions at 0°C, the temperature was returned to room temperature, and the mixture was stirred for 17 hours. After that, water was added at 0°C. Subsequently, TFAA and water were distilled off under reduced pressure. The residue was then purified by MPLC (eluent: A / B=95 / 5>5 / 95), A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA). The eluent was evaporated, and the product was then freeze-dried to obtain compound 1 (982 mg, 3.71 mmol, yield: 6.8%).

[0532] The obtained compound 1 H-NMR and 13 The results of C-NMR analysis are as follows.

[0533] 1 H-NMR (400MHz, DMSO-D6) δ10.17(s,1H),7.98(m,1H),7.74(m,2H).

[0534] 13 C-NMR (100MHz, CD3OD) δ192.0,148.9,134.3,133.2,133.1,127.4,126.1.

[0535] HRMS(ESI - ): [MH] - The calculated value is 262.90137 and the measured value is 262.89719 (-4.17 mmu).

[0536] (2) Synthesis of Compound 2

[0537] [Chemical formula 38]

[0538]

[0539] In a dry flask, 338 mg (1.28 mmol) of compound 1 dissolved in 2.8 mL (12.8 mmol) of triisopropyl orthoformate was added, and the mixture was stirred at 60° C. for 1 hour. Subsequently, the solvent was distilled off under reduced pressure. Subsequently, the resulting solid was purified by column chromatography (silica; ethyl acetate / hexane=0 / 100>20 / 80), and the eluent was evaporated to obtain 357 mg of compound 2 (0.87 mmol, yield: 68%).

[0540] The obtained compound 1 H-NMR, 13 The results of C-NMR and high resolution mass spectrometry (HR-MS) analyses are shown below.

[0541] 1 H-NMR (400MHz, CDCl3) δ 8.22 (m, 1H), 7.68 (m, 2H), 4.76 (sep, J = 5.9Hz, 1H), 3.89 (sep, J = 6.2Hz, 2H), 1.28 (d, J = 5.9Hz, 6H), 1.18 (dd, J = 13.0Hz, 6.2Hz, 12H).

[0542] 13 C-NMR (100MHz, CDCl3) δ141.2,137.0,133.7,128.7,128.5,128.1,97.9,69.3,23.0,22.9,22.5.

[0543] HRMS(ESI + ): [M+Na]+ The calculated value is 431.05038 and the measured value is 431.04944 (-0.94mmu).

[0544] (3) Synthesis of Compound 3

[0545] [Chemical formula 39]

[0546]

[0547] In a dry flask, 213 mg (0.52 mmol) of compound 2 was added, dissolved in dehydrated tetrahydrofuran, and stirred at -78 ° C for 30 minutes under an argon atmosphere. Subsequently, 0.4 mL (0.52 mmol) of 1.3 M sec-butyl lithium was added thereto in portions, and the mixture was stirred at -78 ° C for 30 minutes under an argon atmosphere. Subsequently, 45 mg (0.081 mmol) of 3,6-bis (diphenylmethyleneamino) xanthone dissolved in dehydrated tetrahydrofuran was further added to the reaction solution in portions, and the mixture was then stirred at room temperature for 1.5 hours under an argon atmosphere. TFA and water were then added to the reaction solution, and the mixture was stirred at room temperature for 3.5 hours. Subsequently, the solvent was distilled off under reduced pressure. Subsequently, the obtained solid was purified by MPLC (eluent: A / B=95 / 5>5 / 95, A: 0.1% 100% H2O containing TFA, B: 0.1% 100% CH3CN containing TFA), the eluent was evaporated, and then the product was freeze-dried to obtain 9 mg of compound 3 (0.023 mmol, yield: 28%).

[0548] (4) Synthesis of Compound 4

[0549] [Chemical formula 40]

[0550]

[0551] In a dry flask, 130 mg (0.33 mmol) of compound 3 was added, dissolved in methanol, and cooled to 0° C., then 90 mg (2.38 mmol) of sodium borohydride was added in portions, and the mixture was stirred for 30 minutes. Subsequently, the solvent was distilled off under reduced pressure, and the resulting solid was purified by MPLC (eluent: A / B=95 / 5>5 / 95, A: 100% H2O containing 0.1M TEAA (triethylamine acetate), B: 100% CH3CN containing 0.1M TEAA) and desalted using Sep-Pak C18 (Waters). The eluent was evaporated, and the product was then freeze-dried to obtain 68 mg of compound 4 (0.17 mmol, yield: 52%).

[0552] The obtained compound 1 H-NMR, 13The results of C-NMR and high resolution mass spectrometry (HR-MS) analyses are shown below.

[0553] 1 H-NMR (400MHz, CD3OD) δ7.91(d,J=1.8Hz,1H),7.60(dd,J=8.0,2.1Hz,1H),7.13(d,J=7.8Hz,1H),6 .62(d,J=8.2Hz,2H),6.39(d,J=2.3Hz,2H),6.29(dd,J=8.2,2.3Hz,2H),5.42(s,1H),4.61(s,2H).

[0554] 13 C-NMR (100MHz, CD3OD) δ151.4,147.5,142.7,138.8,130.5,129.8,125.1,124.7,121.2,113.2,110.9,102.0,46.5,7.9.

[0555] HRMS(ESI + ): [M+H] + The calculated value is 399.10147m and the measured value is 399.09779 (-3.68mmu).

[0556] [Synthesis example 2]

[0557] Synthesis of sHMRG-based peptidase and protease probes

[0558] Using the compound obtained by reducing sHMRG (Compound 4), the sHMRG-based peptidase and protease probes (PB101 to PB111) shown in the following Table 1 were synthesized by the following steps.

[0559] [Chemical formula 41]

[0560]

[0561] 1. Compound 4 (11 μmol), DMT-MM (3 μmol) and Boc-protected amino acid or Fmoc-protected amino acid or N-terminally protected peptide (5 μmol) were mixed in a 1.5 mL test tube, and the mixture was dissolved in 30 μL of NMP.

[0562] 2. Stir the reaction mixture at 40°C.

[0563] 3. Chloranil (23 μmol) was added and the reaction mixture was stirred.

[0564] 4. The solution was purified by MPLC (eluent: A / B = 95 / 5 > 5 / 95, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), the eluent was evaporated, and the product was freeze-dried.

[0565] 5. When the raw material is protected by Fmoc, add 50 μL of piperidine and 50 μL of NMP to the obtained solid and stir the reaction mixture solution. When the raw material is not protected by Fmoc, this step is not performed and the process proceeds to step 6.

[0566] 6. Add 1000 μL TFA and stir the reaction mixture.

[0567] 7. The solution was purified by MPLC (eluent: A / B=95 / 5>5 / 95, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), the eluent was evaporated, and the product was freeze-dried to obtain the target compound.

[0568] [Table 1]

[0569] Table 1: sHMRG-based peptidase and protease probes

[0570]

[0571] The instrumental data of the compounds are as follows.

[0572] HPLC conditions: A / B=100 / 0>0 / 100 (5 min), A=0.1% formic acid H2O, B=0.1% formic acid CH3CN (A-sHMRG, M-sHMRG: mixture of diastereomers).

[0573] [Table 2]

[0574]

[0575]

[0576] [Synthesis example 3]

[0577] Synthesis Example 1 of sSiR

[0578] [Chemical formula 42]

[0579]

[0580] (1) Synthesis of Compound 2

[0581] [Chemical formula 43]

[0582]

[0583] In a dry flask, 7.7 g (63.8 mmol) of allyl bromide, 5.0 g (29.0 mmol) of 3-bromoaniline and 8.0 g (58.0 mmol) of potassium carbonate were added, dissolved in 60 mL of acetonitrile, and stirred at 80°C overnight. The temperature was then returned to room temperature, and the mixture was filtered through cotton to remove potassium carbonate. Subsequently, after washing with ethyl acetate, the solvent was distilled off under reduced pressure. Subsequently, the residue was purified by column chromatography (silica; hexane 100%), and the eluent was evaporated to obtain 7.57 g of compound 2 (30.0 mmol, yield: 47%).

[0584] (2) Synthesis of Compound 3

[0585] [Chemical formula 44]

[0586]

[0587] In a dry flask, 7.57g (30.0mmol) of compound 2 was added, dissolved in dehydrated tetrahydrofuran, and stirred at -78°C for 30 minutes under an argon atmosphere. Subsequently, 23.1mL (30.0mmol) of 1.3M sec-butyl lithium was added thereto in portions, and the mixture was stirred at -78°C for 30 minutes under an argon atmosphere. Subsequently, 1794μL (15.02mmol) of dichloromethylsilane was further added to the reaction solution in portions, and the mixture was then stirred at room temperature for 1.5 hours under an argon atmosphere. Subsequently, 10mL of 2N HCl aqueous solution and NaHCO3 aqueous solution were added to neutralize the reaction solution, the reaction solution was extracted with dichloromethane, washed with saturated brine, and then the solvent was distilled off under reduced pressure. Subsequently, the residue was purified by column chromatography (silicon dioxide; dichloromethane / hexane), and the eluent was evaporated to obtain 4.0g of compound 3 (9.93mmol, yield: 66%).

[0588] The obtained compound 1 H-NMR and 13 The results of C-NMR analysis are as follows.

[0589] 1 H-NMR (400MHz, CDCl3) δ7.20-7.16(m,2H),6.88-6.84(m,4H),6.71-6.68(m,2 H), 5.87-5.78 (m, 4H), 5.17-5.11 (m, 8H), 3.89 (d, J = 5.0Hz, 8H), 0.48 (s, 6H).

[0590] 13C-NMR (100MHz, CDCl3) δ148.0,139.0,134.3,128.5,122.3,118.2,116.2,113.3,53.0,-2.3.

[0591] (3) Synthesis of Compound 4

[0592] [Chemical formula 45]

[0593]

[0594] In a dry flask, 1.0 g (2.48 mmol) of compound 3, 769 mg (2.48 mmol) of 4-formylbenzene-1,3-disulfonic acid disodium salt and 5 mL of acetic acid were added, and stirred at 100°C for 1 hour under an argon atmosphere. Then 112 mg (1.86 mmol) of urea was added thereto, and the mixture was stirred for 18 hours. Subsequently, acetic acid was distilled off under reduced pressure, 10 mL of methanol and 94 mg (2.48 mmol) of sodium borohydride were added, and the mixture was stirred at 0°C. Subsequently, the solvent was distilled off under reduced pressure, and the resulting residue was purified by MPLC (eluent: A / B=90 / 10>60 / 40, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), and the eluent was evaporated. 10 mL of methanol and 10 mL of dichloromethane were added to the resulting residue, 610 mg (2.48 mmol) of tetrachlorobenzoquinone was added thereto, and the mixture was stirred at room temperature for 5 hours. Subsequently, the solvent was distilled off under reduced pressure, and the residue was then purified by column chromatography (silica; dichloromethane / methanol), and the eluent was evaporated to obtain 990 mg of compound 4 (1.53 mmol, yield: 62%).

[0595] The obtained compound 1 H-NMR and 13 The results of C-NMR analysis are as follows.

[0596] 1 H-NMR (400MHz, CD3OD) δ8.62(d,J=1.8Hz,1H),8.00(dd,J=7.8,1.8Hz,1H),7.26-7.24(m,3H),7.07(d,J=9.6Hz,2H) ,6.72(dd,J=9.6,2.7Hz,2H),5.96-5.87(m,4H),5.27-5.17(m,8H),4.27(d,J=4.6Hz,8H),0.54(s,3H),0.52(s,3H).

[0597] 13C-NMR (100MHz, CD3OD) δ170.5,153.9,148.4,145.7,144.1,142.7,138.1,1 31.4,130.6,128.7,126.8,125.6,120.9,116.7,113.8,53.2,-2.5,-3.11.

[0598] (4) Synthesis of Compound 5

[0599] [Chemical formula 46]

[0600]

[0601] In a dry flask, 53 mg (0.081 mmol) of compound 4 and 31 mg (0.81 mmol) of sodium borohydride were added, dissolved in 1 mL of methanol, and mixed at room temperature. Subsequently, the solvent was distilled off under reduced pressure, and 1 / 3 of the total amount (equivalent to 0.027 mmol) was used for the next step reaction. 100 μL (1.75 mmol) of acetic acid and 2 mL of water were added to the obtained residue, the mixture was degassed with an aspirator, and then palladium carbon catalyst was added to degas again, and stirred at 90 ° C for 17 hours under an argon atmosphere. Then, the palladium carbon catalyst was removed by Celite filtration, and the solvent was distilled off under reduced pressure. Subsequently, the obtained residue was purified by HPLC (eluent: A / B=90 / 10>50 / 50, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), and the eluent was evaporated to obtain 0.2 mg of compound 5 (0.41 μmol, yield: 1.5%).

[0602] The obtained compound 1 The results of H-NMR analysis are as follows.

[0603] 1 H-NMR (400MHz, CD3OD / D2O=1:1) δ8.48(d,J=2.0Hz,1H),8.01(dd,J=7.6,2.0Hz,1H),7.26(d,J=7.6Hz,1 H), 7.21 (d, J = 2.4Hz, 2H), 6.98 (d, J = 8.4Hz, 2H), 6.59 (dd, J = 8.4, 2.4Hz, 2H), 0.53 (s, 3H), 0.52 (s, 3H).

[0604] [Synthesis Example 4]

[0605] Synthesis Example 2 of sSiR

[0606] [Chemical formula 47]

[0607]

[0608] (1) Synthesis of Compound 7

[0609] [Chemical formula 48]

[0610]

[0611] In a dry flask, 1.1 mL (20.8 mmol) of concentrated sulfuric acid was added, and 6.0 mL (43.7 mmol) of trifluoroacetic anhydride was slowly added thereto at 0°C under an argon atmosphere. Afterwards, the temperature was returned to room temperature, and the mixture was stirred for 3.5 hours. When the solution became uniform, 2.5 mL (20.8 mmol) of 2-methylbenzaldehyde was then added in portions at 0°C, the temperature was returned to room temperature, and the mixture was stirred for 20 hours. Afterwards, water was added at 0°C. Subsequently, trifluoroacetic acid and water were distilled off under reduced pressure. The residue was then purified by MPLC (eluent: A / B=95 / 5>5 / 95, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA). The eluent was evaporated, and the product was then freeze-dried to obtain compound 7 (2.9 g, 14.5 mmol, yield: 70%).

[0612] The obtained compound 1 The results of H-NMR analysis are as follows.

[0613] 1 H-NMR (400MHz, CD3CN) δ10.27(s,1H),8.22(d,J=1.8Hz,1H),7.94(dd,J=8.0,2.1Hz,1H),7.50(d,J=7.8Hz,1H),2.70(s,3H).

[0614] (2) Synthesis of Compound 8

[0615] [Chemical formula 49]

[0616]

[0617] In a dry flask, 1.77 g (4.34 mmol) of compound 3, 868 mg (4.34 mmol) of compound 7, 130 mg (2.17 mmol) of urea and 20 mL of acetic acid were added, and stirred at 90° C. for 17 hours under an argon atmosphere. Subsequently, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (silica; dichloromethane / methanol), and the eluent was evaporated to obtain 1220 mg of compound 8 (2.09 mmol, yield: 48%).

[0618] (3) Synthesis of Compound 9

[0619] [Chemical formula 50]

[0620]

[0621] In a dry flask, 350 mg (0.616 mmol) of compound 8 and 577 mg (3.70 mmol) of N, N-dimethylbarbituric acid were added, dissolved in 20 mL of dichloromethane, and then degassed with an aspirator. Subsequently, 28 mg (0.024 mmol) of tetrakis (triphenylphosphine) palladium (0) was added thereto, the solvent was degassed again with an aspirator, and then the mixture was stirred at 35 ° C. for 1 hour under an argon atmosphere. Subsequently, the solvent was distilled off under reduced pressure, and then the residue was purified by column chromatography (silica; dichloromethane / methanol), and the eluent was evaporated to obtain 60 mg of compound 9 (0.142 mmol, yield: 23%).

[0622] The obtained compound 1 H-NMR and 13 The results of C-NMR analysis are as follows.

[0623] 1 H-NMR (400MHz, CD3OD) δ7.60(d,J=1.8Hz,1H),7.49(dd,J=8.2,1.8Hz,1H),7.15(d,J=8.2Hz,1H),6.99(d,J=2.5 Hz, 2H), 6.79 (d, J = 8.5Hz, 2H), 6.59 (dd, J = 8.5, 2.5Hz, 2H), 5.59 (s, 1H), 2.25 (s, 3H), 0.58 (s, 3H), 0.36 (s, 3H).

[0624] 13 C-NMR (100MHz, CD3OD) δ148.0,145.3,143.1,139.2,138.7,134.6,131.9,130.8,129.1,124.0,120.4,118.4,51.1,20.3,-0.4,-0.8.

[0625] (4) Synthesis of Compound 10

[0626] [Chemical formula 51]

[0627]

[0628] In a dry flask, 211 mg (0.497 mmol) of compound 9, 246 mg (1 mmol) of tetrachlorobenzoquinone, 4 mL of DMF and 4 mL of dichloromethane were added, and the mixture was stirred at room temperature for 5 minutes. Subsequently, the reaction solution was purified by column chromatography (silica; dichloromethane / methanol), and the eluent was evaporated to obtain 168 mg of compound 10 (0.398 mmol, yield: 80%).

[0629] The obtained compound 1 H-NMR and 13 The results of C-NMR analysis are as follows.

[0630] 1 H-NMR (400MHz, CD3OD) δ7.88(dd,J=8.0,1.8Hz,1H),7.53(d,J=1.8Hz,1H),7.46(d,J=8.0Hz,1H),7.18(d ,J=2.5Hz,2H),6.97(d,J=9.4Hz,2H),6.53(dd,J=9.4,2.5Hz,2H),2.05(s,3H),0.50(s,3H),0.49(s,3H) 13 C-NMR (100MHz, CD3OD) δ168.8,157.2,149.0,142.6,142.3,138.7,138.1,130.1,126.9,126.1,123.3,115.7,109.5,18.0,-2.8,-3.1.

[0631] [Synthesis example 5]

[0632] Synthesis Example 3 of sSiR

[0633] [Chemical formula 52]

[0634]

[0635] (1) Synthesis of Compound 11

[0636] [Chemical formula 53]

[0637]

[0638] In a dry flask, 1620 mg (6 mmol) of 4-bromo-3-methylbenzenesulfonyl chloride, 732 mg (6 mmol) of 4-dimethylaminopyridine, 30 mL of 2-propanol and 12 mL of pyridine were added and stirred at room temperature for 2 hours. Subsequently, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (silicon dioxide; ethyl acetate / hexane), and the eluent was evaporated to obtain 1008 mg of compound 11 (3.44 mmol, yield: 57%).

[0639] The obtained compound 1 The results of H-NMR analysis are as follows.

[0640] 1 H-NMR (400MHz, CDCl3) δ7.75(d,J=1.8Hz,1H),7.69(d,J=8.7Hz,1H),7.56(dd, J=8.2, 2.3Hz, 1H), 4.71 (sep, J=5.9Hz, 1H), 2.47 (s, 3H), 1.28 (d, J=5.9Hz, 6H).

[0641] (2) Synthesis of Compound 12

[0642] [Chemical formula 54]

[0643]

[0644] In a dry flask, 667mg (2.27mmol) of compound 11 was added, dissolved in dehydrated tetrahydrofuran, and stirred at -78°C for 30 minutes under an argon atmosphere. Subsequently, 1.75mL (2.27mmol) of 1.3M sec-butyl lithium was added thereto in portions, and the mixture was stirred at -78°C for 30 minutes under an argon atmosphere. Subsequently, 200mg (0.45mmol) of N,N,N',N'-tetraallyldiamino-Si-xanthene dissolved in dehydrated tetrahydrofuran was further added to the reaction solution in portions, and the mixture was then stirred at room temperature for 1.5 hours under an argon atmosphere. 0.1N HCl aqueous solution and water were then added to the reaction solution, and the mixture was stirred at room temperature. Next, the mixture was extracted with dichloromethane, washed with saturated brine, and then dehydrated with sodium sulfate. Subsequently, the solvent was distilled off under reduced pressure, and the residue was purified by column chromatography (silicon dioxide; dichloromethane / methanol), and the solvent was evaporated. Subsequently, the obtained residue was dissolved in methanol and cooled to 0°C, and then 8.3 mg (0.22 mmol) of sodium borohydride was added in portions, and the mixture was stirred. Next, the mixture was extracted with dichloromethane, washed with saturated brine, and then dehydrated with sodium sulfate. After that, the solvent was distilled off under reduced pressure. 78 mg (0.5 mmol) of 1,3-dimethylbarbituric acid and 23 mg (0.02 mmol) of tetrakis (triphenylphosphine) palladium (0) were added to the obtained residue, and the mixture was dissolved in 10 mL of dichloromethane and then stirred at 35°C for 3 hours under an argon atmosphere. Next, the mixture was neutralized with a saturated sodium bicarbonate aqueous solution, then extracted with dichloromethane and washed with saturated brine. The mixture was then dehydrated with sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was purified by column chromatography (silica; ethyl acetate / hexane), and the eluent was evaporated to obtain 6 mg of compound 12 (0.013 mmol, yield: 2.9%).

[0645] The obtained compound 1 The results of H-NMR analysis are as follows.

[0646] 1 H-NMR (400MHz, CDCl3) δ7.66(d,J=1.8Hz,1H),7.58(dd,J=8.2,1.8Hz,1H),7.19(d,J=8.2Hz,1H),6.91(d,J=2.1Hz,2H),6.72(d,J=8.7H z, 2H), 6.56 (dd, J = 8.2, 2.7Hz, 2H), 5.62 (s, 1H), 4.75 (sep, J = 6.4Hz, 1H), 2.35 (s, 3H), 1.27 (d, J = 6.4Hz, 6H), 0.53 (s, 3H), 0.40 (s, 3H).

[0647] [Synthesis Example 6]

[0648] Synthesis of sSiR-based peptidase and protease probes

[0649] Using the compound obtained by reducing sSiR (Compound 9 or Compound 12), the sSiR-based peptidase and protease probes (PB201 to PB211) shown in Table 3 were synthesized by the following steps.

[0650] [Chemical formula 55]

[0651]

[0652] 1. Compound 9 or 12 (11.8 μmol), COMU (4.7 μmol) and Boc-protected amino acid or Fmoc-protected amino acid or N-terminally protected peptide (4.7 μmol) were mixed in a 1.5 mL test tube, and the mixture was dissolved in 60 μL DMF.

[0653] 2. The reaction mixture was stirred at room temperature.

[0654] 3. Chloranil (23.5 μmol) was added and the reaction mixture was stirred.

[0655] 4. The solution was purified by MPLC (eluent: A / B = 95 / 5 > 5 / 95, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), the eluent was evaporated, and the product was freeze-dried.

[0656] 5. When the raw material is protected by Fmoc, add 50 μL of piperidine and 50 μL of NMP to the obtained solid and stir the reaction mixture solution. When the raw material is not protected by Fmoc, this step is not performed and the process proceeds to step 6.

[0657] 6. Add 1000 μL TFA and stir the reaction mixture.

[0658] 7. The solution was purified by MPLC (eluent: A / B=95 / 5>5 / 95, A: 100% H2O containing 0.1% TFA, B: 100% CH3CN containing 0.1% TFA), the eluent was evaporated, and the product was freeze-dried to obtain the target compound.

[0659] [Table 3]

[0660] Table 3: sSiR-based peptidase and protease probes

[0661]

[0662] The instrumental data of the compounds are as follows.

[0663] HPLC conditions: A / B = 95 / 5 > 5 / 95 (5 min), A = 0.1% formic acid H2O, B = 0.1% formic acid CH3CN.

[0664] [Table 4]

[0665]

[0666]

[0667] [Synthesis Example 7]

[0668] [Chemical formula 56]

[0669]

[0670] [Chemical formula 57]

[0671]

[0672] The synthesis method was carried out with reference to Tetrahedron, 2005, 61, 12, 3097-3105.

[0673] 4-Sulfophthalic acid, resorcinol and methanesulfonic acid were mixed, heated and stirred at 130° C. Water was added to the reaction solution, and the precipitated solid was washed with water and dried under reduced pressure to obtain the target compound.

[0674] Synthesis of compound 14

[0675] [Chemical formula 58]

[0676]

[0677] Compound 13 was dissolved in dichloromethane and stirred under argon substitution. Pyridine was added to the reaction solution, and then trifluoromethanesulfonic anhydride was added to the reaction solution. The reaction solution was washed with 2N hydrochloric acid and then extracted with dichloromethane. The solution was dehydrated with sodium sulfate and then evaporated. The residue was purified by MPLC and the eluent was evaporated. Afterwards, the product was freeze-dried under reduced pressure to obtain the target compound.

[0678] Synthesis of compound 15

[0679] [Chemical formula 59]

[0680]

[0681] Compound 14 and triisopropyl orthoformate were dissolved in toluene and stirred to protect the sulfonic acid and triisopropyl. The reaction solution was evaporated, and then the residue, Pd(dba)3-CHCl3, Xantphos, benzophenone imine and cesium carbonate were dissolved in toluene and stirred. Afterwards, TFA was added and stirred for deprotection. After evaporation of the reaction solution, the residue was purified by MPLC, and the eluent was evaporated. Afterwards, the product was freeze-dried under reduced pressure to obtain the target compound.

[0682] Synthesis of compound 16

[0683] [Chemical formula 60]

[0684]

[0685] The synthesis of compound 16 was carried out with reference to J.Am.Chem.Soc.2013,135,16,6002-6005. Compound 15, Fmoc-protected amino acid, HATU and DIEA were dissolved in DMF and stirred to condense the amino acid. Then, piperidine was added and stirred to deprotect the Fmoc group, and then trifluoroacetic acid was added and stirred to deprotect the amino acid side chain. The reaction solution was purified by MPLC, and the eluent was evaporated. Then, the product was freeze-dried under reduced pressure to obtain the target compound.

[0686] [Synthesis example 8]

[0687] [Chemical formula 61]

[0688]

[0689] Wherein MOMCl represents chloromethyl methyl ether (Cl-CH3-O-CH3), and -MOM represents the residue of chloromethyl methyl ether (-CH3-O-CH3).

[0690] Synthesis of compound 17

[0691] [Chemical formula 62]

[0692]

[0693] The synthesis of compounds 17 to 21 was carried out with reference to Nat Commun. 2015, 6, 6463. Compound 13 was dissolved in DMF, and potassium carbonate was added to the reaction solution. Thereafter, chloromethyl methyl ether was added to the reaction solution and stirred. Ethyl acetate and saturated aqueous ammonium chloride solution were added to the reaction solution and stirred. Thereafter, water and 85% phosphoric acid were added to the reaction solution, and the pH of the reaction solution was adjusted to about 3. The ethyl acetate layer was extracted, then washed with saturated brine, and dehydrated with sodium sulfate. After evaporating the reaction solution, the residue was purified by column chromatography, and the eluent was evaporated. Thereafter, the product was dried under reduced pressure to obtain the target compound.

[0694] Synthesis of compound 18

[0695] [Chemical formula 63]

[0696]

[0697] Compound 17 was dissolved in THF, and LAH was then added to the reaction solution. Thereafter, methanol was added to the reaction solution to stop the reaction, and the reaction solution was evaporated. The residue was dissolved in dichloromethane and washed with a Rochelle salt solution. The organic layer was extracted, dehydrated with sodium sulfate, and then evaporated. The residue was dissolved in dichloromethane, tetrachlorobenzoquinone was added and stirred. The reaction solution was filtered, then washed with water, and dehydrated with sodium sulfate. The residue obtained by evaporating the solution was purified by column chromatography, and the eluent was evaporated. Thereafter, the product was dried under reduced pressure to obtain the target compound.

[0698] Synthesis of compound 19

[0699] [Chemical formula 64]

[0700]

[0701] Compound 18 was dissolved in dichloromethane, and pyridine was added to the reaction solution. Then, trifluoromethanesulfonic anhydride dissolved in dichloromethane was added to the reaction solution and stirred. The reaction solution was evaporated, and the obtained residue was purified by column chromatography, and the eluent was evaporated. Then, the product was dried under reduced pressure to obtain the target compound.

[0702] Synthesis of compound 20

[0703] [Chemical formula 65]

[0704]

[0705] Compound 19, Pd2(dba3)-CHCl3, Xantphos, and cesium carbonate were dissolved in toluene and stirred under argon displacement. Benzophenone imine was added and stirred, and then the reaction solution was dissolved in dichloromethane and filtered through Celite. After evaporating the filtrate, the resulting residue was purified by column chromatography and the eluate was evaporated. Thereafter, the product was dried under reduced pressure to obtain the target compound.

[0706] Synthesis of compound 21

[0707] [Chemical formula 66]

[0708]

[0709] Compound 20 was dissolved in dichloromethane, 10% TFA / water was added thereto, and the mixture was stirred. Thereafter, the reaction solution was made alkaline with 1M aqueous NaOH solution and then extracted with dichloromethane. The solution was dehydrated with sodium sulfate and then evaporated. The resulting residue was purified by column chromatography and the eluate was evaporated. Thereafter, the product was dried under reduced pressure to obtain the target compound.

[0710] Synthesis of compound 22

[0711] [Chemical formula 67]

[0712]

[0713] Compound 21, Fmoc-protected amino acid, HATU, and DIEA were dissolved in DMF and stirred to condense the amino acid. Thereafter, piperidine was added and stirred to deprotect the Fmoc group, and then trifluoroacetic acid was added and stirred to deprotect the amino acid side chain. The reaction solution was purified by MPLC and the eluate was evaporated. Thereafter, the product was lyophilized under reduced pressure to obtain the target compound.

[0714] <Measurement of LogP value>

[0715] The relationship between the lipophilicity index (LogP) and the compatibility with the microdevice was studied for the following compounds including the compounds (compounds 4, 5, and 10) containing the fluorescent mother nucleus obtained in the above synthesis examples. The results are shown in Table 5 below.

[0716] [Table 5]

[0717] Table 5: Relationship between the LogP value and the compatibility with the microdevice studied using compounds with a fluorescent mother nucleus

[0718] Applicability of fluorophores to microdevice-based assays

[0719]

[0720] The protocol for measuring logP will be described below.

[0721] (Protocol)

[0722] (1) Add 4 μL of a 10 mM stock solution (calculated based on e = 91,000) to 500 μL of MilliQ and 500 μL of 1-octanol, mix well, and then centrifuge.

[0723] (2) Collect 50 μL of the solution from each layer, mix with 50 μL of MeOH, and analyze the mixture by LC-MS.

[0724] (3) Calculate the concentration ratio based on the area ratio of each peak and calculate LogP.

[0725] (Peak area: unsubstituted product: @600 nM, disubstituted product: @629 nm, trisubstituted product: @638 nm, tetrasubstituted product: @655 nm)

[0726] In Table 5, the compounds are arranged in the order from left to right according to the measured values of the lipophilicity index logP value; however, in the case of compounds with higher lipophilicity (specifically, LogP > 1.01), adsorption to the microdevice results in a doughnut-shaped fluorescence image, indicating that the compound is not suitable for determination using the microdevice.

[0727] The leftmost compound has two sulfonic acids, but due to the substitution of four benzyl groups, its lipophilicity is very high, which also provides a doughnut-shaped fluorescence image, and when measured using a confocal microscope, leakage to the outside of the device is also observed, so its applicability to the microdevice is low.

[0728] The above results have been studied using compounds with a fluorescent parent nucleus, which are structural analogs that exhibit observable fluorescence (compounds having the structure after the fluorescent probe is metabolized by an enzyme), and it is considered that since the compounds of the present invention (fluorescent probes) are structurally similar, they exhibit the same tendency. The compounds of the present invention introduce partial structures such as amino acids and sugar partial structures into the amino group of the xanthene skeleton.

[0729] <Calculation of MiLogP value>

[0730] For the compounds with actual measured LogP values ​​(Table 5), the MiLogP (logP calculated by Molinspiration) values ​​were calculated using the following program: Molinspiration property engine v2022.08 (https: / / www.molinspiration.com / cgi-bin / properties) provided by Molinspiration Cheminformatics. The calculation results are shown in Table 6 below.

[0731] [Table 6]

[0732] Table 6: Relationship between MiLogP values ​​and microdevice compatibility of compounds using fluorescent cores

[0733]

[0734] As shown in Table 6, for the compounds of the present invention, the MiLogP values ​​are almost correlated with the actually measured LogP values. That is, it is considered that the compounds of the present invention can be specified not only by the LogP values ​​but also by the MiLogP values.

[0735] For the fluorescent probes recognized as suitable for enzyme activity assays in microdevices (Examples 1 to 16), MiLogP values ​​were calculated and the results are as follows.

[0736] Ala-sHMRG(PB101):-1.86

[0737] Leu-sHMRG(PB102):-0.55

[0738] Met-sHMRG(PB104):-1.41

[0739] Tyr-sHMRG(PB105):-0.88

[0740] Lys-sHMRG(PB110):-1.62

[0741] gGlu-sHMRG(PB106):-2.26

[0742] GP-sHMRG(PB109):-2.52

[0743] Ac-Ala-sHMRG(PB107):-1.59

[0744] Suc-AAPA-sHMRG(PB111):-3.08

[0745] Ala-sSiR(PB201):0.95

[0746] Trp-sSiR(PB207):2.56

[0747] Glu-sSiR(PB206):0.54

[0748] Arg-sSiR(PB209):0.13

[0749] Ac-GP-sSiR(PB208):0.63

[0750] Ac-LRGG-sSiR(PB203):0.23

[0751] EP-sSiR(PB211):0.28

[0752] Since fluorescent probes are generally recognized to be suitable for enzyme activity measurement in microdevices, it was demonstrated that the MiLogP value can be used as an indicator for determining whether the compound of the present invention is suitable for use as a fluorescent probe in microdevices.

[0753] <Measurement Example>

[0754] Activity measurements in human blood using sHMRG- and sSiR-based peptidase and protease probes

[0755] The following will show the use of sHMRG-based fluorescent probes (X is an oxygen atom) and sSiR-based fluorescent probes (X is Si(R a )(R b )) enzyme activity assay, but those skilled in the art will appreciate that compounds of the present invention in which X is an atom other than an oxygen atom and a silicon atom can also respond to enzyme reactions as fluorescent probes. For example, examples of fluorescent probes in which X is a carbon atom are described in WO 2018 / 151260 A, and examples of fluorescent probes in which X is a germanium atom are described in Communications Chemistry volume 2, Article number: 94 (2019).

[0756] [Example 1]

[0757] Enzyme activity assay using Ala-sHMRG (PB101)

[0758] The enzyme activity assay was performed using Ala-sHMRG having the following structure. The assay protocol is shown below.

[0759] [Chemical formula 68]

[0760]

[0761] Assay protocol

[0762] 1. Dilute the fluorescent probe Ala-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 0.1% BSA, 1 mM DTT, 1 mM Triton X-100,

[0763] 2. Next, the human plasma sample was diluted 500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Ala-sHMRG was 50 μM, and the final concentration of the human plasma sample was 1,000-fold diluted.

[0764] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0765] 4. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. An increase in fluorescence intensity was observed in the wells where the enzyme reacting with Ala-sHMRG was blocked. Figure 2 Fluorescence microscopy images are shown.

[0766] [Example 2]

[0767] Enzyme activity assay using Leu-sHMRG (PB102)

[0768] The enzyme activity assay was performed using Leu-sHMRG having the following structure. The assay protocol is shown below.

[0769] [Chemical formula 69]

[0770]

[0771] Assay protocol

[0772] 1. Dilute the fluorescent probe Leu-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM DTT, and 3 mM Triton X-100.

[0773] 2. Next, the human plasma sample was diluted 2,500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Leu-sHMRG was 50 μM, and the final concentration of the human plasma sample was 5,000-fold diluted.

[0774] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0775] 4. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Leu-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0776] [Example 3]

[0777] Enzyme activity assay using Met-sHMRG (PB104)

[0778] The enzyme activity assay was performed using Met-sHMRG having the following structure. The assay protocol is shown below.

[0779] [Chemical formula 70]

[0780]

[0781] Assay protocol

[0782] 5. Dilute the fluorescent probe Met-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0783] 6. Next, the human plasma sample was diluted 500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Met-sHMRG was 50 μM, and the final concentration of the human plasma sample was 1,000-fold diluted.

[0784] 7. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0785] 8. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Met-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0786] [Example 4]

[0787] Enzyme activity assay using Tyr-sHMRG (PB105)

[0788] Enzyme activity assay was performed using Tyr-sHMRG having the following structure. The assay protocol is shown below.

[0789] [Chemical formula 71]

[0790]

[0791] Assay protocol

[0792] 9. Dilute the fluorescent probe Tyr-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0793] 10. Next, dilute the human plasma sample 500-fold with the above assay buffer and mix it with the diluted probe solution in equal amounts. At this point, the final concentration of Tyr-sHMRG is 50 μM, and the final concentration of the human plasma sample is 1,000-fold diluted.

[0794] 11. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0795] 12. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. An increase in fluorescence intensity was observed in the wells where the enzyme reacting with Tyr-sHMRG was blocked. Figure 2 Fluorescence microscopy images are shown.

[0796] [Example 5]

[0797] Enzyme activity assay using Lys-sHMRG (PB110)

[0798] The enzyme activity assay was performed using Lys-sHMRG having the following structure. The assay protocol is shown below.

[0799] [Chemical formula 72]

[0800]

[0801] Assay protocol

[0802] 1. Dilute the fluorescent probe Lys-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0803] 2. Next, the human plasma sample was diluted 10,000-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Lys-sHMRG was 50 μM, and the final concentration of the human plasma sample was 20,000-fold diluted.

[0804] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0805] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Lys-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0806] [Example 6]

[0807] Enzyme activity assay using gGlu-sHMRG (PB106)

[0808] The enzyme activity assay was performed using gGlu-sHMRG having the following structure. The assay protocol is shown below.

[0809] [Chemical formula 73]

[0810]

[0811] Assay protocol

[0812] 1. Dilute the fluorescent probe gGlu-sHMRG for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0813] 2. Next, dilute the human plasma sample 500-fold with the above assay buffer and mix it with the diluted probe solution in equal amounts. At this point, the final concentration of gGlu-sHMRG is 50 μM, and the final concentration of the human plasma sample is 1,000-fold diluted.

[0814] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0815] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with gGlu-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0816] [Example 7]

[0817] Enzyme activity assay using GP-sHMRG (PB109)

[0818] The enzyme activity assay was performed using GP-sHMRG having the following structure. The assay protocol is shown below.

[0819] [Chemical formula 74]

[0820]

[0821] Assay protocol

[0822] 1. Dilute the fluorescent probe GP-sHMRG for detecting peptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0823] 2. Next, the human plasma sample was diluted 2,500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of GP-sHMRG was 50 μM, and the final concentration of the human plasma sample was 5,000-fold diluted.

[0824] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0825] 4. The microdevice with the blocking solution was incubated at 25°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with GP-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0826] [Example 8]

[0827] Enzyme activity assay using Ac-Ala-sHMRG (PB107)

[0828] The enzyme activity assay was performed using Ac-Ala-sHMRG having the following structure.

[0829] The assay protocol is shown below.

[0830] [Chemical formula 75]

[0831]

[0832] Assay protocol

[0833] 1. Dilute the fluorescent probe Ac-Ala-sHMRG for detecting peptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0834] 2. Next, the human plasma sample was diluted 250 times with the above assay buffer and mixed with the diluted probe solution in equal amounts. At this point, the final concentration of Ac-Ala-sHMRG was 50 μM, and the final concentration of the human plasma sample was 500 times diluted.

[0835] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0836] 4. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Ac-Ala-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0837] [Example 9]

[0838] Enzyme activity assay using Suc-AAPA-sHMRG (PB111)

[0839] The enzyme activity assay was performed using Suc-AAPA-sHMRG having the following structure. The assay protocol is shown below.

[0840] [Chemical formula 76]

[0841]

[0842] Assay protocol

[0843] 1. Dilute the fluorescent probe Suc-AAPA-sHMRG for detecting protease activity to 200 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0844] 2. Next, the human plasma sample was diluted 250 times with the above assay buffer and mixed with the diluted probe solution in equal amounts. At this point, the final concentration of Suc-AAPA-sHMRG was 100 μM, and the final concentration of the human plasma sample was 500 times diluted.

[0845] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0846] 4. The microdevice with blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Suc-AAPA-sHMRG was blocked, an increase in fluorescence intensity was observed. Figure 2Fluorescence microscopy images are shown.

[0847] [Example 10]

[0848] Enzyme activity assay using Ala-siR(PB201)

[0849] The enzyme activity assay was performed using Ala-sSiR having the following structure. The assay protocol is shown below.

[0850] [Chemical formula 77]

[0851]

[0852] Assay protocol

[0853] 1. Dilute the fluorescent probe Ala-siR for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0854] 2. Next, the human plasma sample was diluted 10,000-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Ala-siR was 50 μM and the final concentration of the human plasma sample was 20,000-fold diluted.

[0855] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0856] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Ala-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0857] [Example 11]

[0858] Enzyme activity assay using Trp-sSiR(PB207)

[0859] The enzyme activity assay was performed using Trp-sSiR having the following structure. The assay protocol is shown below.

[0860] [Chemical formula 78]

[0861]

[0862] Assay protocol

[0863] 1. Dilute the fluorescent probe Trp-sSiR for detecting aminopeptidase activity to 100 μM with an assay buffer containing 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0864] 2. Next, the human plasma sample was diluted 500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Trp-sSiR was 50 μM and the final concentration of the human plasma sample was 1,000-fold diluted.

[0865] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0866] 4. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Trp-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0867] [Example 12]

[0868] Enzyme activity assay using Glu-sSiR (PB206)

[0869] The enzyme activity assay was performed using Glu-sSiR having the following structure. The assay protocol is shown below.

[0870] [Chemical formula 79]

[0871]

[0872] Assay protocol

[0873] 1. Dilute the fluorescent probe Glu-sSiR for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0874] 2. Next, the human plasma sample was diluted 500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Glu-siR was 50 μM and the final concentration of the human plasma sample was 1,000-fold diluted.

[0875] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0876] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme that reacts with Glu-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0877] [Example 13]

[0878] Enzyme activity assay using Arg-sSiR(PB209)

[0879] Enzyme activity assay was performed using Arg-sSiR having the following structure. The assay protocol is shown below.

[0880] [Chemical formula 80]

[0881]

[0882] Assay protocol

[0883] 1. Dilute the fluorescent probe Arg-sSiR for detecting aminopeptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0884] 2. Next, dilute the human plasma sample 500-fold with the above assay buffer and mix it with the diluted probe solution in equal amounts. At this point, the final concentration of Arg-siR is 50 μM, and the final concentration of the human plasma sample is 1,000-fold diluted.

[0885] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0886] 4. The microdevice with the blocking solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Arg-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0887] [Example 14]

[0888] Enzyme activity assay using Ac-GP-sSiR(PB208)

[0889] The enzyme activity assay was performed using Ac-GP-sSiR having the following structure. The assay protocol is shown below.

[0890] [Chemical formula 81]

[0891]

[0892] Assay protocol

[0893] 1. Dilute the fluorescent probe Ac-GP-sSiR for detecting peptidase activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0894] 2. Next, the human plasma sample was diluted 10,000-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of Ac-GP-sSiR was 50 μM, and the final concentration of the human plasma sample was 20,000-fold diluted.

[0895] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0896] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Ac-GP-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0897] [Example 15]

[0898] Results of enzyme activity assay using Ac-LRGG-sSiR(PB203)

[0899] The enzyme activity assay was performed using Ac-LRGG-siSiR having the following structure. The assay protocol is shown below.

[0900] [Chemical formula 82]

[0901]

[0902] Assay protocol

[0903] 1. Dilute the fluorescent probe Ac-LRGG-siR for detecting protease activity to 100 μM with assay buffer. The composition of the assay buffer is 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0904] 2. Next, the human plasma sample was diluted 10,000-fold with the above assay buffer and mixed with the diluted probe solution in equal amounts. At this point, the final concentration of Ac-LRGG-sSiR was 50 μM, and the final concentration of the human plasma sample was 20,000-fold diluted.

[0905] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0906] 4. The microdevice with the blocking solution was incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme reacting with Ac-LRGG-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0907] [Example 16]

[0908] Results of enzyme activity assay using EP-siR (PB211)

[0909] The enzyme activity assay was performed using EP-sSiR having the following structure. The assay protocol is shown below.

[0910] [Chemical formula 83]

[0911]

[0912] Assay protocol

[0913] 1. Dilute the fluorescent probe EP-sSiR for detecting protease activity to 60 μM with an assay buffer containing 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 0.1 mM DTT, and 150 μM Triton X-100.

[0914] 2. Next, the human plasma sample was diluted 2,500-fold with the above assay buffer and mixed with an equal amount of the diluted probe solution. At this time, the final concentration of EP-siR was 30 μM and the final concentration of the human plasma sample was 5,000-fold diluted.

[0915] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0916] 4. The microdevice with the blocking solution was incubated at 25°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. In the wells where the enzyme that reacts with EP-siR was blocked, an increase in fluorescence intensity was observed. Figure 2 Fluorescence microscopy images are shown.

[0917] <Example of Detection of Pancreatic Cancer Biomarkers>

[0918] Next, an experiment for detecting pancreatic cancer biomarkers was conducted using the fluorescent probe of the present invention.

[0919] [Example 17]

[0920] Pancreatic disease (pancreatic cancer) diagnostic test using CD13 single molecule enzyme activity

[0921] To detect the single-molecule enzymatic activity of CD13, an experiment was performed as follows to verify the probe with Met bonding as the reaction site (in the general formula (I) or (II), the alanine residue was introduced as -CO-R0 or -CO-R 10 ) can be used to detect pancreatic cancer biomarkers.

[0922] 1. Ala-siR, a CD13 detection probe, was diluted to 100 μM with an assay buffer containing 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0923] 2. Next, the human plasma samples collected from 30 pancreatic cancer patients and 30 healthy subjects were diluted 10,000 times with the above assay buffer and mixed with the diluted Ala-siR solution in equal amounts. At this point, the final concentrations of the Ala-siR probe and human plasma samples were 50 μM and 20,000 times diluted, respectively.

[0924] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0925] 4. The microdevices in the blocking solution were incubated at 37°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. Figure 3 Fluorescence microscopy images are shown.

[0926] 5. As for the number of wells with luminescence of 2,500 AU or higher, i.e. the number of wells considered to be blocked by CD13, a significant increase in the number of wells observed in pancreatic cancer patients was observed compared with healthy subjects. Figure 3 shown.

[0927] As mentioned above, an example of increased activity was detected in the plasma of pancreatic cancer patients, demonstrating that it is possible to distinguish pancreatic cancer patients ( Figure 3 ).

[0928] Specifically, in this example, the CD13 enzyme activity was determined based on the proportion of wells with a luminescence of 2500 AU or more, and a ROC curve for the assay based on CD13 enzyme activity was drawn. When the number of signal wells was 3% minus the threshold, the sensitivity was 27% and the specificity was 100% ( Figure 3 Right). Pancreatic cancer can be identified by such a ROC curve.

[0929] [Example 18]

[0930] Diagnostic test for pancreatic diseases (pancreatic cancer) using DPP4 single-molecule enzyme activity

[0931] To detect the single-molecule enzyme activity of DPP4, an experiment was performed as follows to verify whether the Gly-Pro-bonded probe GP-sHMRG as a reaction site can be used to detect pancreatic cancer biomarkers.

[0932] 1. GP-sHMRG as a DPP4 detection probe was diluted to 100 μM with an assay buffer containing 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 1 mM DTT, and 3 mM Triton X-100.

[0933] 2. Next, human plasma samples collected from 30 pancreatic cancer patients and 30 healthy subjects were diluted 2,500 times with the above assay buffer and mixed with the diluted GP-sHMRG solution in equal amounts. At this point, the final concentrations of the GP-sHMRG probe and human plasma samples were 50 μM and 5,000-fold dilution, respectively.

[0934] 3. The mixed solution of the two is then added to the microdevice, followed by adding sealing oil to seal the solution in each well.

[0935] 4. The microdevices in the blocking solution were incubated at 25°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. Figure 4 Fluorescence microscopy images are shown.

[0936] 5. The histogram drawn based on the fluorescence intensity of each well detected approximates a curve including two normal distributions. When the wells with fluorescence intensity higher than the minimum value of the approximate curve are defined as the high-activity group and the wells with fluorescence intensity lower than the minimum value of the approximate curve are defined as the low-activity group, a significant increase in the ratio of the high-activity group to the sum of these groups was observed in pancreatic cancer patients compared with healthy subjects. The results are shown in Figure 4 shown.

[0937] According to the detection, in the plasma of pancreatic cancer patients, there are examples of changes in the single-molecule activity pattern of the probe bonded with Gly-Pro as the reaction point, which proves that pancreatic cancer patients can be distinguished ( Figure 4 ).

[0938] Specifically, in this example, the DPP4 enzyme activity was determined according to the high activity group ratio, and a ROC curve for determination based on the DPP4 enzyme activity was drawn. When the high activity group ratio was 55%, the threshold was subtracted, the sensitivity was 37%, and the specificity was 97% ( Figure 4Right). Pancreatic cancer can be identified by such a ROC curve.

[0939] <Evaluation of suitability for use in micro devices>

[0940] [Example 19]

[0941] Study on solubility in sealing oil

[0942] The solubility of two types of compounds Glu-siR (PB206 / Example 12) and GP-sHMRG (PB109 / Example 7) and the leftmost compound "Tetrabenzyl" in Table 5 in sealing oil was measured as follows. That is, 1 μL of a 10 mM DMSO solution of the test compound was added to 100 μL of sealing oil hexadecane, and the mixture was sonicated and suspended at room temperature (25°C) for 1 minute. After that, the solution was centrifuged for 30 seconds, 50 μL of the supernatant was collected and mixed with 100 μL of methanol to extract the compound dissolved in hexadecane. After that, the methanol solution of the extracted compound was analyzed by ultra-high performance liquid chromatography (UPLC) (eluent: A / B=95 / 5>5 / 95, A: 100% H2O containing 0.1% formic acid, B: 100% CH3CN containing 0.1% formic acid), and the concentration of the compound in the methanol solution was calculated based on the peak area. When calculating solution concentrations using UPLC peak areas, multiple concentrations of each compound were prepared and measured, and then a calibration curve was prepared ( Figure 5 ), and calculated according to the calibration curve. The peak area of ​​Glu-sSiR was calculated at 500nm, the peak area of ​​GP-sHMRG was calculated at 450nm, and the peak area of ​​the compound "tetrabenzyl" was calculated at 650nm.

[0943] Based on the calibration curve, the solubility was calculated from the analytical results of each compound as follows.

[0944] Glu-siR (PB206): After three tests, the peak area was 1637±524 (mean±standard deviation), so the solubility in hexadecane was calculated to be 0.30±0.096 μM.

[0945] GP-sHMRG (PB109): After three tests, the peak area was 740±74 (mean±standard deviation), so the solubility in hexadecane was calculated to be 0.27±0.026 μM.

[0946] Compound "Tetrabenzyl": After three tests, the peak area was 152015±5430 (mean±standard deviation), so the solubility in hexadecane was calculated to be 2.71±0.097 μM.

[0947] Since Glu-sSiR and GP-sHMRG were confirmed to be suitable for enzyme activity assay in microdevices in Examples 12 and 7, it was shown that solubility in sealing oil can be used as an indicator of suitability for use in microdevices.

[0948] [Example 20]

[0949] Study on the evaluation of adsorption degree on pore wall surface

[0950] For the compound "tetrabenzyl" on the left side of Table 5 and the two compounds "tetraallyl" and "2,4-SO3H" on the right side of Table 5, the degree of adsorption on the pore wall surface was measured as follows. That is, the compounds were diluted with the assay buffer so that the concentration of each compound was 10μM, and then each solution was added to the microdevice, followed by the addition of sealing oil to seal the solution in each well. After incubation at 25°C for 1 hour, images of the compound encapsulated wells were taken with a microscope. The composition of the assay buffer was 100mM HEPES-NaOH (pH 7.4), 1mM MgCl2, 1mM CaCl2, 250μM Triton X-100.

[0951] For the captured image, the fluorescence intensity of the area with a radius of 1.12 μm or less from the center point of the hole (central area) and the area with a radius greater than 1.12 μm and 2.08 μm or less from the center point of the hole (edge ​​area) were measured, and the ratio of the fluorescence intensity of the edge area to the fluorescence intensity of the central area was calculated based on the average value (edge / center) (tetrabenzyl: n=10,153, 2,4-SO3H: n=10,151, tetraallyl: n=10,078). As a result, in the compound "tetrabenzyl", the fluorescence intensity of the central region was 552±60 (mean±standard deviation), the fluorescence intensity of the edge portion was 581±71 (mean±standard deviation), and the ratio (edge / center) was 1.05, while in the compound "2,4-SO3H", the fluorescence intensity of the central region was 1,880±178 (mean±standard deviation), the fluorescence intensity of the edge portion was 1,087±106 (mean±standard deviation), and the ratio (edge / center) was 0.58, and in the compound "tetraallyl", the fluorescence intensity of the central region was 483±33 (mean±standard deviation), the fluorescence intensity of the edge portion was 300±23 (mean±standard deviation), and the ratio (edge / center) was 0.62 ( Figure 6 ).

[0952] As shown in Table 5, since a donut-shaped fluorescence image due to adsorption on the microdevice was observed in the compound “tetrabenzyl”, but such a fluorescence image was not observed in the compounds “tetraallyl” and “2,4-SO3H”, the results indicate that, among the fluorescence intensities of the test compounds observed in the microdevice, the ratio of the fluorescence intensity in the edge region to the fluorescence intensity in the center region can be used as an indicator of the applicability of the microdevice.

[0953] [Example 21]

[0954] Signal-to-Noise Ratio Studies

[0955] For two types of compounds Glu-sSiR (PB206 / Example 12) and GP-sHMRG (PB109 / Example 7), the signal-to-noise ratios of the enzymatic hydrolysates before and after the enzyme reaction were measured in a cuvette and a microdevice, respectively. In the following experiments, the composition of the assay buffer was 100 mM HEPES-NaOH (pH 7.4), 1 mM MgCl2, 1 mM CaCl2, 100 mM DTT, 150 mM Triton X-100.

[0956] (1) Measurement in a cuvette

[0957] The Glu-siR solution was diluted to 10 μM with the assay buffer and then added to a quartz cuvette. The fluorescence spectrum before the enzyme reaction was measured using a fluorimeter (excitation wavelength: 570 nm). After that, aminopeptidase A was added to a concentration of 170 ng / mL in the solution. The solution was incubated at 25°C for 11 hours and then the fluorescence spectrum was measured. The fluorescence spectra before and after the enzyme reaction were compared, and the ratio of the values ​​at the wavelength with the maximum fluorescence intensity was calculated as the signal-to-noise ratio of the enzymatic hydrolyzate. The ratio was 32 times (@616 nm) (see Figure 7 A).

[0958] GP-sHMRG was diluted to 10 μM with assay buffer and then added to a quartz cuvette. The fluorescence spectrum before the enzyme reaction was measured using a fluorimeter (excitation wavelength: 500 nm). After that, dipeptidyl peptidase was added to a concentration of 17 ng / mL in the solution, and the solution was incubated at 25°C for 9 hours, and then the fluorescence spectrum was measured. The fluorescence spectra before and after the enzyme reaction were compared, and the ratio of the values ​​at the wavelength with the maximum fluorescence intensity was calculated as the signal-to-noise ratio of the enzymatic hydrolyzate, which was 481 times (@527 nm) (see Figure 7 B).

[0959] (2) Measurement in micro devices

[0960] For Glu-siR, three types of solutions were prepared: (i) assay buffer only; (ii) 30 μM Glu-siR; and (iii) 30 μM Glu-siR and 0.2 ng / mL aminopeptidase A, and each solution was added to the microdevice, followed by the addition of sealing oil to seal the solution in each well. The microdevice with the sealed solution was incubated at 37°C for 4 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. When the difference between the average values ​​of the fluorescence intensities of the wells in (ii) and (i) was defined as noise, the difference between the average value of the fluorescence intensities of the wells encapsulated with the enzyme in (iii) and the average value of the fluorescence intensities of the wells in (i) was defined as signal, and the ratio of these values ​​was calculated as the signal-to-noise ratio of the enzymatic hydrolysate, the ratio was 8.3 times (see Figure 8 A).

[0961] For GP-sHMRG, three types of solutions were prepared: (iv) assay buffer only, (v) 30 μM GP-sHMRG, and (vi) 30 μM GP-sHMRG and 0.2 ng / mL dipeptidyl peptidase, and each solution was added to the microdevice, followed by the addition of sealing oil to seal the solution in each well. The microdevice with the sealed solution was incubated at 25°C for 2 hours, and then the fluorescence intensity of each well was measured using a fluorescence microscope. When the difference between the average values ​​of the fluorescence intensities of each well in (v) and (iv) was defined as noise, the difference between the average value of the fluorescence intensity of the wells encapsulated with the enzyme in (vi) and the average value of the fluorescence intensity of each well in (iv) was defined as signal, and the ratio of these values ​​was calculated as the signal-to-noise ratio of the enzymatic hydrolysate, the ratio was 287 times (see Figure 8 B).

[0962] (3) Discussion

[0963] When comparing the signal-to-noise ratios of the two assay systems, the ratio of the signal-to-noise ratio in the microdevice to the signal-to-noise ratio in the cuvette was 0.26 for Glu-siR and 0.59 for GP-sHMRG.

[0964] When the compound of the present invention is used as a fluorescent probe, the signal-to-noise ratio in the microdevice is not as good as that in the cuvette, but it is not significantly deteriorated. Since Glu-sSiR and GP-sHMRG are considered suitable for enzyme activity determination in a microdevice in Examples 12 and 7, it is shown that the ratio of the signal-to-noise ratio in the cuvette and the microdevice can be used as an indicator of applicability in a microdevice.

Claims

1. A fluorescent probe for detecting a hydrolase, the fluorescent probe comprising a compound having at least one water-soluble substituent selected from the group consisting of -CO2H, -PO3H2 and -SO3H and represented by the following general formula (II) or a salt thereof: [Chemical formula 1] wherein Ring A is an aromatic ring, which is phenyl or a 5- or 6-membered heteroaryl; Ring Q1 is a ring structure of the following formula (i) or (ii), [Chemical formula 2] Where R 2 , R 4 , R 6 and R0 is defined as follows; Ring Q2 is a ring structure of the following formula (iii), (iv), (v) or (vi), [Chemical formula 3] Where R 3 , R 5 , R 7 , R 8 , R 9 , R 10 and R 11 The following definitions are given; In a ring structure containing an X atom, the bond represented by 1 --- and the key represented by 2 --- each represents a single bond or a double bond, provided that both bonds cannot simultaneously represent a double bond; R is selected from the group consisting of -CO2H, -PO3H2, -SO3H, -L-CO2H, -L-PO3H2 and -L-SO3H, wherein L is a linker; n is an integer from 0 to 5; R 1 represents a monovalent substituent optionally present on the aromatic ring, wherein when two or more monovalent substituents are present, the substituents may be the same or different, Where R 1 With R 1 The carbon atoms on the bonded ring A, the carbon atoms on the ring structure containing the X atom to which the ring A is bonded, and a part of the ring A may form a 5- or 6-membered carbocyclic or heterocyclic ring; R 2 and R 3 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent; R 4 and R 5 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent; R 6 and R 7 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a halogen atom, a water-soluble substituent, or an alkyl group having 1 to 6 carbon atoms and having a water-soluble substituent; R 8 and R 9 Each is independently a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms; an alkyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent, the alkyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent; or an alkenyl group having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which has a water-soluble substituent, the alkenyl group may be substituted with an aryl group, and the aryl group may be substituted with a water-soluble substituent, Where R 8 and R 9 can form a 4- to 7-membered heterocyclic group together, the heterocyclic group may contain a nitrogen atom, R 8 and R 9 is connected to the nitrogen atom and may have a substituent, R 8 or R 9 , or R 8 and R 9 Both, with R 5 or R 7 Together, they can each form a 5- to 7-membered heterocyclic or heteroaryl group containing a nitrogen atom, R 8 or R 9 is connected to the nitrogen atom, and the heterocyclic group or heteroaryl group may have a substituent selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an alkenyl group having 2 to 6 carbon atoms, an alkynyl group having 2 to 6 carbon atoms, an arylalkyl group having 6 to 10 carbon atoms, and an alkyl-substituted alkenyl group having 6 to 10 carbon atoms, The water-soluble substituent may be bonded to these heterocyclic groups and heteroaryl groups directly or through a substituent; R 11 is a hydrogen atom, a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, a substituted or unsubstituted alkenyl group having 1 to 6 carbon atoms; an alkyl group having a water-soluble substituent and having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which may be substituted by an aryl group, and which may be substituted by a water-soluble substituent; or an alkenyl group having a water-soluble substituent and having 1 to 10 carbon atoms, preferably 1 to 6 carbon atoms, which may be substituted by an aryl group, and which may be substituted by a water-soluble substituent); X is selected from oxygen atoms, Si(R a )(R b )、C(R a )(R b )、P(=O)R c 、S(R d )2、Ge(R a )(R b ), tellurium atoms or bismuth atoms, Where R a and R b are each independently a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted aryl group, R c is a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms, or a substituted or unsubstituted phenyl group, R d are the same or different substituted or unsubstituted aryl groups, R a , R b , R c and R d The alkyl, aryl and phenyl groups may be substituted with water-soluble substituents; and R0 and R 10 Each is independently selected from a partial structure of an amino acid, a partial structure of a carboxylic acid, TO-, or a saturated or unsaturated 5- or 6-membered carbocyclic or heterocyclic group, The partial structure of the amino acid is similar to R0 or R 10 The bonded C=O together constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid, The partial structure of the carboxylic acid is similar to R0 or R 10 The bonded C=O together form part of the carboxylic acid, T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar.

2. The fluorescent probe according to claim 1, wherein Q2 represents a ring structure of formula (iii).

3. The fluorescent probe according to claim 1, wherein Q2 represents a ring structure of formula (iv), (v) or (vi). The fluorescent probe according to claim 1 , wherein n is 1 to 3.

5. The fluorescent probe according to claim 1 or 2, wherein R 2 To R 9 and R a , R b , R c and R d At least one of the following is a water-soluble substituent, an alkyl group having a water-soluble substituent and having 1 to 6 carbon atoms, an aryl group having a water-soluble substituent, and a phenyl group having a water-soluble substituent.

6. The fluorescent probe according to claim 1 or 2, wherein X is an oxygen atom or Si(R a )(R b ).

7. The fluorescent probe according to claim 4, wherein R is -SO3H or -L-SO3H.

8. The fluorescent probe according to claim 1 or 2, wherein the hydrolase is a peptide bond hydrolase selected from aminopeptidase, peptidase, protease or amidase. 9 . The fluorescent probe according to claim 1 , wherein the compound or a salt thereof has a MiLogP value of 2.56 or less. 10 . The fluorescent probe according to claim 1 or 2 , wherein the MiLogP value of the fluorescent core of the compound or the salt thereof is less than 2.

47. 11 . The fluorescent probe according to claim 1 or 2 , wherein the MiLogP value of the compound or the salt thereof is 2.56 or lower, and the MiLogP value of the fluorescent core of the compound or the salt thereof is less than 2.

47.

12. The fluorescent probe according to claim 1 or 9, which is a fluorescent probe for a microdevice.

13. The fluorescent probe according to claim 1 or 2, wherein the compound represented by general formula (II) or a salt thereof is a compound represented by any one of the following general formulas (Ia) to (Ie) or a salt thereof: [Chemical formula 4] Where R 4 To R 9 , R a , R b and R0 are as defined in the general formula (II).

14. The fluorescent probe according to claim 1 or 2, wherein the compound represented by general formula (II) or a salt thereof is a compound represented by the following general formula (If) or a salt thereof: [Chemical formula 5] Where R 4 To R 9 and R0 are as defined in the general formula (II).

15. The fluorescent probe according to claim 1 or 2, wherein the compound or its salt is a compound or its salt selected from the following compounds: [Chemical formula 6] 16. A method for diagnosing pancreatic cancer or a method for predicting the possibility that a test subject from which a biological sample is derived suffers from pancreatic cancer, the method comprising using the fluorescent probe according to claim 1 or 2 to detect the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (II).

17. A method for diagnosing pancreatic cancer or a method for predicting the possibility that a test subject from which a biological sample is derived suffers from pancreatic cancer, the method comprising using the fluorescent probe according to claim 1 or 2 to detect the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 In the general formula (II), -Pro-Xaa, Pro represents a proline residue, and Xaa represents an amino acid residue, such as glycine, serine or glutamic acid.

18. A method for diagnosing pancreatic cancer or a method for predicting the likelihood that a test subject from which a biological sample is derived has pancreatic cancer, the method comprising: Using the fluorescent probe according to claim 1 or 2, detecting the single-molecule enzyme activity of CD13 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 is an alanine, lysine, arginine or methionine residue in the general formula (II); and Using the fluorescent probe according to claim 1 or 2, detecting the single-molecule enzyme activity of DPP4 in a biological sample obtained from a subject by a microdevice, wherein -CO-R0 and optionally -CO-R 10 In the general formula (II), -Pro-Xaa, Pro represents a proline residue, and Xaa represents an amino acid residue, such as glycine, serine or glutamine.

19. The method of claim 16 or 17, wherein the biological sample is a biological sample of a pancreatic cancer patient, a patient suspected of having pancreatic cancer, or a healthy subject.

20. A compound represented by any one of the following formulae (1) to (5) or a salt thereof: [Chemical formula 7] in R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-, The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid, The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid. T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar.

21. A compound represented by the following formula (6) or a salt thereof: [Chemical formula 8] in R0 is selected from the partial structure of an amino acid, the partial structure of a carboxylic acid or TO-, The partial structure of the amino acid together with the C=O to which R0 is bonded constitutes an amino acid, an amino acid residue, a peptide or a part of an amino acid, The partial structure of the carboxylic acid together with the C=O to which R0 is bonded constitutes a part of the carboxylic acid. T is a sugar, a sugar portion, or a sugar with a self-cleavable linker, and The sugar partial structure of T, together with O to which T is bonded, constitutes a sugar or a part of a sugar.

22. Use of a compound represented by the following general formula (III) or a salt thereof for producing a compound or a salt thereof according to claim 1 or 2: [Chemical formula 9] Wherein ring A, ring Q2, X, n, R, R 1 , R 2 , R 4 and R 6 As defined in general formula (II), the bond Represents a single bond or a double bond.

23. Use of a compound represented by the following general formula (IV) or a salt thereof for producing a compound or a salt thereof according to claim 1 or 2: [Chemical formula 10] Among them, ring A, X, n, R and R 1 To R 9 As defined in general formula (II).

24. A compound represented by the following general formula (V) or a salt thereof: [Chemical formula 11] in Ring A, R, R 8 and R 9 As defined in general formula (II), n is 1 or 2, R 1 is a hydrogen atom, an unsubstituted alkyl group having 1 to 4 carbon atoms, or an alkyl group having 1 to 4 carbon atoms substituted with a hydroxyl group, R 2 To R 7 is a hydrogen atom, R 8 and R 9 are each independently a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 4 carbon atoms, and X is an oxygen atom or Si(R a )(R b ), R a and R b As defined in formula (II).

25. The compound or salt thereof according to claim 24, which is a compound or salt thereof selected from the following compounds: [Chemical formula 12] 26. Use of the compound according to claim 24 or 25 or a salt thereof for producing the compound according to claim 1 or 2 or a salt thereof.

27. A method for selecting a fluorescent probe, the method comprising: Prepare the fluorescent probe according to claim 1 or 2; as well as Select a fluorescent probe that meets the following conditions: (A) in a solubility measurement test in sealing oil (25°C), the solubility of the fluorescent probe and its hydrolysate in hexadecane is 0.3 μM or less; (B) in a fluorescence intensity measurement test in a microdevice, the ratio of the fluorescence intensity of the pore edge area to the fluorescence intensity of the pore center area of ​​the hydrolysate of the fluorescent probe is 0.6 or less, and (C) in a fluorescent probe enzymatic hydrolysis test in a microdevice and a cuvette, the ratio of the signal-to-noise ratio (first ratio) of the hydrolysate in the microdevice to the signal-to-noise ratio (second ratio) of the hydrolysate in the cuvette is 0.4 or greater.

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