Fluorescent probe for detecting ENPP activity

Abstract

[Problem] To provide a novel fluorescent probe. [Solution] The compound represented by general formula (I), or a salt thereof.

Description

[Technical field]

[0001] The present invention relates to a fluorescent probe for detecting ENPP and its use. Specifically, the present invention relates to a compound, a fluorescent probe for detecting ENPP, a microdevice, and a method for detecting the enzyme activity of ENPP in a biological sample. [Background technology]

[0002] Methods for detecting enzyme activity at the single-enzyme level using microdevices have been widely used to clarify individual biochemical parameters of enzymes and to use them as reporter proteins to detect the presence of specific proteins with high sensitivity. Furthermore, by applying a recently developed microdevice-compatible fluorescent probe for detecting phosphatase activity (Patent Document 1), it has been possible to separate and detect multiple phosphatases in biological samples and to find differences in activity patterns between healthy individuals and diseased patients, which is expected to lead to the establishment of a new method for diagnosing pathological conditions.

[0003] However, existing phosphatase activity detection fluorescent probes have an enzyme recognition site structure that is a phosphate monoester, and the enzyme species whose activity can be detected are limited, which means that their performance for the purpose of discovering new disease biomarkers is limited.

[0004] On the other hand, ENPP (ectonucleotide pyrophosphatase / phosphodiesterase family) is an enzyme that hydrolyzes substrates such as extracellular nucleotide triphosphates, and the existence of seven subtypes has been confirmed. Some of them, such as ENPP2, also known as autotaxin, have been reported to be associated with diseases, but the functions of many of them are unknown.

[0005] To date, there have been no reports of detecting activity in blood other than ENPP2, so it is hoped that if it is possible to detect minute amounts of ENPP activity that may be present in biological samples, it could lead to the discovery of a new biomarker. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2018 / 159810A1 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides a novel fluorescent probe and an intermediate thereof, and more specifically, an object of the present invention is to provide a fluorescent probe for detecting ENPP. [Means for solving the problem]

[0008] In order to solve the above problems, the inventors investigated the design of new fluorescent probes by introducing a structure that can serve as an enzyme substrate into the phosphate monoester site, which was the enzyme recognition site in conventional microdevice-compatible fluorescent probes. As a result, they found that these fluorescent probes are metabolized to ENPP, which uses nucleic acid as a substrate, resulting in an increase in fluorescence intensity, which led to the completion of the present invention.

[0009] That is, the present invention provides: [1] A compound represented by the following general formula (I) or a salt thereof: JPEG0007628233000001.jpg70140 (in the formula, R 1 represents 1 to 2 monovalent substituents present on a benzene ring, each of which is an electron-donating group, and R 1 When multiple are present, they may be the same or different; R 2represents 1 to 2 monovalent substituents present on a benzene ring, each of which has an anionic functional group at its terminal; R 2 When multiple are present, they may be the same or different; R 3 and R 4 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R 5 and R 6 each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms; R 7 and R 8 each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; X is a silicon atom, a phosphorus atom, a germanium atom, or a tin atom; Z is an oxygen atom or N + R 9 R 10 and Here, R 9 and R 10 each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R 9 and R 10 Let's get together and 9 and R 10 may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R 9 or R 10 Alternatively, R 9 and R 10 Both of these are R 3 or R 7 Together with R 9 or R 10is bonded to a nitrogen atom, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; Y is a single bond, -O-(CH2) n1 -, -O-(CH2) n2 -Ar1-, -NH-(CH2) n3 - or -NH-(CH2) n4 -Ar2-, Here, n1, n2, n3, and n4 each independently represent an integer from 1 to 10. Ar1 and Ar2 each independently represent a substituted or unsubstituted arylene group; JPEG0007628233000002.jpg23107 is It is represented by RS- where R is an organic base, S is a partial structure of a sugar or its derivative, or a single bond. [2] A compound represented by the following general formula (II) or a salt thereof: JPEG0007628233000003.jpg63134 (in the formula, R 1a represents 1 to 2 monovalent substituents present on a benzene ring, each of which is an electron-donating group, and R 1a When multiple are present, they may be the same or different; R 2a represents 1 to 2 monovalent substituents present on a benzene ring, each of which has an anionic functional group at its terminal; R 2a When multiple are present, they may be the same or different; R 3a and R 4a each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R 5aand R 6a each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms; R 7a and R 8a each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; Z a is an oxygen atom or N + R 9a R 10a and Here, R 9a and R 10a each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R 9a and R 10a Let's get together and 9a and R 10a may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which R 9a or R 10a Alternatively, R 9a and R 10a Both of these are R 3a or R 7a Together with R 9a or R 10a is bonded to a nitrogen atom, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; Y a is a single bond, -O-(CH2) n1 -, -O-(CH2) n2 -Ar1-, -NH-(CH2) n3 - or -NH-(CH2) n4 -Ar2-, Here, n1, n2, n3, and n4 each independently represent an integer from 1 to 10. Ar1 and Ar2 each independently represent a substituted or unsubstituted arylene group; JPEG0007628233000004.jpg1291 is It is represented by RS- where R is an organic base, S is a partial structure of a sugar or its derivative, or a single bond. [3] The compound or salt thereof according to [1] or [2], wherein the sugar or derivative thereof of S is ribose, deoxyribose, or a derivative thereof. [4] The organic base of R is a nucleic acid base or its derivative, (CH3)3N + The compound or salt thereof according to any one of [1] to [3], selected from the group consisting of -C2H4-, (CH3CH2)2N-, and R2N- (R is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and may be the same or different). [5] The compound or salt thereof according to [4], wherein the nucleic acid base is selected from the group consisting of adenine, thymine, cytosine, guanine and uracil. [6] The compound or salt thereof according to any one of [1] to [5], wherein the anionic functional group is any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphate group. [7]R 1 is an alkoxy group having 1 to 10 carbon atoms. [8] A fluorescent probe for detecting ENPP, comprising the compound according to any one of [1] to [7]. [9] A test kit for ENPP enzyme activity, comprising the compound according to any one of [1] to [7].

[10] A kit comprising the compound according to any one of [1] to [7] and a plate having microwells containing the compound.

[11] A method for measuring the enzyme activity of ENPP, comprising contacting the compound according to any one of [1] to [7] with ENPP.

[12] The method according to

[11] , wherein the contact is carried out in the presence of serum.

[13] The kit according to

[10] , comprising two or more types of fluorescent probes for detecting ENPP, each of which has different reaction sites in one well and has different fluorescent wavelengths.

[14] A method for detecting the enzyme activity of ENPP3, comprising contacting ENPP3 with a compound represented by the following (A) and / or a compound represented by the following (B). JPEG0007628233000005.jpg155159

[15] The method according to

[14] , wherein the contact is contact between both the compound (A) and the compound (B) and ENPP3. This provides: Effect of the Invention

[0010] According to the present invention, a fluorescent probe for detecting ENPP can be provided. ENPP is known to have seven subtypes, but the presence of activity in blood has not been reported except for ENPP2, known as autotaxin, and many of the associations with diseases remain unclear. The fluorescent probe of the present invention is capable of measuring the activity of ENPP in a biological sample (e.g., body fluid). The fluorescent probe of the present invention is advantageous in that it can be evaluated based on the activity of ENPP, not on the amount of protein, and therefore can take into account physiological functions. In addition, the fluorescent probe of the present invention is expected to newly detect minute amounts of ENPP activity that may be present in biological samples, leading to the discovery of a new biomarker. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 is a perspective view illustrating a microdevice according to an embodiment of the present invention. [Diagram 2] An example of a microscope image of a microdevice to which an enzyme and a fluorescent probe have been added is shown. [Figure 3A] FIG. 1 shows a schematic diagram of a method for separating and detecting ENPP in a biological sample at the single molecule level by using the kit of the present invention. [Figure 3B]FIG. 1 shows a schematic diagram of a method for separating and detecting ENPP in a biological sample at the single molecule level by using the kit of the present invention. [Figure 4] 1 shows the results of in vitro fluorescence assay of compounds (4)-(8). [Figure 5A] 1 shows the results of measuring a human plasma sample using an ENPP probe on a microdevice. [Figure 5B] Fluorescence microscope images of a combination of compounds (4) and (8) are shown. [Figure 6A] The results of measuring pancreatic cancer patient samples using an ENPP probe on a microdevice are shown. [Figure 6B] The results of measuring pancreatic cancer patient samples using an ENPP probe on a microdevice are shown. [Figure 7A] The results of measuring purified ENPP enzyme using an ENPP probe in a microdevice are shown. [Figure 7B] The results of measuring purified ENPP enzyme using an ENPP probe in a microdevice are shown. [Figure 8] The results of measurement of plasma samples using a plate reader are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In this specification, the term "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0013] In the present specification, "alkyl" may be any of linear, branched, and cyclic aliphatic hydrocarbon groups, or a combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but for example, the alkyl group may have 1 to 6 carbon atoms (C 1~6 ), 1 to 10 carbon atoms (C 1~10 ), 1 to 15 carbon atoms (C 1~15 ), carbon number 1~20 (C 1~20 When the number of carbon atoms is specified, it means "alkyl" having the number of carbon atoms in that range. For example, C 1~8Alkyl includes methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. In this specification, an alkyl group may have one or more optional substituents. Examples of such substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl. When an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl portion of other substituents (e.g., an alkoxy group, an arylalkyl group, and the like) that contain an alkyl portion.

[0014] In the present specification, when a functional group is defined as "optionally substituted", the type of the substituent, the substitution position, and the number of the substituent are not particularly limited, and when it has two or more substituents, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, and an oxo group. These substituents may further have a substituent. Examples of such a substituent include, but are not limited to, a halogenated alkyl group and a dialkylamino group.

[0015] In the present specification, "aryl" may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, or may be an aromatic heterocycle containing one or more heteroatoms (e.g., oxygen, nitrogen, or sulfur atoms) as ring constituent atoms. In this case, it may be called "heteroaryl" or "heteroaromatic". In either case of a monocyclic or condensed ring, aryl may be bonded at any possible position. Non-limiting examples of monocyclic aryl include phenyl group (Ph), thienyl group (2- or 3-thienyl group), pyridyl group, furyl group, thiazolyl group, oxazolyl group, pyrazolyl group, 2-pyrazinyl group, pyrimidinyl group, pyrrolyl group, imidazolyl group, pyridazinyl group, 3-isothiazolyl group, 3-isoxazolyl group, 1,2,4-oxadiazol-5-yl group, or 1,2,4-oxadiazol-3-yl group. Non-limiting examples of fused polycyclic aryls include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3-dihydroinden-2-yl, 2-anthryl, 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-dihydrobenzothiophen-1-yl, 2,3-dihydrobenzothiophen-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, or thioxanthenyl. In this specification, an aryl group may have one or more optional substituents on its ring. Examples of the substituents include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono- or di-substituted amino group, a substituted silyl group, or an acyl group. When an aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents (such as an aryloxy group or an arylalkyl group) that contain an aryl moiety.

[0016] In the present specification, the term "alkoxy group" refers to a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include saturated alkoxy groups that are linear, branched, cyclic, or a combination thereof. Suitable examples include methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, s-butoxy, t-butoxy, cyclobutoxy, cyclopropylmethoxy, n-pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy, and cyclopentylmethyloxy.

[0017] In the present specification, "alkylene" refers to a divalent group consisting of a linear 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, 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, 2,2-di-n-propyltrimethylene, and the like.

[0018] 1. A compound represented by general formula (I) or a salt thereof One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof: JPEG0007628233000006.jpg70140

[0019] In the general formula (I), R1 represents one or two monovalent substituents present on a benzene ring and is an electron-donating group. R 1 When multiple are present, they may be the same or different.

[0020] In the present specification, the "electron-donating group" may be any substituent that can donate electrons to a benzene ring. Specific examples include, but are not limited to, a hydroxyl group, an alkoxy group having 1 to 10 carbon atoms, an amino group, an alkylamino group having 1 to 10 carbon atoms, and the like. R 1 The number of R is 1 or 2, and preferably 2. 1 When there are two R, they may be the same or different. 1 When there are two of them, they are preferably the same for ease of synthesis.

[0021] R 1 The alkoxy group having 1 to 10 carbon atoms in the above formula may have a structure in which a linear or branched alkyl group having 1 to 10 carbon atoms is bonded to an oxygen atom. Specific examples of the alkoxy group having 1 to 10 carbon atoms include a methoxy group, an ethoxy group, an n-propoxy group, an isopropoxy group, an n-butoxy group, an isobutoxy group, a sec-butoxy group, a tert-butoxy group, an n-pentoxy group, an isopentoxy group, a neopentoxy group, a tert-pentoxy group, a 1-methylbutoxy group, an n-hexoxy group, a 2-methylpentoxy group, a 3-methylpentoxy group, a 2,2-dimethylbutoxy group, ... a sec-butoxy group, a tert-butoxy group, an n-pentoxy group, an isopentoxy group, a tert-pentoxy group, a 1-methylbutoxy group, an n-hexoxy group, a 2-methylpentoxy group, a 3-methylpentoxy group, a 2,2-dimethylbutoxy group, a methoxy group, an isopentoxy group, a 2,2-dimethylbutoxy group, a methoxy group, an isopentoxy group, a 2,2-dimethylbutoxy group, a methoxy group, an isopentoxy group, a 2,2-dimethylbutoxy group, a methoxy group, group, 2,3-dimethylbutoxy group, n-heptoxy group, 2-methylhexoxy group, 3-methylhexoxy group, 2,2-dimethylpentoxy group, 2,3-dimethylpentoxy group, 2,4-dimethylpentoxy group, 3,3-dimethylpentoxy group, 3-ethylpentoxy group, 2,2,3-trimethylbutoxy group, n-octoxy group, isooctoxy group, 2-ethylhexoxy group, nonynoxy group, and decyloxy group. 1 The alkoxy group having 1 to 10 carbon atoms in the formula (I) is preferably a straight-chain alkoxy group, and more preferably a methoxy group or an ethoxy group.

[0022] R 1 The alkylamino group having 1 to 10 carbon atoms in the formula (I) may have a structure in which a linear or branched alkyl group having 1 to 10 carbon atoms is bonded to an amino group. Specific examples of the alkylamino group having 1 to 10 carbon atoms include a methylamino group, an ethylamino group, an isopropylamino group, a dimethylamino group, a diethylamino group, and a diisopropylamino group. Among these, R 1 The alkylamino group having 1 to 10 carbon atoms in the formula (I) is preferably a straight-chain group, and more preferably a methylamino group or an ethylamino group. Among them, in the general formula (2), R 1 Since these have high hydrophilicity, a linear alkoxy group having 1 to 10 carbon atoms or a linear alkylamino group having 1 to 10 carbon atoms is preferred, and a methylamino group, an ethylamino group, a methoxy group, or an ethoxy group is more preferred.

[0023] In addition, in the general formula (1), R 1 It is preferable that there are two R in the benzene ring. 1 The positions of are preferably ortho to each other.

[0024] In the general formula (I), R 2 represents one or two monovalent substituents present on a benzene ring and is a group having an anionic functional group at its terminal. R 2 When multiple are present, they may be the same or different.

[0025] Among these, the "group having an anionic functional group at its terminal" in general formula (I) is preferably a group consisting of only anionic functional groups, since it is easy to synthesize. The anionic functional group is preferably any one selected from the group consisting of a carboxy group, a sulfonic acid group, and a phosphate group, and is particularly preferably a sulfonic acid group. R 2 The number of R is 1 or 2, and preferably 1. 2When there are two R, they may be the same or different. 2 When there are two of them, they are preferably the same for ease of synthesis.

[0026] Among them, the maximum absorption wavelengths of the compound of general formula (I) (non-dissociated type (neutral type)) and the compound after removal of the moiety containing the phosphate group (dissociated type (anionic type)) are significantly different from each other. 1 2 pieces, R 2 In a benzene ring having one R 1 4th place, another R 1 When ranked 6th, R 2 is preferably located at the 3-position or 5-position, and more preferably at the 3-position.

[0027] In a preferred aspect of the compounds of general formula (I), R 1 It has two methoxy groups as R 2 It is preferable that the alkyl group has one sulfonic acid group.

[0028] In a preferred aspect of the compound of formula (I), R 1 It has two methoxy groups at the 4th and 6th positions, and R 2 It is preferable that the alkyl group has a sulfonic acid group at the 3-position.

[0029] In the general formula (I), R 3 and R 4 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. R 3 or R 4 When R represents an alkyl group, the alkyl group may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R 3 or R 4 The alkyl group represented by R may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. 3 and R 4are preferably each independently a hydrogen atom or a halogen atom, and R 3 and R 4 are both hydrogen atoms, or R 3 and R 4 It is more preferable that both of are fluorine atoms or chlorine atoms.

[0030] In the general formula (I), R 5 and R 6 are each independently an alkyl group or an aryl group having 1 to 6 carbon atoms. R 5 and R 6 are each preferably independently an alkyl group having 1 to 3 carbon atoms, and R 5 and R 6 More preferably, both R are methyl groups. 5 and R 6 The alkyl group represented by may contain one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc., and for example, R 5 or R 6 The alkyl group represented by the formula (I) may be a halogenated alkyl group, a hydroxyalkyl group, a carboxyalkyl group, or the like. R 5 or R 6 When is an aryl group, the aryl group may be either a monocyclic aromatic group or a condensed aromatic group, and the aryl ring may contain one or more ring-constituting heteroatoms (e.g., nitrogen atom, oxygen atom, sulfur atom, etc.). The aryl group is preferably a phenyl group. One or more substituents may be present on the aryl ring. The substituents may be, for example, one or more halogen atoms, carboxy groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc.

[0031] In the general formula (I), R 7 and R 8 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R 3 and R 4 The same as that explained for R.7 and R 8 It is preferred that both are hydrogen atoms, both are chlorine atoms, or both are fluorine atoms.

[0032] In general formula (I), X is a silicon atom, a phosphorus atom, a germanium atom or a tin atom, and is preferably a silicon atom.

[0033] In the general formula (I), Z is an oxygen atom or N + R 9 R 10 is

[0034] Z is N + R 9 R 10 If R 9 and R 10 are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms is preferably a straight chain group, and more preferably a methyl group or an ethyl group.

[0035] Also, R 9 and R 10 Let's get together and 9 and R 10 may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which it is bonded. Also, R 9 or R 10 Alternatively, R 9 and R 10 Both of these are R 4 or R 8 Together with R 9 or R 10 may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded. The ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms. Examples of the heterocyclyl or heteroaryl thus formed include, but are not limited to, pyrrolidine, piperidine, hexamethyleneimine, pyrrole, imidazole, pyrazole, oxazole, thiazole, and the like.

[0036] When Z is an oxygen atom, Y is a single bond, -O-(CH2) n1 - or -O-(CH2) n2 -Ar1- is preferred, Z is N + R 9 R 10 When Y is a single bond, -NH-(CH2) n3 - or -NH-(CH2) n4 It is preferred that it is -Ar2-.

[0037] In the general formula (I), Y is a single bond, -O-(CH2) n1 -, -O-(CH2) n2 -Ar1-, -NH-(CH2) n3 - or -NH-(CH2) n4 -Ar2-. In Y, the bond opposite to the alkylene group of -O- or -NH- is bonded to a carbon atom constituting the hetero three-membered ring in the above general formula (I). n1 -, -Ar1-, -(CH2) n3 The bond opposite to the oxygen atom (O), amino group (NH) or alkylene group of - or -Ar2- is bonded to the phosphate group in the above general formula (I). n1, n2, n3, and n4 each independently represent an integer of 1 to 10. Ar1 and Ar2 each independently represent a substituted or unsubstituted arylene group.

[0038] n1, n2, n3, and n4 are each the number of repeats of the alkylene group in Y. n1, n2, n3, and n4 are preferably integers of 1 to 8, more preferably integers of 1 to 6, further preferably integers of 1 to 4, and particularly preferably integers of 1 to 2, because they have high hydrophilicity.

[0039] The unsubstituted arylene group in Ar1 and Ar2 is preferably one having 6 to 14 carbon atoms, and specific examples thereof include a phenylene group, a naphthylene group, etc. Among them, the unsubstituted arylene group in Ar1 and Ar2 is preferably a phenylene group. Examples of the substituent on the arylene group include a halogen atom and an alkyl group having 1 to 10 carbon atoms. The halogen atom is preferably a chlorine atom, a bromine atom or an iodine atom. The alkyl group having 1 to 10 carbon atoms is preferably a straight-chain group, and more preferably a methyl group or an ethyl group.

[0040] In the general formula (I), JPEG0007628233000007.jpg20102 is It is a partial structure of a nucleoside, which is composed of an organic base and a sugar bonded by a glycosidic bond, and is represented as RS-. Here, R is an organic base and S represents the sugar portion of the nucleoside.

[0041] ENPP is an enzyme that hydrolyzes substrates such as nucleotide triphosphates, but its mechanism of action differs from that of alkaline phosphatase, which cleaves the phosphate structure itself, in that ENPP recognizes the structure of nucleic acids and cleaves the phosphate bond. Focusing on this point, the present invention introduces a new structure that can become an enzyme substrate to the phosphate monoester site, which was the enzyme recognition site in conventional microdevice-compatible fluorescent probes. As a result, the fluorescent probe of the present invention is metabolized into ENPP, which uses nucleic acid as a substrate, and this can lead to an increase in fluorescence intensity.

[0042] In addition, in the general formula (I), JPEG0007628233000008.jpg20107 is It may also be an organic base that is an amine or an ammonium salt, in which case R in RS- is an organic base that is an amine or an ammonium salt, and S represents a single bond (i.e., R is directly bonded to the phosphate ester).

[0043] Here, the organic base of R is preferably a nucleic acid base or a derivative thereof; + The aryl group is selected from the group consisting of an amino group or an ammonium group represented by —C2H4—, a diethylamino group ((CH3CH2)2N—), and an amino group (R2N—). R in RN- is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and the two R may be the same or different. Preferably, one or both of R are a hydrogen atom or a methyl group, and more preferably, both of R are a hydrogen atom or a methyl group.

[0044] Here, when the organic base of R is the above-mentioned amino group or ammonium group, S is a single bond. In the fluorescent probe of the present invention having such an organic base introduced therein, as shown in JPEG0007628233000009.jpg19101, it is possible for the probe to be recognized by ENPP as having a structure similar to that of nucleic acid, leading to the cleavage of the phosphate bond and an increase in fluorescence intensity.

[0045] The nucleic acid base is preferably selected from the group consisting of adenine, thymine, cytosine, guanine and uracil.

[0046] S represents the sugar portion of a nucleoside and is a partial structure of a sugar or a derivative thereof. The sugar or derivative thereof represented by S is preferably ribose, deoxyribose, or a derivative thereof. When the organic base of R is the above amino group or ammonium group, S is a single bond.

[0047] Ribose, deoxyribose, or a derivative thereof is linked to an organic base at the 1' position and to a phosphate at the 5' position.

[0048] In one preferred aspect of the invention, RS- is selected from: JPEG0007628233000010.jpg50159(CH3)3N + -C2H4- (CH3CH2)2N- R2N- (R is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and may be the same or different).

[0049] 2. Compound of general formula (II) or its derivative Another embodiment of the present invention is a compound represented by the following general formula (II) or a salt thereof: JPEG0007628233000011.jpg66140

[0050] In the general formula (II), R 1a represents 1 to 2 monovalent substituents present on a benzene ring, each of which is an electron-donating group, and R 1a When multiple are present, they may be the same or different. R 1a The details of R in general formula (I) are as follows: 1 is the same as:

[0051] In the general formula (II), R 2a represents 1 to 2 monovalent substituents present on a benzene ring, each of which has an anionic functional group at its terminal; R 2a When multiple are present, they may be the same or different. R 2a The details of R in general formula (I) are as follows: 2 is the same as:

[0052] In the general formula (II), R 3a and R 4a are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. R 3a and R 4a The details of R in general formula (I) are as follows: 3 and R 4 is the same as:

[0053] In the general formula (II), R 7a and R 8a are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom. R 7a and R 8a The details of R in general formula (I) are as follows: 7 and R 8 is the same as:

[0054] In the general formula (II), Z a is an oxygen atom or N + R 9a R 10a It is. Here, R 9a and R 10a are each independently a hydrogen atom or an alkyl group having 1 to 10 carbon atoms. The alkyl group having 1 to 10 carbon atoms is preferably a straight chain group, and more preferably a methyl group or an ethyl group.

[0055] Also, R 9a and R 10a Let's get together and 9a and R 10a may form a 4- to 7-membered heterocyclyl containing the nitrogen atom to which it is bonded. Also, R 9a or R 10a Alternatively, R 9a and R 10a Both of these are R 4a or R 8a Together with R 9a or R 10a may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded. The ring may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms. Examples of the heterocyclyl or heteroaryl thus formed include, but are not limited to, pyrrolidine, piperidine, hexamethyleneimine, pyrrole, imidazole, pyrazole, oxazole, thiazole, and the like.

[0056] In the general formula (II), Y a is a single bond, -O-(CH2) n1 -, -O-(CH2) n2 -Ar1-, -NH-(CH2) n3 - or -NH-(CH2) n4 -Ar2-, where n1, n2, n3 and n4 each independently represent an integer of 1 to 10. Ar1 and Ar2 each independently represent a substituted or unsubstituted arylene group. Details of n1, n2, n3 and n4, Ar1 and Ar2 are the same as those of n1, n2, n3 and n4, Ar1 and Ar2 in general formula (I).

[0057] In the general formula (II), JPEG0007628233000012.jpg20106 is It is represented by RS- where R is an organic base, S is a partial structure of a sugar or a derivative thereof, or a single bond. The details of R and S are the same as those explained in general formula (I).

[0058] The compound represented by general formula (I) or (II) may have one or more asymmetric carbons depending on the type of the substituent, and may have stereoisomers such as optical isomers or diastereoisomers. Pure stereoisomers, any mixture of stereoisomers, racemates, etc. are all included in the scope of the present invention. In addition, the compound represented by general formula (I) or (II) or its salt may exist as a hydrate or solvate, and all of these substances are included in the scope of the present invention. The type of solvent that forms the solvate is not particularly limited, and examples thereof include solvents such as ethanol, acetone, and isopropanol.

[0059] The methods for producing representative compounds of the present invention are specifically shown in the Examples of this specification. Therefore, those skilled in the art can produce the compounds of the present invention by appropriately selecting reaction raw materials, reaction conditions, reaction reagents, etc. based on these explanations, and modifying or altering these methods as necessary.

[0060] 3. Fluorescent probe for detecting ENPP One embodiment of the present invention is a fluorescent probe for detecting ENPP, comprising a compound represented by general formula (I) or a salt thereof.

[0061] Another embodiment of the present invention is a fluorescent probe for detecting ENPP, which contains a compound represented by general formula (II) or a salt thereof.

[0062] Hereinafter, the compound represented by general formula (I) or a salt thereof and the compound represented by general formula (II) or a salt thereof will be collectively referred to as the compound of the present invention or a salt thereof.

[0063] Another aspect of the present invention is a method for detecting ENPP activity in a cell, comprising the steps of (a) introducing a compound of the present invention or a salt thereof into a cell, and (b) measuring the fluorescence emitted by the compound or a salt thereof reacting with ENPP in the cell. The compound of the present invention or a salt thereof has the characteristic of being substantially non-fluorescent or only weakly fluorescent in an environment in the absence of aldehyde hydrogenase 1A1, but emitting strong fluorescence in an environment in the presence of ENPP.

[0064] The method of the present invention may further include observing the fluorescence response using a fluorescence imaging means. The means for observing the fluorescence response may be a fluorometer having a wide measurement wavelength, but the fluorescence response may also be visualized using a fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image. By using the fluorescence imaging means, the fluorescence response can be visualized two-dimensionally, so that the ENPP can be instantly visually recognized. As the fluorescence imaging device, a device known in the technical field may be used. In some cases, it is also possible to detect the reaction between the measurement sample and the fluorescent probe by a change in the ultraviolet-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).

[0065] The method of using the fluorescent probe of the present invention is not particularly limited, and it can be used in the same manner as conventionally known fluorescent probes. Usually, the compound of the present invention or a salt thereof is dissolved in an aqueous medium such as physiological saline or a buffer solution, or a mixture of a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide with an aqueous medium, and the solution is added to an appropriate buffer solution containing cells or tissues to measure the fluorescence spectrum. The fluorescent probe of the present invention may be used in the form of a composition in combination with an appropriate additive. For example, it can be combined with additives such as a buffer, a solubilizing agent, or a pH adjusting agent.

[0066] The cell sample to be measured in the above step (a) may be cells expressing ENPP, but when such cells are cancer cells or cancer tissues expressing ENPP, the detection method of the present invention can detect or visualize the cancer cells or cancer tissues. That is, the fluorescent probe of the present invention, the composition containing the fluorescent probe, and the detection method of the present invention can also be used for predicting or diagnosing cancer.

[0067] In this specification, the term "cancer tissue" means any tissue containing cancer cells. The term "tissue" should be interpreted in the broadest sense, including a part or the whole of an organ, and should not be interpreted in any restrictive sense. As the cancer tissue, tissue that highly expresses ENPP is preferable. In addition, in this specification, the term "diagnosis" should be interpreted in the broadest sense, including confirming the presence of cancer tissue in any living body site with the naked eye or under a microscope.

[0068] In the detection method of the present invention, it is preferable to use an ENPP detection kit containing the above-mentioned fluorescent probe. In the kit, the fluorescent probe of the present invention is usually prepared as a solution, but it can also be provided as a composition in an appropriate form such as a powder mixture, a lyophilized product, a granule, a tablet, or a liquid, and can be applied by dissolving it in distilled water for injection or an appropriate buffer solution when used.

[0069] The kit may also contain other reagents, etc., as necessary. For example, additives such as solubilizing agents, pH regulators, buffers, and isotonicity agents can be used, and the amounts of these additives can be appropriately selected by those skilled in the art.

[0070] By providing the ENPP-detecting fluorescent probe of the present invention in the well of a microdevice described below, it can be used in a method for detecting the enzyme activity of ENPP in a biological sample. That is, one preferred embodiment of the present invention is a fluorescent probe for detecting ENPP, which contains the compound of the present invention and is used in a microdevice.

[0071] Another preferred embodiment of the present invention is a fluorescent probe for detecting ENPP, which contains the compound of the present invention and is used in an enzyme activity detection method using a microdevice.

[0072] 4. Microdevices A microdevice according to one embodiment of the present invention includes the above-mentioned fluorescent probe for detecting ENPP.

[0073] According to the microdevice of this embodiment, the enzyme activity of ENPP in a biological sample can be detected with high quantitative accuracy and sensitivity.

[0074] The material of the microdevice is not particularly limited, and examples thereof include glass materials, silicon, plastics including dendritic polymers or copolymers, etc. Examples of glass materials include soda lime glass, Pyrex glass, Vycor glass, quartz glass, etc. Examples of resin polymers include poly(vinyl chloride), poly(vinyl alcohol), poly(methyl methacrylate), poly(vinyl acetate-co-maleic anhydride), poly(dimethylsiloxane) monomethacrylate, cyclic olefin polymers, fluorocarbon polymers, polystyrene, polypropylene, polyethyleneimine, etc. Examples of copolymers include poly(vinyl acetate-co-maleic anhydride), poly(styrene-co-maleic anhydride), poly(ethylene-co-acrylic acid), or derivatives thereof, etc. The shape of the microdevice may be, for example, a multiwell plate in which any number of wells (e.g., microwells) are arranged, as shown in Fig. 1. The number of wells per plate may be, for example, from 1 to 10 million, for example, from 10 to 500,000, for example, about 100,000.

[0075] The pore size of the wells of the microdevice of this embodiment may be, for example, 10 nm or more and 10 μm or less, for example, 100 nm or more and 10 μm or less, for example, 1 μm or more and 10 μm or less. The depth of the wells of the microdevice of this embodiment may be, for example, 10 nm or more and 1 μm or less, for example, 100 nm or more and 800 μm or less, for example, 200 nm or more and 700 nm or less. When the pore size and depth are within the above ranges, a single molecule of ENPP can be captured in the well, and the enzymatic activity of each single molecule of ENPP in a biological sample can be detected.

[0076] The microdevice of this embodiment may include one type of the above-mentioned fluorescent probe for detecting ENPP per well. This makes it possible to detect the fluorescence intensity of one type of ENPP-detecting fluorescent probe for ENPP1 molecules in a biological sample and compare the enzymatic activity of ENPP1 molecules.

[0077] Furthermore, the microdevice of this embodiment may have different reaction sites in one well and may be equipped with two or more of the above-mentioned ENPP-detecting fluorescent probes with different fluorescent wavelengths. In this specification, "having different reaction sites" means that the cleavage sites by ENPP are different, i.e., the length from the parent nucleus of the fluorescent compound (e.g., rhodamine skeleton) to the phosphate group hydrolyzed by ENPP is different, or the organic base and its derivative, or the sugar and its derivative are different. Specifically, examples of two types of fluorescent probes for detecting ENPP that have different reaction sites and different fluorescence wavelengths include the compounds shown below. JPEG0007628233000013.jpg192149

[0078] It is known that there are subtypes of ENPP, including ENPP1, ENPP2, ENPP3, ENPP4, ENPP5, ENPP6, and ENPP7. The characteristics of each subtype are as follows: JPEG0007628233000014.jpg87140

[0079] By providing one well with two or more of the above-mentioned fluorescent probes for detecting ENPP, each of which has different reaction sites and different fluorescent wavelengths, it is possible to classify ENPP subtypes based on the pattern of fluorescence intensity. Furthermore, by using a microdevice having different reaction points in one well and equipped with two or more of the above-mentioned ENPP detection fluorescent probes with different fluorescent wavelengths, ENPP fluorescent assays of biological samples from healthy and diseased subjects can be performed, and the method can be applied to the discovery of disease-specific ENPP subtypes and to disease diagnosis methods.

[0080] Furthermore, the amount of fluorescent probe for detecting ENPP contained in one well of the microdevice of this embodiment may be, for example, 100 nM or more and 100 μM or less, for example, 1 μM or more and 100 μM or less, for example, 10 μM or more and 100 μM or less. In the method of using the microdevice of this embodiment, first, a solution containing a biological sample is added to the microdevice, and then, sealing oil is dripped onto the well of the microdevice to seal the ENPP in the biological sample.

[0081] 5. ENPP enzyme activity test kit Another embodiment of the invention is a kit for testing the enzymatic activity of ENPP, comprising a compound of the invention.

[0082] Another embodiment of the invention is a kit comprising a compound of the invention and a plate with microwells containing said compound.

[0083] In one preferred aspect of the kit of the present invention, one well is provided with two or more types of ENPP-detecting fluorescent probes having different reaction sites and different fluorescent wavelengths.

[0084] The plate used in the kit of the present invention has any number of microwells (eg, 1 to 10 million, eg, 10 to 500,000, eg, about 1 million, etc.) arranged therein.

[0085] In the kit of the present invention, the microwells are preferably capable of capturing ENPP1 molecules. Specifically, the plate used in the kit of the present invention has a structure in which millions of tiny wells are arranged, and by adding a sufficiently diluted enzyme solution to these wells, conditions are created in which less than one molecule of enzyme is present in each well, and the activity of the enzyme can be detected as a signal. Figure 2 shows an example of an image in which an enzyme is detected in this way. This image is a microscope image of a microdevice to which an enzyme and a fluorescent probe have been added, and the bright spots are wells that contain the enzyme, and the rest are wells that do not contain the enzyme.

[0086] By using the kit of the present invention, it is possible to separate and detect ENPP in a biological sample at the single molecule level, and a specific method for this is described below. First, the biological sample is diluted sufficiently and added to the microdevice, creating a state in which there is less than one molecule of enzyme per well. Then, multiple colored fluorescent probes with different reactivity to the enzymes are reacted simultaneously, and the increase in fluorescence at multiple wavelengths in each well is measured, and the enzyme contained in the well is identified from the combination. For example, a well with a significant increase in red fluorescence is thought to contain Isozyme3, which is highly reactive with the red probe (see Figure 3A).

[0087] Next, the fluorescence intensity at each wavelength for each of the hundreds of thousands of wells in the microdevice was measured and plotted on a scatter diagram to visualize the activity pattern of the enzyme in the biological sample. Since one well contains one enzyme molecule, it is possible to quantify the number of enzyme molecules in the biological sample by counting the number of plots that make up a cluster (see Figure 3B).

[0088] Using this analytical method, it is possible to separate and detect ENPP in biological samples at the single molecule level, and identify patient-specific clusters or overall patterns, which can be used for diagnosis.

[0089] 6. Method for detecting ENPP enzyme activity in biological samples The method for detecting the enzymatic activity of ENPP according to one embodiment of the present invention is a method using the above-mentioned compound. The method for detecting the enzymatic activity of ENPP according to one embodiment of the present invention can detect the enzymatic activity of ENPP in a biological sample. According to the present invention, the method for detecting the enzymatic activity of ENPP in a biological sample according to one embodiment of the present invention can be a method using the above-mentioned microdevice.

[0090] According to the present invention, there is provided a method for detecting the enzymatic activity of ENPP, comprising contacting a sample (e.g., a biological sample isolated from a subject, a biopsy sample, a body fluid sample, an aqueous solution) that contains or may contain ENPP with the above-mentioned compound. In one embodiment, the biological sample may be a blood sample (e.g., a serum sample or a plasma sample). The method may further comprise measuring the fluorescence of the compound after contacting the compound with the aqueous solution. If the compound emits fluorescence, the presence or absence of the fluorescence indicates the presence of the activity of ENPP in the aqueous solution, and the intensity of the fluorescence indicates the intensity of the activity of ENPP in the aqueous solution.

[0091] One embodiment of the invention is a method of measuring the enzymatic activity of an ENPP, comprising contacting an ENPP with a compound of the invention.

[0092] In one preferred aspect of the method for measuring the enzymatic activity of ENPP of the present invention, the contacting is carried out in the presence of serum.

[0093] The present invention also provides a method for predicting the possibility that a subject has cancer, comprising contacting a biological sample obtained from the subject with the above-mentioned compound, and detecting the fluorescence of the compound after contact, wherein the detection of fluorescence indicates the possibility that the subject from which the biological sample is derived has a cancer that expresses ENPP. In one embodiment, the prediction method of the present invention is an in vitro method. In one embodiment, the prediction method of the present invention is a method that can be industrially applied (or is not a medical practice). In one embodiment, the cancer can be pancreatic cancer.

[0094] One embodiment of the present invention is a method for detecting the enzyme activity of ENPP3, comprising contacting ENPP3 with a compound of the following (A) and / or a compound of the following (B): JPEG0007628233000015.jpg165170

[0095] In one aspect of the method for detecting the enzymatic activity of ENPP3 of the present invention, the contacting is contacting ENPP3 with both the above compound (A) and compound (B).

[0096] According to the detection method of this embodiment, the enzyme activity of ENPP in a biological sample can be detected. The detection method of this embodiment will be described in detail below.

[0097] [Process 1] First, a solution containing a biological sample is added to the microdevice equipped with the above-mentioned fluorescent probe for detecting ENPP. Examples of the biological sample include those exemplified in the above-mentioned "fluorescent probe for detecting ENPP." The pH of the solution containing the biological sample may be a value close to that in a living body, specifically, for example, from 6.0 to 8.0. The protein concentration of the biological sample may be, for example, from 1 pM to 100 pM, for example, from 10 pM to 100 pM. Methods for measuring the protein concentration of a biological sample include, for example, methods utilizing antibody-antigen reactions (e.g., ELISA method, etc.) and colorimetric methods utilizing reactions between proteins and reagents (e.g., bicinchoninic acid (BCA) method, Bradford method, Lowry method, Biuret method, etc.). The biological sample may be diluted to the above-mentioned concentration using various aqueous solvents, etc. Examples of the aqueous solvent include, but are not limited to, water, saline, phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline, etc.

[0098] [Process 2] Next, sealing oil is dropped to seal the ENPP in the biological sample in the well of the microdevice equipped with the above-mentioned ENPP detection fluorescent probe. The sealing oil may be any known oil that is usually used for sealing samples in microdevices, such as fluorine-based oil (FC-40, etc.).

[0099] [Process 3] Next, the fluorescence in the wells of the microdevice is detected using a fluorescence scanner, etc. The enzyme activity can be evaluated from the detected fluorescence intensity. Furthermore, when a microdevice having different reaction sites in one well and two or more of the above-mentioned fluorescent probes for detecting ENPP with different fluorescent wavelengths is used, it is advantageous to compare the enzymatic activity of ENPP in biological samples from healthy subjects and subjects with a disease, in order to discover ENPP subtypes found specifically in diseases and to apply this to methods for diagnosing diseases.

[0100] In particular, when a microdevice equipped with a red fluorescent probe containing the following compound (sTM-dCMP) among the compounds of general formula (I) and a green fluorescent probe containing the following compound (sTG-mdTMP) among the compounds of general formula (II) is used, by comparing the enzymatic activity of ENPP in biological samples derived from healthy subjects and diseased subjects, it is possible to discover ENPP3, which is found specifically in pancreatic cancer, and provide a method for diagnosing pancreatic cancer (or a method for predicting the possibility that the subject from whom the biological sample is derived has pancreatic cancer). JPEG0007628233000016.jpg200170 EXAMPLES

[0101] The present invention will be described below with reference to examples, but the present invention is not limited thereto.

[0102] [Samples and measurement methods] Reagents and solvents for organic synthesis were supplied by Tokyo Chemical Industry Co., Ltd. (TCI), Wako Pure Chemical Industries, Ltd., or Aldrich Chemical Co., and were used without further purification. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a JEOL JMN-LA400 instrument. Mass spectra were measured on a JEOL JMS-T100LP AccuTOFTM LC-plus4G.

[0103] [Synthesis Example 1] Synthesis of compound (4) Compound (4) was synthesized according to the following scheme. JPEG0007628233000017.jpg47159

[0104] 5-O-Tosylthymidine was synthesized according to reference (1). 31 mg (0.078 mmol) of 5-O-Tosylthymidine dissolved in 0.5 mL of N,N-dimethylformamide, 8.4 mg (0.016 mmol) of compound (1), and 27 μL (0.16 mmol) of N,N-diisopropylethylamine (DIEA) were added to a dry flask and stirred at 50°C for 48 hours under nitrogen substitution. The solvent was then distilled off under reduced pressure. The resulting solid was then purified by HPLC (A / B=99 / 1→0 / 100 for 30 minutes, A: 0.1M triethylamine acetate (TEAA), B: 0.1M TEAA / 80% acetonitrile / 20% water, then A / B=90 / 10→0 / 100 for 30 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1% TFA / acetonitrile) to obtain 0.8 mg (0.0011 mmol, 6.7% yield) of orange solid compound (4). Compound (1) was synthesized in accordance with the description in Patent Document 1 (WO2018 / 159810A1). The obtained compound 1The analysis results by \(^1\)H-NMR and high-resolution Mass Spectrometry (HR-MS) are shown below. 1 \(^1\)H-NMR (500 MHz, CD3OD): δ 1.87 (s, 3H), 2.01 - 2.18 (m, 2H), 3.83 (d, 3H, J = 2.3 Hz), 3.93 (d, 1H, J = 10.5 Hz), 4.08 - 4.20 (m, 5H), 4.33 - 4.39 (m, 1H), 5.95 (d, 2H, J = 13.8 Hz), 6.16 - 6.22 (m, 1H), 7.00 (s, 1H), 7.32 (dd, 1H, J = 2.3, 9.2 Hz), 7.37 (d, 1H, J = 2.1 Hz), 7.41 - 7.46 (m, 1H), 7.63 (s, 1H), 7.76 (d, 1H, J = 2.2 Hz), 7.85 (d, 1H, J = 5.6 Hz), 7.87 - 7.93 (m, 2H). HRMS (ESI - ): Calcd. for [M-H] - , 761.1054, Found, 761.1037 (-1.7 mmu).

[0105] Reference (1) M. Kawaguchi, T. Okabe, S. Okudaira, H. Nishimasu, R. Ishitani, H. Kojima, O. Nureki, J. Aoki, T. Nagano, Screening and X-ray crystal structure-based optimization of autotaxin (ENPP2) inhibitors, using a newly developed fluorescence probe. ACS Chem. Biol. 8, 1713 - 1721 (2013).

[0106] [Synthesis Example 2] Synthesis of compound (5) Compound (5) was synthesized according to the following scheme. JPEG0007628233000018.jpg59159

[0107] In a dried flask, 30 mg (0.067 mmol) of compound (2), 1 mg (0.0082 mmol) of N,N-dimethylaminopyridine (DMAP), and 2 drops of triethylamine were placed and dissolved in tertiary butyl alcohol and water. Next, 28 mg (0.067 mmol) of guanosine 5'-monophosphate disodium salt and 25 mg (0.13 mmol) of EDCI were added, and the mixture was heated under reflux for 20 minutes. Next, 28 mg (0.067 mmol) of guanosine 5'-monophosphate disodium salt and 25 mg (0.13 mmol) of EDCI were added three times every 20 minutes, and the reaction solution was heated under reflux for 4 hours. Next, the solvent was distilled off under reduced pressure. The resulting solid was then purified by HPLC (A / B = 90 / 10 to 0 / 100 in 30 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1% TFA / acetonitrile) to give 28 mg (0.036 mmol, 54% yield) of compound (5) as an orange solid. Compound (2) was synthesized in accordance with the description in Patent Document 1 (WO2018 / 159810A1). The obtained compound 1 The results of analysis by H-NMR and high-resolution mass spectrometry (HR-MS) are shown below. 1H-NMR (500 MHz, DMSO-d6): δ 3.76 & 3.77 (s, 3H), 3.97 (s, 3H), 4.03-4.22 (m, 5H), 4.52-4.56 (m, 1H), 5.74-5.77 (m, 1H), 6.77 (s, 1H), 6.80-6.98 (m, 3H), 7.14-7.26 (m, 2H), 7.33-7.42 (m, 2H), 7.57-7.68 (m, 2H), 8.62 & 8.65 (s, 1H), 11.04 (brs, 1H), 11.13 (brs, 1H). HRMS (ESI + ): Calcd. for [M+H] + , 774.1119, Found, 774.1119 (+0.0 mmu).

[0108] [Synthesis Example 3] Synthesis of compound (6) Compound (6) was synthesized according to the following scheme. JPEG0007628233000019.jpg64159

[0109] In a dried flask, 20 mg (0.044 mmol) of compound (2), 1 mg (0.0082 mmol) of N,N-dimethylaminopyridine (DMAP), and 2 drops of triethylamine were placed and dissolved in tertiary butyl alcohol, water, and 2N aqueous sodium hydroxide solution. Then, 7.2 mg (0.022 mmol) of 2'-deoxycytidine 5'-monophosphate and 8.4 mg (0.044 mmol) of EDCI were added, and the mixture was heated and refluxed for 20 minutes. Then, 7.2 mg (0.022 mmol) of 2'-deoxycytidine 5'-monophosphate and 8.4 mg (0.044 mmol) of EDCI were added three times every 20 minutes, and the reaction solution was heated and refluxed for 4 hours. Then, the solvent was distilled off under reduced pressure. The resulting solid was then purified by HPLC (A / B = 90 / 10 to 0 / 100 in 30 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1% TFA / acetonitrile) to give 15 mg (0.021 mmol, 47% yield) of compound (6) as an orange solid. The obtained compound 1 The results of analysis by H-NMR and high-resolution mass spectrometry (HR-MS) are shown below. 1 H-NMR (500 MHz, DMSO-d6): δ 2.08-2.25 (m, 2H), 3.78 (s, 3H), 3.92-4.02 (m, 1H), 3.97 (s, 3H), 4.04-4.10 (m, 1H), 4.10-4.17 (m, 1H), 4.18-4.25 (m, 1H), 5.96-6.10 (m, 2H), 6.85 (dd, 1H, J = 1.7, 9.6 Hz), 6.86-6.94 (m, 2H), 7.27-7.44 (m, 3H), 7.60 (d, 1H, J = 9.1 Hz), 7.68 (dd, 1H, J = 2.1, 8.5 Hz), 8.01-8.12 (m, 1H), 8.53-8.65 (m, 1H), 9.56 (d, 1H, J = 3.3 Hz). HRMS (ESI - ): Calcd. for [M-2H] 2- , 716.0713, Found, 716.0719 (+0.6 mmu).

[0110] [Synthesis Example 4] Synthesis of compound (7) Compound (7) was synthesized according to the following scheme. JPEG0007628233000020.jpg62159

[0111] In a dried flask, 18 mg (0.038 mmol) of compound (3), 1 mg (0.0082 mmol) of N,N-dimethylaminopyridine (DMAP), and 2 drops of triethylamine were placed and dissolved in tertiary butyl alcohol and water. Next, 16 mg (0.038 mmol) of guanosine 5'-monophosphate disodium salt and 14 mg (0.076 mmol) of EDCI were added, and the mixture was heated under reflux for 20 minutes. Next, 16 mg (0.038 mmol) of guanosine 5'-monophosphate disodium salt and 14 mg (0.076 mmol) of EDCI were added three times every 20 minutes, and the reaction solution was heated under reflux for 4 hours. Next, the solvent was distilled off under reduced pressure. The resulting solid was then purified by HPLC (A / B = 90 / 10 to 0 / 100 in 30 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1% TFA / acetonitrile) to obtain 3 mg (0.0037 mmol, 9.6% yield) of compound (7) as an orange solid. The obtained compound 1 The results of analysis by H-NMR and high-resolution mass spectrometry (HR-MS) are shown below. 1H-NMR (500 MHz, DMSO-d6): δ 0.48 (s, 3H), 0.59 (s, 3H), 3.73 (s, 3H), 4.02 (s, 3H), 4.18-4.39 (m, 4H), 4.48-4.54 (m, 1H), 5.97 (brs, 1H), 6.04 (d, 1H, J = 1.8 Hz), 6.69 (d, 1H, J = 1.8 Hz), 6.74 (dd, 1H, J = 2.3, 9.0 Hz), 7.08 (d, 1H, J = 2.6 Hz), 7.23 (dd, 1H, J = 1.8, 8.8 Hz), 7.28 (d, 1H, J = 9.0 Hz), 7.35 (d, 1H, J = 8.9 Hz), 7.56 (s, 1H), 9.04 (brs, 1H). HRMS (ESI + ): Calcd. for [M+H] + , 816.1408, Found, 816.1406 (-0.2 mmu).

[0112] [Synthesis Example 5] Synthesis of compound (8) Compound (8) was synthesized according to the following scheme. JPEG0007628233000021.jpg58159

[0113] In a dried flask, 11 mg (0.022 mmol) of compound (3), 1 mg (0.0082 mmol) of N,N-dimethylaminopyridine (DMAP), and 2 drops of triethylamine were placed and dissolved in tertiary butyl alcohol, water, and 2N aqueous sodium hydroxide solution. Then, 3.6 mg (0.011 mmol) of 2'-deoxycytidine 5'-monophosphate and 4.2 mg (0.022 mmol) of EDCI were added, and the mixture was heated and refluxed for 20 minutes. Then, 3.6 mg (0.011 mmol) of 2'-deoxycytidine 5'-monophosphate and 4.2 mg (0.022 mmol) of EDCI were added three times every 20 minutes, and the reaction solution was heated and refluxed for 4 hours. Then, the solvent was distilled off under reduced pressure. The resulting solid was then purified by HPLC (A / B = 90 / 10 to 0 / 100 in 30 minutes, A: 0.1% trifluoroacetic acid (TFA) / water, B: 0.1% TFA / acetonitrile) to give 12 mg (0.016 mmol, 71% yield) of compound (8) as an orange solid. The obtained compound 1 The results of analysis by H-NMR and high-resolution mass spectrometry (HR-MS) are shown below. 1H-NMR (500 MHz, DMSO-d6): δ 0.49 & 0.51 (s, 3H), 0.60 & 0.60 (s, 3H), 2.13-2.20 (m, 1H), 2.30-2.38 (m, 1H), 3.73 & 3.74 (s, 3H), 4.02 & 4.02 (s, 3H), 4.07-4.12 (m, 1H), 4.13-4.19 (m, 1H), 4.22-4.30 (m, 1H), 4.34-4.40 (m, 1H), 5.90-5.96 (m, 1H), 6.03-6.07 (m, 1H), 6.13-6.18 (m, 1H), 6.68-6.75 (m, 2H), 7.07-7.10 (m, 1H), 7.20-7.24 (m, 1H), 7.28-7.32 (m, 1H), 7.34-7.39 (m, 1H), 7.58-7.62 (m, 1H), 8.13 (d, 1H, J = 8.1 Hz). HRMS (ESI - ): Calcd. for [M-2H] 2- , 758.1003, Found, 758.1019 (+1.6 mmu).

[0114] [Example 1] In vitro ENPP fluorescence assay First, compounds (4)-(8) were each diluted with an assay buffer to 10 μmol / L. The composition of the assay buffer was Tris-HCl buffer (pH 9.0), 5.0 mmol / L magnesium chloride, and 100 mmol / L sodium chloride. Next, ENPP1, ENPP2, and ENPP3 were added to each well to a concentration of 1 μmol / L, and dispensed into a multiwell plate. The fluorescence intensity was then measured using a plate reader. The results are shown in FIG. 4.

[0115] [Example 2] Measurement of human plasma samples using ENPP probes in a microdevice First, one of the compounds (4)-(8), which are green (4), (5), and (6), and one of the compounds (7) and (8), which are red, were selected and diluted with the assay buffer to 100 μmol / L. The composition of the assay buffer was 100 mmol / L Tris-HCl buffer (pH 9.3), 1.0 mmol / L magnesium chloride. Next, a human plasma sample was diluted 500 times and added, and dispensed into the wells of a microdevice consisting of a multi-well plate. The fluorescence intensity of each well was then measured using a fluorescence microscope. Figure 5A shows a scatter plot of the fluorescence intensity of each well for all combinations performed in this experiment. Figure 5B shows a fluorescence microscope image of the combination of compounds (4) and (8).

[0116] [Example 3] Measurement of pancreatic cancer patient samples using ENPP probes in microdevices First, compounds (4) and (8) were each diluted with assay buffer to 100 μmol / L. The composition of the assay buffer was 100 mmol / L Tris-HCl buffer (pH 9.3), 1.0 mmol / L magnesium chloride. Next, plasma samples collected from 31 pancreatic cancer patients and 14 healthy subjects were diluted 500-fold and added, and dispensed into the wells of a microdevice consisting of a multi-well plate. The fluorescence intensity of each well was then measured using a fluorescence microscope. The data plotted on a scatter plot of the fluorescence intensity of each well was subjected to cluster analysis by fitting to a multivariate normal distribution using variational Bayesian estimation, and the data could be classified into three clusters. An example of one sample is shown in Figure 6A. The number of plots corresponding to cluster 1, i.e., the number of enzymes, was significantly increased in pancreatic cancer patients compared to healthy subjects. The results are shown in Figure 6B.

[0117] [Example 4] Measurement of purified ENPP enzyme using ENPP probe in microdevice First, compounds (4) and (8) were each diluted with an assay buffer to 100 μmol / L. The composition of the assay buffer was 100 mmol / L Tris-HCl buffer (pH 9.3), 1.0 mmol / L magnesium chloride. Next, purified enzymes ENPP1 and ENPP3 were added and dispensed into wells of a microdevice consisting of a multi-well plate. The fluorescence intensity of each well was then measured using a fluorescence microscope. The data obtained by plotting the fluorescence intensity of each well on a scatter plot was applied to the cluster analysis performed in Example 3. The results are shown in FIG. 7A for ENPP1 and in FIG. 7B for ENPP3. Since only ENPP3 has a plot corresponding to cluster 1, it is presumed that the enzyme that was significantly increased in pancreatic cancer patients in Example 3 was ENPP3.

[0118] [Example 5] Measurement of plasma samples using a plate reader First, compound (4) was diluted with assay buffer to 10 μmol / L. The composition of the assay buffer was Tris-HCl buffer (pH 8.3), 1.0 mmol / L magnesium chloride, and 0.5% CHAPS. Next, 25 μg / mL of ENPP2 and human serum were added to the wells to give a 10-fold dilution, and the mixture was dispensed into a multi-well plate. Then, the fluorescence intensity was measured using a plate reader. The results were as shown in FIG. 8. As shown in FIG. 8, in the multi-well plate, the measured intensity of ENPP activity in the presence of serum was lower than that in the absence of serum. In contrast, as described above, the measurement method using the microdevice was able to measure ENPP activity well even in the presence of serum. This shows that it is advantageous to use a microdevice and a probe suitable for use with it to detect ENPP activity in blood.

Claims

1. A compound represented by the following general formula (I) or a salt thereof: 【Chemistry 1】 (In the formula, R １ is one or two monovalent substituents present on the benzene ring, which are electron donating groups; R １ When multiple are present, they may be the same or different; R ２ is one to two sulfonic acid groups present on a benzene ring; R ３ and R ４ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ５ and R ６ each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms; R ７ and R ８ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; X is a silicon atom or a germanium atom; Z is an oxygen atom or N ＋ R ９ R １０ and Here, R ９ and R １０ each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ９ and R １０ Let's get together and ９ and R １０ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which is attached, R ９ Or R １０ Alternatively, R ９ and R １０ Both of these are R ３ Or R ７ Together with ９ Or R １０ may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; Y is a single bond, —O—(CH ２ ) ｎ１ -, -O-(CH ２ ) ｎ２ -Ar １ -, -NH-(CH ２ ) ｎ３ -, or -NH-(CH ２ ) ｎ４ -Ar ２ - and Here, n1, n2, n3, and n4 each independently represent an integer from 1 to 10; Ar １ and Ar ２ each independently represents a substituted or unsubstituted arylene group; 【Chemistry 2】 teeth, It is represented by R-S-, where R is an organic base; S is a partial structure of a sugar or a derivative thereof, or a single bond.

2. A compound represented by the following general formula (II) or a salt thereof: 【Chemistry 3】 (In the formula, R １ａ is one or two monovalent substituents present on the benzene ring, which are electron donating groups; R １ａ When multiple are present, they may be the same or different; R ２ａ is one to two sulfonic acid groups present on a benzene ring; R ３ａ and R ４ａ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R ５ａ and R ６ａ each independently represents an alkyl group or an aryl group having 1 to 6 carbon atoms; R ７ａ and R ８ａ each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; Z ａ is an oxygen atom or N ＋ R ９ａ R １０ａ and Here, R ９ａ and R １０ａ each independently represents a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, R ９ａ and R １０ａ Let's get together and ９ａ and R １０ａ may form a 4- to 7-membered heterocyclyl containing a nitrogen atom to which is attached, R ９ａ Or R １０ａ Alternatively, R ９ａ and R １０ａ Both of these are R ３ａ Or R ７ａ Together with ９ａ Or R １０ａ may form a 5-7 membered heterocyclyl or heteroaryl containing a nitrogen atom to which is bonded, and may contain 1 to 3 additional heteroatoms selected from the group consisting of oxygen atoms, nitrogen atoms and sulfur atoms as ring-constituting members, and the heterocyclyl or heteroaryl may be substituted with 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 aralkyl group having 6 to 10 carbon atoms, or an alkyl-substituted alkenyl group having 6 to 10 carbon atoms; Y ａ is a single bond, -O-(CH ２ ) ｎ１ -, -O-(CH ２ ) ｎ２ -Ar １ -, -NH-(CH ２ ) ｎ３ -, or -NH-(CH ２ ) ｎ４ -Ar ２ - and Here, n1, n2, n3, and n4 each independently represent an integer from 1 to 10. Ar １ and Ar ２ each independently represents a substituted or unsubstituted arylene group; 【Chemistry 4】 teeth, It is represented by R-S-, where R is an organic base; S is a partial structure of a sugar or a derivative thereof, or a single bond.

3. 3. The compound or salt thereof according to claim 1 or 2, wherein the sugar or derivative thereof of S is ribose, deoxyribose, or a derivative thereof.

4. The organic base of R is a nucleic acid base or a derivative thereof, (CH ３ ) ３ N ＋ -C ２ H ４ -, (CH ３ CH ２ ) ２ N- and R ２ The compound or salt thereof according to any one of claims 1 to 3, wherein R is a hydrogen atom or an alkyl group having 1 to 10 carbon atoms, and may be the same or different.

5. The compound or salt thereof according to claim 4, wherein the nucleic acid base is selected from the group consisting of adenine, thymine, cytosine, guanine and uracil.

6. R １ The compound or salt thereof according to any one of claims 1 and 3 to 5, wherein is an alkoxy group having 1 to 10 carbon atoms.

7. The compound or salt thereof according to claim 2, wherein R 1a is an alkoxy group having 1 to 10 carbon atoms.

8. A fluorescent probe for detecting ENPP, comprising the compound according to any one of claims 1 to 7 or a salt thereof.

9. A test kit for the enzyme activity of ENPP, comprising the compound or salt thereof according to any one of claims 1 to 7.

10. A kit comprising a plate having microwells containing the compound or salt thereof according to any one of claims 1 to 7.

11. A method for measuring the enzymatic activity of ENPP, comprising contacting the compound according to any one of claims 1 to 7 or a salt thereof with ENPP in vitro.

12. The method of claim 11 , wherein the contacting is performed in the presence of serum.

13. The kit according to claim 10, comprising two or more types of fluorescent probes for detecting ENPP, each of which has different reaction sites in one well and has different fluorescent wavelengths.

14. A method for detecting the enzyme activity of ENPP3, comprising contacting ENPP3 in vitro with a compound of (A) below and / or a compound of (B) below. 【Chemistry 5】

15. The method according to claim 14, wherein the contacting is contacting both the compound (A) and the compound (B) with ENPP3.

16. A method for predicting the likelihood that a subject has cancer, comprising contacting a biological sample obtained from the subject in vitro with a compound or salt thereof described in any one of claims 1 to 7, and detecting fluorescence of the compound or salt thereof after the contact, wherein the detection of fluorescence indicates the likelihood that the subject from which the biological sample was derived has a cancer that expresses ENPP.

17. The method of claim 16, wherein the ENPP is ENPP3.

18. The method described in claim 16 or 17, wherein the cancer is pancreatic cancer.

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