JP2018140971A - Near-infrared fluorescent probe for peptidase activity detection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent probe that can detect peptidase activity highly expressed in a cancer cell by fluorescence response, and has excellent tissue permeability, and a cancer diagnostic composition containing the probe.SOLUTION: The present invention provides a fluorescent probe for peptidase activity detection, typified by a compound of the formula, and a cancer diagnostic composition containing the probe. The composition is used for cancer surgical treatment or cancer test, is suitable for retractor surgery, endoscopic surgery, or endoscopy, and is used for observing fluorescence response by fluorescent imaging means.SELECTED DRAWING: None

Description

  The present invention relates to a fluorescent probe for detecting peptidase activity. More specifically, the present invention relates to a novel fluorescent probe capable of detecting peptidase activity such as aminopeptidase by fluorescence in the near infrared region, a composition for cancer diagnosis containing the probe, and a detection method using the fluorescent probe. .

  With the number of cancer patients and deaths increasing year by year, the development of treatment methods continues to be expected. At present, one of the most reliable cancer treatments is the early detection of cancer and its reliable surgical removal, but the complete removal of cancer tissue that is difficult to see completely. Is difficult and causes recurrence.

  On the other hand, in cancer cells, increased expression of γ-glutamyltransferase (GGT), a peptidase (protease), has been observed, and it has been reported that this increased expression is related to drug resistance. Therefore, it can be expected that detection of γ-glutamyltransferase will lead to the development of a diagnostic method for identifying cancer cells and cancer tissues with high accuracy.

  The present inventors have so far developed a fluorescent probe group capable of detecting the activity of γ-glutamyltransferase based on a fluorescent dye exhibiting intramolecular spirocyclization equilibrium (Non-patent Document 1, etc.). However, the absorption / emission wavelength of such a conventional fluorescent probe is 550 nm or less, and although it can be detected with high sensitivity to cancer cells and the like present on the tissue surface, living organisms such as lymph node metastasis can be used. There is a restriction that it cannot be applied to cancer cells existing in tissues or inside organs.

  In general, the near infrared region of 650 nm to 900 nm is called a “biological window”, and it is known that light absorption by water and light scattering by tissue are small. In addition, since autofluorescence of living tissue decreases as it is excited at a longer wavelength, it is considered that imaging with a lower background and higher contrast is possible. However, under the present circumstances, a fluorescent probe that can detect peptidase activity by such a fluorescence response in the near-infrared region and is excellent in cell penetration has not been developed.

Y. Urano et al., Sci. Transl. Med., 2011, vol. 3, pp. 110ra119

  Therefore, an object of the present invention is to provide a novel fluorescent probe that can detect peptidase activity highly expressed in cancer cells with a longer-wavelength fluorescence response and has excellent tissue permeability. is there. Another object of the present invention is to provide a system capable of detecting and visualizing cancer cells existing in a living organization or an organ with high sensitivity in surgical extraction operations and the like.

  As a result of intensive studies to solve the above problems, the present inventors have obtained a fluorescent molecule having a structure in which a group cleaved by a peptidase is introduced into a silicon rhodamine skeleton and having optimized intramolecular spirocyclization characteristics. It was found that a fluorescent probe that is colorless and non-fluorescent before contact with the target peptidase, but exhibits a fluorescence response in the near-infrared region, can be obtained by reaction with the peptidase. In addition, as a result of applying the fluorescent probe to clinical specimens, it has been found that cancer cells and tissues expressing the target peptidase can be detected and visualized rapidly. Based on this knowledge, the present invention has been completed.

〔式中、
Ｘは、Ｓｉ（Ｒ^ａ）（Ｒ^ｂ）を表し（ここで、Ｒ^ａ及びＲ^ｂは、それぞれ独立に水素原子、又はアルキル基を表す）；
Ｙは、Ｃ_０-Ｃ_３アルキレン基を表し、当該アルキレン基はハロゲン原子又はハロアルキルで置換されていてもよく；
Ｒ^１は、水素原子、シアノ基、それぞれ置換されていてもよいアルキル基、カルボキシル基、エステル基、アルコキシ基、アミド基、及びアジド基よりなる群から独立に選択される１〜４個の同一又は異なる置換基を表し；
Ｒ^２は、Ｒ^３と一緒になって、それらが結合する窒素原子及び炭素原子を含む５〜８員のヘテロ環構造を形成し；
Ｒ^４は、Ｒ^５と一緒になって、それらが結合する窒素原子及び炭素原子を含む５〜８員のヘテロ環構造を形成し；
Ｒ^６、Ｒ^７、Ｒ^８及びＲ^９は、それぞれ独立に水素原子、ハロゲン原子、ヒドロキシル基、アルキル基、スルホ基、カルボキシル基、エステル基、アミド基、又はアジド基を表し；
Ｒ^１０は、アミノ酸由来のアシル残基を表す。〕；
＜２＞Ｒ^２とＲ^３が一緒になって形成する前記ヘテロ環構造が６員環であり、Ｒ^４とＲ^５が一緒になって形成する前記ヘテロ環構造が６員環である、上記＜１＞に記載のペプチダーゼ活性検出用蛍光プローブ；
＜３＞Ｒ^ａ及びＲ^ｂが、いずれもＣ_１-Ｃ_４アルキル基である、上記＜１＞又は＜２＞に記載のペプチダーゼ活性検出用蛍光プローブ；
＜４＞Ｒ^６、Ｒ^７、Ｒ^８及びＲ^９が、いずれも水素原子である、上記＜１＞〜＜３＞のいずれかに記載のペプチダーゼ活性検出用蛍光プローブ；
＜５＞前記ペプチダーゼが、γ-グルタミルトランスペプチダーゼ、ジペプチジルペプチダーゼＩＶ（ＤＰＰ−ＩＶ）、又はカルパインである、上記＜１＞〜＜４＞のいずれかに記載のペプチダーゼ活性検出用蛍光プローブ；
＜６＞Ｒ^１０が、γ-グルタミル基である、上記＜１＞に記載のペプチダーゼ活性検出用蛍光プローブ；
＜７＞Ｒ^１０が、プロリン残基を含む、上記＜１＞に記載のペプチダーゼ活性検出用蛍光プローブ；及び
＜８＞式（Ｉ）で表される化合物が以下の構造を有する、上記＜１＞に記載のペプチダーゼ活性検出用蛍光プローブ

を提供するものである。 That is, the present invention in one aspect,
<1> A fluorescent probe for detecting peptidase activity, comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
X represents Si (R ^a ) (R ^b ) (where R ^a and R ^b each independently represents a hydrogen atom or an alkyl group);
Y represents a C ₀ -C ₃ alkylene group, and the alkylene group may be substituted with a halogen atom or haloalkyl;
R ¹ is ¹ to 4 identically independently selected from the group consisting of a hydrogen atom, a cyano group, an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group. Or represents a different substituent;
R ² together with R ³ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached;
R ⁴ together with R ⁵ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached;
R ⁶ , R ⁷ , R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group;
R ¹⁰ represents an amino acid-derived acyl residue. ];
<2> The heterocyclic structure formed by combining R ² and R ³ is a 6-membered ring, and the heterocyclic structure formed by combining R ⁴ and R ⁵ is a 6-membered ring. The fluorescent probe for detecting peptidase activity according to <1>;
<3> The fluorescent probe for detecting peptidase activity according to the above <1> or <2>, wherein R ^a and R ^b are both C ₁ -C ₄ alkyl groups;
<4> The fluorescent probe for detecting peptidase activity according to any one of <1> to <3>, wherein R ⁶ , R ⁷ , R ⁸ and R ⁹ are all hydrogen atoms;
<5> The fluorescent probe for detecting peptidase activity according to any one of <1> to <4>, wherein the peptidase is γ-glutamyltranspeptidase, dipeptidylpeptidase IV (DPP-IV), or calpain;
<6> The fluorescent probe for detecting peptidase activity according to <1>, wherein R ¹⁰ is a γ-glutamyl group;
<7> The fluorescent probe for detecting peptidase activity according to <1> above, wherein R ¹⁰ contains a proline residue; and <8> the compound represented by formula (I) has the following structure: > Fluorescent probe for detecting peptidase activity

Is to provide.

In another aspect, the present invention provides:
<9> A cancer diagnostic composition comprising the fluorescent probe for detecting peptidase activity according to any one of <1> to <8>above;
<10> The composition for cancer diagnosis as described in <9> above, which is used for cancer surgical treatment or cancer examination; and <11> the cancer surgical treatment or cancer examination is open surgery, endoscopic surgery, Or the composition for cancer diagnosis as described in said <10> which is an endoscopy is provided.

In a further aspect, the present invention also relates to a method for detecting or visualizing a target cell in which a specific peptidase is expressed, specifically,
<12> A method for detecting or visualizing a target cell in which a specific peptidase is expressed using the fluorescent probe for detecting a peptidase activity according to any one of <1> to <8>above;
<13> a step of bringing the fluorescent probe into contact with the target cell in vitro; and a step of observing a fluorescent response due to a reaction between the peptidase specifically expressed in the target cell and the fluorescent probe. And the method according to <12>above;
<14> The method according to <13>, comprising observing the fluorescence response using a fluorescence imaging means; and <15> the method according to <12>, wherein the target cell is a cancer cell. Is to provide.

In a further aspect, the present invention also relates to an apparatus comprising means for observing a fluorescence response by the above-described fluorescent probe for detecting peptidase activity, specifically,
<16> a fluorescence imaging means for observing a fluorescence response due to a reaction between the peptidase specifically expressed in the target cell and the peptidase activity detection fluorescent probe according to any one of <1> to <8> above, apparatus;
<17> The apparatus according to <16>, wherein the apparatus is an endoscope or an in vivo fluorescence imaging apparatus; and <18> the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8. A kit is provided.

  The fluorescent probe of the present invention is colorless and non-fluorescent before contact with the target peptidase, but can detect the fluorescence response in the near infrared region specifically and on / off by reaction with the peptidase. In particular, to show the fluorescence response in the near-infrared region, which is about 100 nm longer than conventional fluorescent probes, to visualize cancer cells that exist in the deep part of the body, such as lymph node metastasis, which was difficult in the past It has the effect of being able to.

  In addition, by applying the fluorescent probe of the present invention to intraoperative or preoperative imaging, various cancers can be identified accurately, rapidly, and with high sensitivity. As a result, accurate diagnosis of cancer becomes possible, microscopic cancer at the time of endoscopy with relatively small invasion can be detected at an early stage, and it is useful for radical treatment with chemoradiotherapy. Moreover, it is suitable for evaluation of a resection stump by applying it at the time of surgical operation such as ESD or laparotomy, and it is possible to perform radical treatment without any residual. It can be said that the medical and industrial utility value and economic effect of the present invention are extremely large.

FIG. 1 is an absorption and fluorescence spectrum of Compound 1 (HMJSiR). FIGS. 1A and 1B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 1C is a graph showing the pH dependence of absorbance and fluorescence intensity. FIG. 2 is an absorption and fluorescence spectrum of Compound 2 (gGlu-HMJSiR) which is a fluorescent probe of the present invention. 2A and 2B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 2C is a graph showing the pH dependence of absorbance and fluorescence intensity. FIG. 2D is an absorption spectrum of Compound 2 normalized with respect to the absorption spectrum of Compound 1. FIG. 3 is an absorption and fluorescence spectrum of Compound 3 (HMSiR600) of Comparative Example. 3A and 3B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 3C is a graph showing the pH dependence of absorbance and fluorescence intensity. FIG. 4 is an absorption and fluorescence spectrum of Compound 4 (HMSiR620h) of Comparative Example. 4A and 4B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 4C is a graph showing the pH dependence of absorbance and fluorescence intensity. FIG. 5 shows changes in fluorescence intensity with time when γ-glutamyltranspeptidase (GGT) was added to Compound 2, which is the fluorescent probe of the present invention, and addition of γ-glutamyltranspeptidase (GGT) and an inhibitor (GGsTop). The time change of the fluorescence intensity at the time of performing is shown. FIG. 6 is a fluorescence confocal imaging image of GGT activity in living cells using Compound 2, which is the fluorescent probe of the present invention. FIG. 7 is a two-color imaging image showing the intracellular localization of Compound 2 in SHIN3 cells. FIG. 8 is a fluorescence imaging image in the mesentery of a model mouse having peritoneal metastasis using Compound 2 which is the fluorescent probe of the present invention. FIG. 9 is a fluorescence imaging image of a model mouse model of a subcutaneous tumor using Compound 2, which is the fluorescent probe of the present invention.

  Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention.

1. Definition

  In the present specification, the “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

In the present specification, “alkyl” may be any of an aliphatic hydrocarbon group composed of linear, branched, cyclic, or a combination thereof. The number of carbon atoms of the alkyl group is not particularly limited, for example, 1-20 carbon atoms _{(C 1-20),} 1 to 15 carbon atoms _{(C 1 to 15),} 1 to 10 carbon atoms _{(C 1 to 10} ). When the number of carbons is specified, it means “alkyl” having the number of carbons within the range. For example, C _1-8 alkyl 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 are included. In the present specification, the alkyl group may have one or more arbitrary substituents. Examples of such a substituent 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, and acyl. When the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing an alkyl moiety (eg, an alkoxy group, an arylalkyl group, etc.).

  In the present specification, when a functional group is defined as “may be substituted”, the type of substituent, the substitution position, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different. Examples of the substituent group 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 include, but are not limited to, a halogenated alkyl group, a dialkylamino group, and the like.

  In the present specification, “alkenyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon double bond. For example, as non-limiting examples, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,3-butanedienyl, 1-pentenyl, 2-pentenyl, 3-pentenyl 4-pentenyl, 1,3-pentanedienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl and 1,4-hexanedienyl). The double bond may be either cis or trans conformation.

  In the present specification, “alkynyl” refers to a linear or branched hydrocarbon group having at least one carbon-carbon triple bond. For example, non-limiting examples include ethynyl, propynyl, 2-butynyl and 3-methylbutynyl.

  In the present specification, “cycloalkyl” refers to a monocyclic or polycyclic non-aromatic ring system composed of the above alkyl. The cycloalkyl can be unsubstituted or substituted by one or more substituents, which can be the same or different, and non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclopentyl, cyclohexyl Non-limiting examples of polycyclic cycloalkyl include 1-decalinyl, 2-decalinyl, norbornyl, adamantyl and the like. In addition, the cycloalkyl may be a heterocycloalkyl containing one or more hetero atoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as ring-constituting atoms. Any —NH in the heterocycloalkyl ring may be protected, for example as a —N (Boc) group, —N (CBz) group and —N (Tos) group, a nitrogen atom in the ring or The sulfur atom may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide. For example, non-limiting examples of monocyclic heterocycloalkyl include diazapanyl, piperidinyl, pyrrolidinyl, piperazinyl, morpholinyl, thiomorpholinyl, thiazolidinyl, 1,4-dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, lactam and Examples include lactones.

  As used herein, “cycloalkenyl” refers to a monocyclic or polycyclic non-aromatic ring system containing at least one carbon-carbon double bond. The cycloalkenyl may be unsubstituted or substituted by one or more substituents, which may be the same or different, and non-limiting examples of monocyclic cycloalkenyl include cyclopentenyl, cyclohexenyl And cyclohepta-1,3-dienyl, and non-limiting examples of polycyclic cycloalkenyl include norbornylenyl and the like. In addition, the cycloalkyl may be a heterocycloalkenyl which may be a heterocycloalkenyl containing one or more heteroatoms (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring-constituting atom. Atoms may be oxidized to the corresponding N-oxide, S-oxide or S, S-dioxide.

  In the present specification, “alkylene” is a divalent group consisting of a linear or branched saturated hydrocarbon, such as 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-methyltrimethyl , Tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyltetramethylene, 2,2-dimethyltetramethylene, 2,2-di-n-propyl Trimethylene and the like.

  In the present specification, “aryl” may be either a monocyclic or condensed polycyclic aromatic hydrocarbon group, and a hetero atom (for example, an oxygen atom, a nitrogen atom, or a sulfur atom) as a ring constituent atom Etc.) may be an aromatic heterocyclic ring. In this case, it may be referred to as “heteroaryl” or “heteroaromatic”. Whether aryl is a single ring or a fused ring, it can be attached at all possible positions. 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-oxadiazole-3 -An yl group etc. are mentioned. Non-limiting examples of fused polycyclic aryl include 1-naphthyl, 2-naphthyl, 1-indenyl, 2-indenyl, 2,3-dihydroinden-1-yl, 2,3 -Dihydroinden-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group, indolyl group, Isoindolyl group, phthalazinyl group, quinoxalinyl group, benzofuranyl group, 2,3-dihydrobenzofuran-1-yl group, 2,3-dihydrobenzofuran-2-yl group, 2,3-dihydrobenzothiophen-1-yl group, 2 , 3-Dihydrobenzothiophen-2-yl group, benzothiazolyl group, benzimidazolyl group, fluorenyl group, thioxanthenyl group, etc. And the like. In the present specification, an aryl group may have one or more arbitrary substituents on the ring. Examples of the substituent 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, and acyl. When the aryl group has two or more substituents, they may be the same or different. The same applies to the aryl moiety of other substituents containing the aryl moiety (for example, an aryloxy group and an arylalkyl group).

  In the present specification, the “alkoxy group” is a structure in which the alkyl group is bonded to an oxygen atom, and examples thereof include a saturated alkoxy group that is linear, branched, cyclic, or a combination thereof. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n- Pentyloxy, cyclopentyloxy, cyclopropylethyloxy, cyclobutylmethyloxy, n-hexyloxy, cyclohexyloxy, cyclopropylpropyloxy, cyclobutylethyloxy or cyclopentylmethyloxy are preferred Take as an example.

  As used herein, “amide” includes both RNR′CO— (when R = alkyl, alkaminocarbonyl-) and RCONR′- (where R = alkyl, alkylcarbonylamino-).

  As used herein, “ester” includes both ROCO— (in the case of R = alkyl, alkoxycarbonyl-) and RCOO— (in the case of R = alkyl, alkylcarbonyloxy-).

  As used herein, the term “ring structure”, when formed by a combination of two substituents, means a heterocyclic or carbocyclic ring, such ring being saturated, unsaturated, or aromatic. be able to. Accordingly, it includes cycloalkyl, cycloalkenyl, aryl, and heteroaryl as defined above.

  In the present specification, the term “heterocyclic structure” is synonymous with a heterocyclic ring, and means a monocyclic heterocycle having one or more heteroatoms arbitrarily selected from O, S and N in the ring. And such rings can be saturated, unsaturated, or aromatic. In addition, these monocyclic hetero rings may further include, for example, a ring (polycyclic hetero ring) in which one or two 3- to 8-membered rings are condensed. Examples of the non-aromatic hetero ring include a piperidine ring, a piperazine ring, and a morpholine ring. Examples of the aromatic hetero ring include a pyridine ring, a pyrimidine ring, a pyrrole ring, and an imidazole ring. Other examples include julolidine and indoline.

  In the present specification, a specific substituent can form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will recognize a specific substituent, for example, hydrogen. It can be understood that the bonds are formed. Therefore, when it is described that specific substituents together form a ring structure, those skilled in the art can understand that the ring structure can be formed by an ordinary chemical reaction and can be easily generated. Both such ring structures and their process of formation are within the purview of those skilled in the art. Moreover, the said heterocyclic structure may have arbitrary substituents on the ring.

2. Fluorescent probe for detecting peptidase activity of the present invention In one aspect, the fluorescent probe for detecting peptidase activity of the present invention comprises a compound having a structure represented by the following formula (I).

In the above formula (I), X represents Si (R ^a ) (R ^b ). Here, R ^a and R ^b each independently represent a hydrogen atom or an alkyl group. The alkyl group can be a C ₁ -C ₄ alkyl group. When R ^a and R ^b are alkyl groups, they can have one or more substituents, and examples of such substituents include alkyl groups, alkoxy groups, halogen atoms, hydroxyl groups, carboxyl groups, You may have 1 or 2 or more amino groups, sulfo groups, etc. R ^a and R ^b are preferably each a C ₁ -C ₄ alkyl group, more preferably a methyl group (in which case X is Si (CH ₃ ) ₂ ). In some cases, R ^a and R ^b may be bonded to each other to form a ring structure. For example, when R ^a and R ^b are both alkyl groups, R ^a and R ^b can be bonded to each other to form a spirocarbocycle. The ring formed is preferably about 5 to 8 membered ring, for example.

Y represents a C ₀ -C ₃ alkylene group. The alkylene group may be substituted with a halogen atom or haloalkyl. In the case of a C ₀ alkylene group, Y means a direct bond. The alkylene group may be a linear alkylene group or a branched alkylene group. For example, in addition to a methylene group (—CH ₂ —), an ethylene group (—CH ₂ —CH ₂ —), a propylene group (—CH ₂ —CH ₂ —CH ₂ —), a branched alkylene group such as —CH ( CH ₃ ) —, —CH ₂ —CH (CH ₃ ) —, —CH (CH ₂ CH ₃ ) — and the like can also be used. Among these, a methylene group or an ethylene group is preferable, and a methylene group is more preferable.

R ¹ represents a hydrogen atom, a cyano group, or an optionally substituted alkyl group (for example, the “optionally substituted alkyl group” may include haloalkyl such as fluoroalkyl), a carboxyl group, 1 to 4 identical or different substituents independently selected from the group consisting of an ester group, an alkoxy group, an amide group, and an azide group. Moreover, when it has two or more substituents on the benzene ring, they may be the same or different. R ¹ is more preferably a hydrogen atom, a lower alkyl group or a lower alkoxy group. A hydrogen atom is particularly preferred.

R ² together with R ³ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached. That is, the heterocyclic structure forms a heterocyclic structure in which R ² and R ³ are connected to each other, and the nitrogen atom to which R ² is bonded and the carbon atom to which R ³ is bonded are ring member atoms. . Preferably, the heterocyclic structure is a 6-membered ring. Further, the hetero ring structure can further contain heteroatoms other than the nitrogen atom to which R ² is attached, it ring member atoms of the nitrogen atom to which R ² is attached are both carbon atoms Is preferred.

Similarly, R ⁴ together with R ⁵ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached. That is, the heterocyclic structure forms a heterocyclic structure in which R ⁴ and R ⁵ are connected to each other so that the nitrogen atom to which R ⁴ is bonded and the carbon atom to which R ⁵ is bonded are ring members. . Preferably, the heterocyclic structure is a 6-membered ring. Further, the hetero ring structure can further contain heteroatoms other than the nitrogen atom to which R ⁴ is bonded, that ring member atoms of the nitrogen atom to which R ⁴ is bonded are both carbon atoms Is preferred.

Preferably, both the heterocyclic structure formed by combining R ² and R ³ and the heterocyclic structure formed by combining R ⁴ and R ⁵ are both 6-membered rings.

R ⁶ , R ⁷ , R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group. R ⁶ , R ⁷ , R ⁸ and R ⁹ are preferably all hydrogen atoms.

R ¹⁰ represents an amino acid-derived acyl residue. The said acyl residue means the acyl group which is the remaining partial structure which removed the hydroxyl group from the carboxyl group of the amino acid. That is, the carbonyl moiety of the acyl residue derived from an amino acid and NH of the formula (I) are linked to the silicon rhodamine skeleton by forming an amide bond.

  In the present specification, “amino acid” may be any compound as long as it is a compound having both an amino group and a carboxyl group, and includes natural and non-natural compounds. It may be any of neutral amino acids, basic amino acids, or acidic amino acids. In addition to amino acids that themselves function as transmitters such as neurotransmitters, bioactive peptides (in addition to dipeptides, tripeptides, tetrapeptides, An amino acid that is a constituent component of a polypeptide compound such as an oligopeptide or a protein can be used. As the amino acid, an optically active amino acid is preferably used. For example, as the α-amino acid, either D- or L-amino acid may be used, but it may be preferable to select an optically active amino acid that functions in a living body.

As will be described later, R ¹⁰ is a site cleaved by reaction with a target peptidase. The target peptidase can be γ-glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), or calpain. Therefore, when the target peptidase is γ-glutamyl transpeptidase, R ¹⁰ is preferably a γ-glutamyl group. When the target peptidase is dipeptidyl peptidase IV, R ¹⁰ is preferably an acyl group containing a proline residue. When the target peptidase is calpain, R ¹⁰ can be, for example, an acyl group containing a cysteine residue, or Suc-Leu-Leu-Val-Tyr (Suc-) known in the art as a calpain substrate. LLVY) or AcLM can also be used.

が挙げられる。ただし、これに限定されるものではない。 As a specific example of the compound of the formula (I) representative as a fluorescent probe for detecting peptidase activity of the present invention,

Is mentioned. However, it is not limited to this.

  The compound represented by the above formula (I) may exist as a salt. Examples of such salts include base addition salts, acid addition salts, amino acid salts and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, and acid addition salt. Examples thereof include mineral acid salts such as hydrochloride, sulfate and nitrate, and organic acid salts such as carboxylate, methanesulfonate, paratoluenesulfonate, citrate and oxalate. . Examples of amino acid salts include glycine salts. However, it is not limited to these salts.

  The compound represented by the formula (I) may have one or more asymmetric carbons depending on the type of substituent, and there are stereoisomers such as optical isomers or diastereoisomers. There is a case. Pure forms of stereoisomers, any mixture of stereoisomers, racemates, and the like are all within the scope of the present invention.

  The compound represented by the formula (I) or a salt thereof may exist as a hydrate or a solvate, and any of these substances is included in the scope of the present invention. Although the kind of solvent which forms a solvate is not specifically limited, For example, solvents, such as ethanol, acetone, isopropanol, can be illustrated.

  The above-described fluorescent probe may be used as a composition by blending additives usually used in the preparation of reagents as required. For example, additives such as solubilizers, pH adjusters, buffers, and tonicity agents can be used as additives for use in a physiological environment, and the amount of these can be appropriately selected by those skilled in the art. is there. These compositions can be provided as a composition in an appropriate form such as a powder-form mixture, a lyophilized product, a granule, a tablet, or a liquid.

  Moreover, when detecting a peptidase activity using the fluorescent probe of this invention, or when using for cancer diagnosis as mentioned later, it can also be used as a form of the kit containing the said fluorescent probe. In the kit, the fluorescent probe of the present invention is usually prepared as a solution. However, for example, it is provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid. It can also be applied by dissolving in distilled water for injection or an appropriate buffer. The kit may contain the above-described additives as necessary.

  In the examples of the present specification, production methods for representative compounds included in the compounds of the present invention represented by the formula (I) are specifically shown. Any compound included in the formula (I) can be easily produced by referring to, and appropriately selecting starting materials, reagents, reaction conditions and the like as necessary.

3. Fluorescence emission mechanism of fluorescence probe of the present invention Hereinafter, the fluorescence emission mechanism in the fluorescence probe for detecting peptidase activity of the present invention will be described.

As shown in the following scheme, the compound represented by the above formula (I) has a structure in which the upper part of the silicon rhodamine skeleton having a structure in which the central atom of rhodamine is substituted from O to Si is closed to form a spiro ring. At physiological pH (around pH 7.4), the fluorescent probe itself is substantially non-absorbing and non-fluorescent (the fluorescence response is off). In contrast, when the acyl residue derived from the amino acid of R ¹⁰ is hydrolyzed by a peptidase and cleaved from the silicon rhodamine skeleton, a compound of the formula (II) is generated. The compound of the formula (II) accumulates in lysosomes (pH 4.0 to 5.0) of intracellular acidic vesicles, thereby rapidly opening the spiro ring and showing strong fluorescence.

That is, the compound represented by the formula (I) hardly emits fluorescence when irradiated with excitation light of, for example, about 500 to 650 nm in a pH environment in a living body, but the cleavage caused by the reaction with peptidase occurs. The ring compound (II) has a property of emitting extremely strong fluorescence under the same conditions. Therefore, cells which have taken up a fluorescent probe of the formula (I) is, if not expressing cleavable peptidases hydrolyze the R ¹⁰ is preferably a ring-opening compound (II) does not generate a fluorescent Although no substance is produced in the cell, when such a peptidase is present, a ring-opening compound (II) is produced and strong fluorescence is obtained.
Therefore, the presence of the target peptidase is observed by the on / off change of the fluorescence intensity, thereby detecting the presence of a cancer cell or the like that expresses the peptidase.

  In addition, in the compound represented by the formula (I), a structure having a silicon atom at X which is the 10th element of the xanthene ring and having a hetero ring at one of the ring parts is used. Fluorescence emission due to the ring opening of can be made into fluorescence in the near-infrared region having a fluorescence peak wavelength of 650 nm to 900 nm. This makes it possible to visualize cancer cells and the like existing in the deep part of the living body, such as lymph node metastasis, which was difficult in the past.

  When the fluorescent probe of the present invention is applied to living cells, the ring-opening compound (II) hydrolyzed by the peptidase will accumulate in the lysosome of the cell, and 1) the maximum absorption wavelength Is increased by about 100 nm, 2) the fluorescence quantum yield and the molar extinction coefficient are increased, and 3) the spirocyclization equilibrium is shifted due to the low pH in the lysosome to change from the closed ring structure to the open ring structure, and the fluorescence response is changed. can get. In addition, 4) since the fluorescence of the ring-opening compound (II) decreases outside the lysosome, the background signal emitted from the probe leaked from the cell is suppressed, and high-sensitivity detection is possible.

4). Peptidase activity detection method using the fluorescent probe of the present invention According to such a luminescence mechanism, a target cell expressing a specific peptidase can be specifically detected or visualized using the fluorescent probe of the present invention. In particular,
A) contacting the fluorescent probe and the target cell in vivo or in vitro; and
B) By observing a fluorescence response due to the reaction between the peptidase specifically expressed in the target cell and the fluorescent probe, only the target cell expressing the specific peptidase is specifically detected in the near infrared region. It can be detected or visualized as a fluorescent signal. In this specification, the term “detection” should be interpreted in the broadest sense including measurement for various purposes such as quantitative and qualitative.

  As mentioned above, the specific peptidase can preferably be γ-glutamyl transpeptidase, dipeptidyl peptidase IV (DPP-IV), or calpain. However, it is not limited to these. The target cell is preferably a cancer cell.

  In addition, the method of the present invention can further include observing the fluorescence response using fluorescence imaging means. As the means for observing the fluorescence response, a fluorometer having a wide measurement wavelength can be used. However, the fluorescence response can be visualized by using a fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image. By using the means of fluorescence imaging, the fluorescence response can be visualized in two dimensions, so that it is possible to instantly recognize the target cell expressing the peptidase. As the fluorescence imaging apparatus, an apparatus known in the technical field can be used. In some cases, the reaction between the peptidase and the fluorescent probe can be detected by a change in the ultraviolet-visible absorption spectrum (for example, a change in absorbance at a specific absorption wavelength).

  In the above step A), as means for bringing the fluorescent probe of the present invention into contact with the peptidase specifically expressed in the target cell, typically, a solution containing the fluorescent probe is added, applied or sprayed. However, it can be appropriately selected depending on the application. Further, when the fluorescent probe of the present invention is applied to diagnosis or assistance in an animal individual or detection of a specific cell or tissue, the fluorescent probe and a peptidase expressed in the target cell or tissue are brought into contact with each other. There is no particular limitation, and for example, administration means common in the art such as intravenous administration can be used.

  The application concentration of the fluorescent probe of the present invention is not particularly limited. For example, a solution having a concentration of about 0.1 to 100 μM can be applied.

  Moreover, the light irradiation performed to a target cell can irradiate the said cell with light directly or via a waveguide (optical fiber etc.). As the light source, any light source can be used as long as it can irradiate light including the absorption wavelength of the fluorescent probe of the present invention after being subjected to enzymatic cleavage. It can be selected as appropriate.

  As the fluorescent probe of the present invention, the compound represented by the above general formula (I) or a salt thereof may be used as it is, but if necessary, a composition containing additives usually used in the preparation of reagents. It may be used as For example, additives such as a solubilizer, pH adjuster, buffer, and isotonic agent can be used as an additive for using the reagent in a physiological environment. Is possible. These compositions are generally provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid, but distilled water for injection or an appropriate buffer at the time of use. It can be dissolved and applied in

5. Cancer Diagnostic Composition Containing the Fluorescent Probe of the Present Invention As described above, cancer cells and cancer tissues expressing a specific peptidase can be detected and visualized by using the fluorescent probe of the present invention. Therefore, in another aspect, the present invention also relates to a cancer diagnostic composition comprising the above-described fluorescent probe for detecting peptidase activity.

  As used herein, the term “cancer tissue” means any tissue containing cancer cells. The term “tissue” must be interpreted in the broadest sense including part or all of an organ, and should not be limitedly interpreted in any way. The cancer diagnostic composition of the present invention has an action of detecting peptidase specifically expressed strongly in cancer tissue, typically γ-glutamyltransferase. A tissue that highly expresses γ-glutamyltransferase is preferred. Further, in this specification, the term “diagnosis” should be interpreted in the broadest sense, including confirming the presence of cancer tissue at an arbitrary biological site under the naked eye or under a microscope.

  The cancer diagnostic composition of the present invention can be used, for example, during surgery or examination. In this specification, the term “surgery” means, for example, craniotomy with open wound, thoracotomy, laparotomy, or skin surgery, as well as gastroscope, colonoscope, laparoscope, thoracoscope, etc. Includes any surgery applied for the treatment of cancer, including microscopic surgery. In addition, the term “examination” refers to examinations using endoscopes such as gastroscopes and colonoscopes, treatments such as excision and collection of tissues associated with examinations, and tissues separated and collected from living bodies. This includes inspections performed on

  The cancer that can be diagnosed by the composition for cancer diagnosis of the present invention is not particularly limited, and includes any malignant tumor including sarcoma, but preferably used for diagnosis of solid cancer. As one preferred embodiment, for example, the cancer diagnostic composition of the present invention is applied to a part or the whole of a surgical field under the naked eye or under a microscope by an appropriate method such as spraying, applying, or injecting, After 10 seconds to several minutes, the application site can be irradiated with light having a wavelength of about 500 nm. When cancer tissue is included in the application site, the tissue emits fluorescence. Therefore, the tissue is identified as cancer tissue and is excised together with surrounding tissues including the tissue. For example, in the surgical treatment of typical cancers such as gastric cancer, lung cancer, breast cancer, colon cancer, liver cancer, gallbladder cancer, pancreatic cancer, etc., in addition to making a definitive diagnosis on cancerous tissue that can be visually confirmed, lymph nodes, etc. It is possible to diagnose infiltration and metastasis of the lymph tissue and surrounding organs and tissues, and it is possible to make a quick diagnosis during the operation to determine the resection range.

  As another preferred embodiment, for example, the composition for cancer diagnosis of the present invention is applied to an examination site by an appropriate method such as spraying, applying, or injecting in a gastroscope or colonoscopy, When the application site is irradiated with light having a wavelength of about 500 nm after 10 seconds to several minutes and a fluorescent tissue is confirmed, it can be identified as a cancer tissue. When a cancer tissue can be confirmed by endoscopy, it can be subjected to examination resection or therapeutic resection.

  The composition for cancer diagnosis of the present invention may be blended with the above-mentioned additives usually used for the preparation of reagents as necessary.

6). The apparatus using the fluorescent probe of the present invention The present invention is for observing a fluorescence response due to a reaction between a peptidase specifically expressed in a target cell and the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8. It also relates to an apparatus comprising fluorescence imaging means.

  Preferably, the device can be an endoscope or an in vivo fluorescence imaging device.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not limited by these.

[Reagents, equipment, etc.]
The organic solvents used in the reactions shown below were all dehydrated grades. Commercial raw materials were purchased from reagent manufacturers (Wako Pure Chemical Industries, Ltd., Tokyo Chemical Industry Co., Ltd., Kanto Chemical Co., Ltd., Sigma-Aldrich Co. Ltd., Acros Co. Ltd.).

The following apparatus and column were used for purification by high performance liquid chromatography.
・ Pump: PU-2080 and PU-2087 (JASCO Corporation)
・ Detector: MD-2010 (JASCO Corporation)
Column: Inertsil ODS-3 (10 x 250 mm or 20 x 250 mm, GL Science Inc.)

In the separation and purification by HPLC, the following solvents A, B and C were used and purified by mixing them in an arbitrary composition unless otherwise specified.
A: Purified water (containing 1% acetonitrile, 0.1% trifluoroacetic acid)
B: Acetonitrile (including 1% purified water)
C: Purified water (including 0.2M acetic acid-triethylamine)
For HPLC separation, the flow rate is 25 ml / min (pump: PU-2087, column: 20 x 250 mm), 5 ml / min (pump: PU-2080, column: 10 x 250 mm), 1 ml / min, respectively. (Pump: PU-2080, column: 4.6 x 200 mm). Purification by medium pressure column chromatography was performed using YFLC-Al580 (Yamazen Co., Ltd.).

  NMR measurement was performed using AVANCE III 400 Nanobay (Bruker, Co. Ltd.) (400 MHz for 1H NMR, 101 MHz for 13C NMR). Mass spectrometric measurements were performed using MicrOTOF (ESI-TOF, Bruker, Co. Ltd.). For high-resolution MS (HRMS) measurement, sodium formate was used as an external standard.

1. Synthesis of fluorescent probes

[Synthesis of Compound 1 (Natural Core Compound)]
Compound 1 (HMJSiR) serving as a mother nucleus of the fluorescent probe molecule of the present invention was synthesized according to the following scheme 1. Based on the compound 1, by introducing a specific acyl residue (amino acid residue) that can be cleaved with a target peptidase at a site corresponding to R ¹⁰ in the formula (I), various types of peptidases are used. The fluorescent probe molecule can be obtained.

[Synthesis of A1]
8-Bromo-1,2,3 synthesized according to literature (MW Kryman, G. a. Schamerhorn, JE Hill, BD Calitree, KS Davies, MK Linder, TY Ohulchanskyy, MR Detty, Organometallics 2014, 33, 2628-2640) , 5,6,7-hexahydropyrido [3,2,1-ij] quinolone (14.1 g, 49.8 mmol, 1.0 equiv) and literature ADDIN CSL_CITATION {"citationItems": [{"id": "ITEM-1", " itemData ": {" DOI ":" 10.1038 / nchem.2648 "," ISSN ":" 1755-4330 "," author ": [{" dropping-particle ":""," family ":" Umezawa "," given ":" Keitaro "," non-dropping-particle ":""," parse-names ": false," suffix ":""},{" dropping-particle ":""," family ":" Yoshida "," given ":" Masafumi "," non-dropping-particle ":""," parse-names ": false," suffix ":""},{" dropping-particle ":""," family " : "Kamiya", "given": "Mako", "non-dropping-particle": "", "parse-names": false, "suffix": ""}, {"dropping-particle": "", "family": "Yamasoba", "given": "Tatsuya", "non-dropping-particle": "", "parse-names": false, "suffix": ""}, {"dropping-particle" : "", "family": "Urano", "given": "Yasuteru", "non-dropping-particle": "", "parse-names": false, "suffix": ""}], "container -title ":" Nature Chemistry "," id ":" ITEM-1 "," issued ": {" date-parts ": [[" 2016 "," 11 "," 7 "]]}," publisher " : "Nature Publishing Group", "title": "Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics", "type": "article-journal"}, "uris": ["http: //www.mendeley.com/documents/?uuid=22725b53-b465-4869-81c0-219780ee56fd "]}]," mendeley ": {" formattedCitation ":" ^[11] "," plainTextFormattedCitation ":" [11] "," previouslyFormattedCitation ":" ^[14] "}," properties ": {" noteIndex ": 0}," schema ":" https://github.com/citation-style-language/schema/raw/master/ csl-citation.json "} ^[ 2-bromo-4- (N, N-diallylamino) phenyl) methanol (14.8 g, 58.7 mmol, 1.2 eq) synthesized according to ^[11] into 40 ml of dehydrated dichloromethane under an argon atmosphere Dissolved. The solution was cooled to 0 ° C. and 20.0 ml (160 mmol, 3.2 eq) of boron trifluoride diethyl etherate was added dropwise. The reaction mixture was stirred at room temperature for 69 hours and then quenched with saturated aqueous sodium bicarbonate. Extracted with dichloromethane three times, the combined organic layer was washed with saturated brine, dried over sodium sulfate, and removed under reduced pressure. The resulting residue was purified by silica gel flash column chromatography (normal hexane / dichloromethane), 17.99 g of a yellow oil (34.8 mmol, 70.0%) was obtained.
¹ H NMR (400 MHz, CDCl ₃ ): δ 7.05 (d, J = 2.6 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 6.67-6.64 (m, 2H), 5.99-5.90 (m , 2H), 5.32-5.27 (m, 4H), 4.10 (s, 2H), 3.99-3.98 (m, 4H), 3.21-3.15 (m, 4H), 2.95 (t, J = 6.7 Hz, 2H), 2.75 (t, J = 6.5 Hz, 2H), 2.13-2.01 (m, 4H); ¹³ C NMR (101 MHz, CDCl ₃ ): δ 148.0, 143.0, 133.6, 130.6, 128.7, 127.1, 126.8, 125.6, 125.5 , 121.1, 120.7, 116.2, 115.9, 111.6, 77.4, 52.7, 50.0, 49.5, 40.9, 29.4, 27.5, 22.3, 21.9; HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ 515.06689, Found, 515.06920 (+ 2.3 mmu).

[Synthesis of A2]
250.0 mg (484.2 μmol, 1.0 equivalent) of Compound A1 was dissolved in 15 ml of dehydrated tetrahydrofuran and cooled to −78 ° C. under an argon atmosphere. 1.2 ml (1.1 M, 1.21 mmol, 2.5 equivalents) of sec-butyllithium was added dropwise, and the mixture was stirred at −78 ° C. for 5 minutes in an argon atmosphere. To this solution, 88 μl (0.726 mmol, 1.5 equivalents) of dichlorodimethylsilane was added and stirring was continued at 0 ° C. for 2 hours. The reaction was quenched with 1 M dilute hydrochloric acid and neutralized with saturated aqueous sodium bicarbonate. The aqueous layer was extracted three times with ethyl acetate, and the combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by aminated silica gel flash column chromatography (normal hexane / dichloromethane) to obtain a colorless liquid. This was dissolved in 10 ml of acetone, cooled to −15 ° C., and 153.0 mg (0.968 mmol, 2.0 equivalents) of potassium permanganate was added in 6 portions over 60 minutes. The reaction mixture was diluted with dichloromethane, filtered through celite, and purified by silica gel flash column chromatography (normal hexane / ethyl acetate) to obtain 33.0 mg of a pale yellow solid (77.0 μmol, 15.9% over 2 steps).
¹ H NMR (400 MHz, CDCl ₃ ): δ 8.31 (d, J = 8.6 Hz, 1H), 8.09 (s, 1H), 6.82-6.79 (m, 2H), 5.93-5.84 (m, 2H), 5.23 -5.19 (m, 4H), 4.03-4.02 (m, 4H), 3.30-3.26 (m, 4H), 2.92 (t, J = 6.2 Hz, 2H), 2.82 (t, J = 6.3 Hz, 2H), 2.06-1.93 (m, 4H), 0.49 (s, 6H); ¹³ C NMR (101 MHz, CDCl ₃ ): δ 185.4, 150.3, 145.3, 141.5, 135.3, 133.3, 131.3, 130.2, 129.7, 129.1, 124.7, 122.9, 116.7, 114.7, 113.5, 52.8, 50.6, 50.0, 29.0, 283.3, 22.0, 21.7, -0.1; HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ 429.23567, Found 429.23595 (+0.3 mmu).

[Synthesis of Compound A3 and Compound 1]
106.1 mg (0.436 mmol, 11.0 equivalents) of 1-bromo-2-[(1,1-dimethylethoxy) methyl] -benzene was dissolved in 10 ml of dehydrated tetrahydrofuran and cooled to −78 ° C. under an argon atmosphere. 357 μl (1.0 M, 0.357 mmol, 9.0 eq) of sec-butyllithium was added dropwise, and the mixture was stirred at −78 ° C. under an argon atmosphere for 5 minutes. To this solution, 17.0 mg of compound A2 (40.0 μmol, 1.0 equivalent) dissolved in 5 ml of dehydrated tetrahydrofuran was added dropwise, and stirring was continued at −78 ° C. for 60 minutes. The reaction was quenched with 1M dilute hydrochloric acid and neutralized with saturated aqueous sodium bicarbonate. The aqueous layer was extracted 3 times with dichloromethane, and the combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in 10 ml of methanol and cooled to 0 ° C. To this solution was added sodium borohydride (1.8 mg, 48 μmol, 1.2 eq) and stirred at 0 ° C. for 10 minutes. The mixture was concentrated under reduced pressure, water was added, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. To the resulting residue was added 1,3-dimethylbarbituric acid (92.8 mg, 0.595 mmol, 15.0 equiv), tetrakis (triphenylphosphine) palladium (0) (9.2 mg, 8.0 μmol, 0.2 equiv) and deoxygenated dichloromethane ( 2 ml) was added and the mixture was heated to reflux under an argon atmosphere for 1 hour. The reaction mixture was diluted with dichloromethane, washed with saturated aqueous sodium sulfate and saturated brine, dried over sodium sulfate, and concentrated in vacuo. The obtained residue was dissolved in 10 ml of dichloromethane, 38.7 mg of chloranil (0.157 mmol, 3.9 equivalents) was added, and the mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by short silica chromatography to remove chloranil and concentrated under reduced pressure to obtain an intermediate compound A3. To the residue was added 4 ml of a 1: 1 mixture of trifluoroacetic acid and dichloromethane, and the mixture was stirred at room temperature for 9 hours. The reaction mixture was diluted with dichloromethane and the solvent was removed under reduced pressure. It was then dissolved in 4 ml of a 1: 1 mixture of methanol and water. The reaction solution was stirred at room temperature for 12 hours, methanol was removed under reduced pressure, and the remaining solution was filtered and purified by preparative HPLC to obtain 5.4 mg of blue powder (12.3 μmol, 30.8% in 4 steps).
¹ H NMR (400 MHz, (CD ₃ ) ₂ CO): δ 7.30 (d, J = 7.6 Hz, 1H), 7.15 (dt, J = 7.6, 1.2 Hz, 1H), 7.08 (dt, J = 7.6, 1.2 Hz, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H), 6.82 (s, 1H), 6.77 (d, J = 7.6 Hz, 1H), 6.57 (dd, J = 8.4, 2.4 Hz, 1H), 5.46 (s, 2H), 3.16-3.09 (m, 4H), 2.97-2.93 (m, 2H, partially overlapped with the H ₂ O peak), 2.62-2.49 (m, 2H), 2.02-1.94 (m, 2H), 1.86-1.81 (m, 2H), 0.64 (s, 3H), 0.48 (s, 3H); ¹³ C NMR (101 MHz, (CD ₃ ) ₂ CO): δ 150.6, 147.3, 142.2, 141.0, 140.8, 137.7, 134.6, 129.1, 129.0, 128.0, 127.7, 127.3, 126.8, 124.2, 123.9, 122.1, 118.6, 117.0, 94.1, 74.6, 50.9, 50.4, 30.1, 29.1, 23.2, 22.7, 1.6, 1.5; HRMS (ESI ⁺ ): Calcd for [M] ⁺ 439.22002, Found, 439.21920 (-0.8 mmu).

[Synthesis of Compound 2 (fluorescent probe molecule)]
Compound 2 (gGlu-HMJSiR), which is a fluorescent probe molecule of the present invention, was synthesized from Compound 1 according to Scheme 2 below. Compound 2 is a compound in which a γ-glutamyl group is introduced at the site corresponding to R ¹⁰ in formula (I), but by introducing an arbitrary acyl group according to the target peptidase as described above. Other fluorescent probes can be synthesized similarly.

Intermediate A3 (10.8 mg, 21.8 μmol, 1.0 equiv), 1-tert-butyl N- (tert-butoxycarbonyl) -L-glutamate (660.6 mg, 2.18 mmol, 100.0 equiv), HATU (827.9 mg, 2.18 mmol, 100.0 equivalents) and N, N-diisopropylethylamine (378.5 μl, 2.18 mmol, 100.0 equivalents) were added to 5 ml of dehydrated N, N-dimethylformamide. The mixture was stirred at room temperature under an argon atmosphere for 23 hours. The reaction solvent was removed under reduced pressure, the residue was dissolved in 5 ml of a 4: 1 mixture of trifluoroacetic acid and dichloromethane, and the solution was stirred for 8 hours. The solvent was removed under reduced pressure and the residue was purified by preparative HPLC to give 1.5 mg of red powder (2.6 μmol, 12.1%).
¹ H NMR (400 MHz, CD ₃ OD + TFA): δ8.13 (d, 1H, J = 2.4 Hz), 7.73-7.65 (m, 3H), 7.57 (dd, 1H, J = 9.0, 2.0 Hz) , 7.31-7.29 (m, 1H), 6.85-6.82 (m, 2H), 5.15 (q, 2H, J = 11.5 Hz), 4.08 (t, 1H, J = 6.6 Hz), 3.84-3.78 (m, 4H ), 3.12-3.09 (m, 2H), 2.77-2.73 (m, 2H), 2.55-2.51 (m, 2H), 2.34-2.22 (m, 2H), 2.20-2.14 (m, 2H), 2.00-1.94 (m, 2H), 0.69 (s, 3H), 0.65 (s, 3H); HRMS (ESI ⁺ ): Calcd for [M + H] ⁺ 568.26261, Found, 568.26163 (-1.0 mmu).

[Synthesis of Compound 3 (Comparative Example)]
Compound 3 (HMSiR600) as a comparative example was synthesized according to the following scheme 3.

N, N, N ′, N′-tetraallyldiamino-Si-xanthone A4 was published in literature (T. Egawa, Y. Koide, K. Hanaoka, T. Komatsu, T. Terai, T. Nagano, Chem. Commun. 2011). , 47, 4162.). 70 μl (0.360 mmol, 5.0 eq) 1-bromo-2-[(1,1-dimethylethoxy) methyl] -benzene in 10 ml dry tetrahydrofuran was cooled to −78 ° C. under an argon atmosphere. 0.35 ml (1.0 M, 0.350 mmol, 4.9 equivalents) of sec-butyllithium was added dropwise, and the mixture was stirred at −78 ° C. under an argon atmosphere for 10 minutes. To this solution, 27.8 mg (72.1 μmol, 1.0 equivalent) of Compound A4 in 10 ml of dry tetrahydrofuran was added dropwise, and stirring was continued at −78 ° C. for 30 minutes. The reaction mixture was then warmed to room temperature and stirred for 15 hours under an argon atmosphere. The reaction was quenched with 1 M dilute hydrochloric acid, diluted with dichloromethane, and the aqueous layer was extracted twice with dichloromethane. The combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in 8 ml of methanol and cooled to 0 ° C. To this solution was added sodium borohydride (3.3 mg, 86.5 μmol, 1.2 equivalents), and the mixture was stirred at 0 ° C. for 10 minutes. The mixture was concentrated under reduced pressure, water was added, and the aqueous layer was extracted 3 times with ethyl acetate. The combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated in vacuo. To the green residue obtained, 1,3-dimethylbarbituric acid (135.1 mg, 0.865 mmol, 12.0 equiv), tetrakis (triphenylphosphine) palladium (0) (8.3 mg, 7.21 μmol, 0.1 equiv) and deoxygenated dichloromethane (20 ml) The mixture was stirred at room temperature for 15 hours under an argon atmosphere. Then, 19.5 mg of chloranil (79.3 μmol, 1.1 eq) was added to the reaction mixture and stirred at room temperature for 1 hour. The reaction mixture was purified by silica chromatography on a short column to remove chloranil and concentrated under reduced pressure. 2 ml of trifluoroacetic acid was added to the residue and stirred at room temperature for 16 hours. The solvent was evaporated and then dissolved in 10 ml of a 1: 1 mixture of methanol and water. The solution was stirred at room temperature for 17 hours, the methanol was evaporated and the remaining solution was filtered and purified by preparative HPLC to give 4.24 mg of blue powder (8.95 μmol, 12.4% over 4 steps).
¹ H NMR (400 MHz, (CD ₃ ) ₂ CO, closed form): δ 7.34 (d, 1H, J = 7.4 Hz), 7.25-7.22 (m, 1H), 7.19-7.16 (m, 1H), 6.94 -6.89 (m, 5H), 6.56 (d, 1H, J = 2.6 Hz), 6.54 (d, 1H, J = 2.6 Hz), 5.31 (s, 2H), 0.52 (s, 3H), 0.41 (s, ¹³ C NMR (101 MHz, CD ₃ OD + TFA): δ 152.1, 146.6, 139.0, 136.9, 131.9, 130.4, 129.3, 128.9, 128.3, 125.2, 124.3, 123.1, 93.3, 75.4, -0.1,- 0.7; HRMS (ESI ⁺ ) Calcd for [M] ⁺ , 359.15742, Found, 359.15690 (-0.52 mmu).

[Synthesis of Compound 4 (Comparative Example)]
Compound 4 (HMSiR620h) as a comparative example was synthesized according to the following scheme 4.

N, N-diallyl-N ′, N′-dimethylamino-Si-xanthone (A5) was synthesized according to Example 1 of WO2014 / 106957. 1000 μl (5.14 mmol, 25.7 equivalents) of 1-bromo-2-[(1,1-dimethylethoxy) methyl] -benzene in 10 ml of dehydrated tetrahydrofuran was cooled to −78 ° C. under an argon atmosphere. sec-Butyllithium 5.0 ml (0.99 M, 4.95 mmol, 24.8 equivalents) was added dropwise, and the mixture was stirred at −78 ° C. under an argon atmosphere for 5 minutes. To this solution, 75.4 mg (200 μmol, 1.0 equivalent) of Compound A5 in 10 ml of dehydrated tetrahydrofuran was added dropwise, and stirring was continued at −78 ° C. for 30 minutes. The reaction mixture was then warmed to room temperature and stirred for 21 hours under an argon atmosphere. The reaction was quenched with 1 M dilute hydrochloric acid and neutralized with saturated aqueous sodium bicarbonate. The aqueous layer was extracted 3 times with dichloromethane, and the combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated under reduced pressure. The residue was dissolved in 15 ml of methanol and cooled to 0 ° C. To this solution was added sodium borohydride (52.8 mg, 1.40 mmol, 14.3 eq) and stirred at 0 ° C. for 10 minutes. The mixture was concentrated under reduced pressure, water was added, and the aqueous layer was extracted twice with ethyl acetate. The combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated in vacuo. To the resulting residue, 1,3-dimethylbarbituric acid (316.0 mg, 2.02 mmol, 20.7 equiv), tetrakis (triphenylphosphine) palladium (0) (45.4 mg, 39.3 μmol, 0.4 equiv) and degassed dichloromethane (10 ml) was added and the mixture was stirred at room temperature under an argon atmosphere for 41 hours. The reaction mixture was evaporated, saturated aqueous sodium sulfate was added, and the mixture was extracted 3 times with dichloromethane. The combined organic layers were washed with saturated brine, dried over sodium sulfate, and concentrated in vacuo. The obtained residue was dissolved in 10 ml of dichloromethane, 154.7 mg of chloranil (0.629 mmol, 3.1 equivalents) was added, and the mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica chromatography on a short column to remove chloranil and concentrated under reduced pressure. 4 ml of trifluoroacetic acid was added to the residue and stirred at room temperature for 2 hours. The reaction mixture was diluted with dichloromethane and concentrated in vacuo. The remaining residue was purified by preparative HPLC to give 20.3 mg of blue powder (52.4 μmol, 26.2% over 3 steps).
¹ H NMR (400 MHz, (CD ₃ ) ₂ CO): δ 7.36 (d, J = 7.2 Hz, 1H), 7.24 (dt, J = 7.2, 1.2 Hz, 1H), 7.18 (dt, J = 7.6, 1.2 Hz, 1H), 7.06 (d, J = 8.8 Hz, 1H), 7.00 (d, J = 2.8 Hz, 1H), 6.97-6.95 (m, 2H), 6.90 (d, J = 7.2 Hz, 1H) , 6.65 (dd, J = 8.8, 2.8 Hz, 1H), 6.57 (dd, J = 8.8, 2.8 Hz, 1H), 5.34 (s, 2H), 2.92 (s, 6H), 0.57 (s, 3H), 0.45 (s, 3H); ¹³ C NMR (101 MHz, (CD ₃ ) ₂ CO): δ 149.7, 148.9, 147.3, 140.8, 140.5, 139.3, 135.0, 134.8, 129.4, 129.3, 128.0, 127.7, 124.5, 122.2 , 119.1, 117.1, 116.6, 114.8, 93.4, 73.7, 40.53, 0.4, -0.3; HRMS (ESI ⁺ ): Calcd for [M] ⁺ 387.18879, Found, 387.18872 (-0.1 mmu).

2. Absorption / fluorescence spectrum measurement of compounds 1-4 The absorption spectrum and the fluorescence spectrum of compounds 1-4 synthesized in Example 1 were measured.

[Compound 1]
Absorption / fluorescence spectra of Compound 1 (HMJSiR) dissolved in 0.2 M sodium phosphate buffer at various pHs containing 0.5% DMSO as a co-solvent were obtained. The results are shown in FIG. FIGS. 1A and 1B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 1C is a graph showing the pH dependence of absorbance and fluorescence intensity.
Experimental conditions:
Excitation wavelength of fluorescence spectrum: 580 nm, excitation / fluorescence slit width: 2.5 nm, photomultiplier tube voltage: 700V

[Compound 2]
Absorption / fluorescence spectra of compound 2 (gGlu-HMJSiR) dissolved in 0.2 M sodium phosphate buffer at various pHs containing 0.1% DMSO as a co-solvent were obtained. The results are shown in FIG. 2A and 2B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 2C is a graph showing the pH dependence of absorbance and fluorescence intensity. FIG. 2D is an absorption spectrum of Compound 2 normalized with respect to the absorption spectrum of Compound 1.
Experimental conditions:
Excitation wavelength of fluorescence spectrum: 500 nm, excitation / fluorescence slit width: 5.0 nm, photomultiplier tube voltage: 700 V

[Compound 3]
Absorption / fluorescence spectra of compound 3 (HMSiR600) dissolved in 0.2 M sodium phosphate buffer at various pHs containing 0.8% DMSO as a co-solvent were obtained. 3A and 3B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 3C is a graph showing the pH dependence of absorbance and fluorescence intensity.
Experimental conditions:
Excitation wavelength of fluorescence spectrum: 596 nm, excitation / fluorescence slit width: 2.5 nm, photomultiplier tube voltage: 700V

[Compound 4]
Absorption / fluorescence spectra of compound 4 (HMSiR620h) dissolved in 0.2 M sodium phosphate buffer at various pHs containing 0.12% or less acetonitrile and 0.68% DMSO as co-solvents were obtained. 4A and 4B are an absorption spectrum and a fluorescence spectrum, respectively, and FIG. 4C is a graph showing the pH dependence of absorbance and fluorescence intensity.
Experimental conditions: Excitation wavelength of fluorescence spectrum: 600 nm, Excitation / fluorescence slit width: 2.5 nm, Photomultiplier tube voltage: 700 V

Table 1 shows the absorption maximum wavelength (λ _abs ), fluorescence maximum wavelength (λ _fl ), quantum yield (Φ _fl ), and spirocyclization equilibrium constant (pK _cycl) obtained for compounds 1 to 4. In the table, HMSiR (650) is a compound having the following structure, and each value is S. Uno, M. Kamiya, T. Yoshihara, K. Sugawara, K. Okabe, MC Tarhan, H. Fujita, Based on T. Funatsu, Y. Okada, S. Tobita, et al., Nat. Chem. 2014, 6, 681-9.

As a result, in the range of pH 4.0-5.0 corresponding to the pH in lysosome, Compound 1 exhibits 100% ring-opened body and thus has large absorption and strong fluorescence, whereas Compound 3 and Compound 4 are The close proximity of the two pK _{cycls revealed} that there was no pH present in 100% ring-opened form and only relatively weak fluorescence was shown. Compound 2 is present in a colorless and non-fluorescent closed ring structure near physiological pH 7.4, but in addition to that, the maximum absorption wavelength is greatly shortened, so that when excited at the maximum absorption wavelength of Compound 1, it becomes more fluorescent. Was shown to be suppressed.

3. Enzyme assay of fluorescent probe γ-glutamyltranspeptidase (GGT) was added to compound 2 which is the fluorescent probe of the present invention, and the change in fluorescence intensity was measured. The results are shown in FIG. The arrow in the figure indicates the GGT addition time.
Experimental conditions:
Compound 2 (gGlu-HMJSiR) was dissolved to a concentration of 1 μM in 2.5 ml of 0.2 M sodium phosphate buffer containing 0.1% DMSO as a cosolvent, and 10 μM GGsTop was further added in the experiment in which an inhibitor was added. . The solution maintained at 37 ° C. was stirred with a magnetic stirrer, and the fluorescence intensity was measured. In experiments other than negative control, 5 mU GGT was added 30 seconds after the start of measurement. The fluorescence intensity at 662 nm was measured for a total of 1800 seconds and plotted as a function of elapsed time. The excitation wavelength was 637 nm, the slit width was 5.0 nm for both excitation and fluorescence, and the photomultiplier tube voltage was 700 V. After the experiment in which only GGT was added, several drops of concentrated hydrochloric acid were added to adjust the pH to 5, and the fluorescence intensity was measured again to calculate the fluorescence increase rate due to pH change.

  As a result, it was shown that compound 2 became a substrate for GGT, and this reaction was suppressed by the presence of GGsTop, an inhibitor of GGT. In the pH 7.4 system, the fluorescence increase rate was about 145 times, but after reacting with the enzyme for 30 minutes, when the pH of the system was lowered to 5, the fluorescence increased further by about 5 times, and the total fluorescence increase rate Reached over 700 times. This result shows that Compound 2, which is the fluorescent probe of the present invention, can detect the presence of GGT in living cells as a fluorescence response of 662 nm in the near infrared region.

4). Live cell imaging Compound 2 (gGlu-HMJSiR) was applied to 4 types of cancer cells with different γ-glutamyltranspeptidase (GGT) activities, and live cell imaging was performed. Specifically, SHIN3 human ovarian cancer-derived cells, A549 human lung cancer-derived cells with high GGT activity, and H226 human lung cancer-derived cells with low GGT activity, NMuMG normal mouse mammary epithelial cells Using. The results are shown in FIG. The scale bar represents 100 μm.

  When observed with a confocal fluorescence microscope 5 minutes after the application of Compound 2, strong fluorescence was observed from SHIN3 and A549, and only very weak fluorescence was observed from H226 and NMuMG. In addition, since the fluorescence observed from SHIN3 and A549 was suppressed by simultaneously applying GGT inhibitor GGsTop, the fluorescence response was expressed by GGT expressed in these cells and compound 2 which is the fluorescent probe of the present invention. It was shown to be based on the reaction of

Next, a co-staining experiment with LysoTracker Green was performed in order to investigate the intracellular localization of Compound 1 produced by γ-glutamyl group when Compound 2 reacted with GGT. The results are shown in FIG. The scale bar represents 5 μm.
Experimental conditions:
50 nM LysoTracker Green was added to SHIN3 cells incubated for 5 minutes after application of 1 μM compound 2, and fluorescence images were acquired in two colors immediately using a confocal fluorescence microscope.

  As a result of two-color imaging, the fluorescence observed in both the green and red channels colocalized well. This shows that Compound 2 reacts with GGT to produce Compound 1, and that Compound 1 accumulates in the lysosome in the cell, thereby further increasing the fluorescence intensity in a low pH environment.

5. In vivo imaging using ovarian cancer peritoneal seeding model mouse Compound 2 was used to perform fluorescence spectrum imaging in the mesentery of a model mouse having peritoneal metastasis.

Experimental conditions:
Fluorescence spectrum imaging was performed using a mouse model in which SHIN3 cells were seeded intraperitoneally. SHIN3-seeded model mice were established by intraperitoneal injection of 2 × 10 ⁶ SHIN3 cells suspended in 300 μl of PBS (−) into 7-8 week old female nude mice. Experiments were performed 7-10 days after injection when peritoneal seeding reached a size of about 1 mm. The mice were anesthetized by intraperitoneal injection of ketamine (2 mg / animal) and xylazine (0.2 mg / animal), and the abdominal skin was incised about 1 cm. The intestine was pulled out from the incision and placed on a black rubber plate to spread the mesentery. A solution of Compound 2 (100 μM) dissolved in 100 μl of PBS (−) was dropped onto the spread mesentery. Fluorescent images, Maestro ^TM Ex In-Vivo Imaging using System (CRi Inc.), blue- filter setting (excitation, 435 to 480 nm; emission, 490 nm long-pass) and yellow-filter setting (excitation, 576 to 621 nm; emission, 635 nm long-pass). A wavelength of 660 nm was cut out.

  The results are shown in FIG. Five minutes after the addition of Compound 2, a minute cancer on the mesentery was visualized with fluorescence, and the fluorescence intensity continued to rise for 60 minutes. Even 5 minutes after administration, small tumors on the mesentery could be observed with sufficient contrast from the background. The background is mainly autofluorescence from stool containing alfalfa remaining in the intestine, and does not cause a problem in human application.

6). In vivo imaging using a subcutaneous tumor model mouse In order to verify that Compound 2 is capable of imaging tumors deeper in the living body and that different GGT activity intensities can be distinguished in vivo, A mouse model of subcutaneous tumor was created and fluorescence imaging was performed. Two types of cancer cells, A549 cells with strong GGT activity and H226 cells with weak GGT activity, were injected into model mice, and tumors with different GGT activities were created in one individual.

Experimental conditions:
Subcutaneous tumor model mice were created by subcutaneously injecting ⁷ × 10 female nude mice with 5 × 10 ⁶ A549 cells and 1.5 × 10 ⁷ H226 cells suspended in 100 μl of PBS (−). . A549 cells were injected into the left flank of the mice, and H226 cells were injected approximately 1 cm away from the caudal side. Experiments were performed 7 days after the injection when the long sides of both tumors reached approximately 5 mm. Mice were anesthetized with an intraperitoneal injection of ketamine (2 mg / animal) and xylazine (0.2 mg / animal). 40 μl of 1 mM Compound 2 solution diluted with PBS (-) was injected intratumorally into each tumor, and 75 minutes after I2 Lumina XR in vivo imager before Compound 2 injection, immediately after injection, and 5 minutes after injection Fluorescence images were acquired every 5 minutes until and 120 minutes later.

   The results are shown in FIG. When compound 2 was injected intratumorally into each tumor, the fluorescent signal was already detectable in the A549 cell-derived tumors immediately after injection and continued to increase during the experimental time. The fluorescence from tumors derived from H226 cells was relatively weak and became detectable only 15 minutes after injection. This result was in good agreement with the difference in GGT activity detected in cultured cell imaging.

  The above results demonstrate that the microprobe expressing the peptidase can be detected with high sensitivity and speed as a fluorescence response in the near infrared region even in vivo by the fluorescent probe of the present invention.

Claims (18)

A fluorescent probe for detecting peptidase activity, comprising a compound represented by the following formula (I) or a salt thereof:

[Where,
X represents Si (R ^a ) (R ^b ) (where R ^a and R ^b each independently represents a hydrogen atom or an alkyl group);
Y represents a C ₀ -C ₃ alkylene group, and the alkylene group may be substituted with a halogen atom or haloalkyl;
R ¹ is ¹ to 4 identically independently selected from the group consisting of a hydrogen atom, a cyano group, an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group. Or represents a different substituent;
R ² together with R ³ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached;
R ⁴ together with R ⁵ forms a 5-8 membered heterocyclic structure containing the nitrogen and carbon atoms to which they are attached;
R ⁶ , R ⁷ , R ⁸ and R ⁹ each independently represent a hydrogen atom, a halogen atom, a hydroxyl group, an alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, or an azide group;
R ¹⁰ represents an amino acid-derived acyl residue. ] The heterocyclic structure formed by R ² and R ^{3 taken} together is a 6-membered ring, and the heterocyclic structure formed by R ⁴ and R ^{5 taken} together is a 6-membered ring. The fluorescent probe for detecting peptidase activity as described. The fluorescent probe for detecting peptidase activity according to claim 1 or 2, wherein R ^a and R ^b are both C ₁ -C ₄ alkyl groups. The fluorescent probe for detecting peptidase activity according to any one of claims 1 to 3, wherein R ⁶ , R ⁷ , R ⁸ and R ⁹ are all hydrogen atoms.   The fluorescent probe for detecting peptidase activity according to any one of claims 1 to 4, wherein the peptidase is γ-glutamyltranspeptidase, dipeptidylpeptidase IV (DPP-IV), or calpain. The fluorescent probe for detecting peptidase activity according to claim 1, wherein R ¹⁰ is a γ-glutamyl group. R ¹⁰ comprises proline residues, peptidase activity detecting fluorescent probe of claim 1.
The fluorescent probe for detecting peptidase activity according to claim 1, wherein the compound represented by formula (I) has the following structure.

  A cancer diagnostic composition comprising the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8.   The composition for cancer diagnosis of Claim 9 used for a cancer surgical treatment or a cancer test | inspection.   The cancer diagnostic composition according to claim 10, wherein the cancer surgical treatment or cancer examination is a wound surgery, an endoscopic surgery, or an endoscopic examination.   A method for detecting or visualizing a target cell in which a specific peptidase is expressed, using the fluorescent probe for detecting a peptidase activity according to any one of claims 1 to 8.   A step of contacting the fluorescent probe with the target cell in vitro; and a step of observing a fluorescent response due to a reaction between the peptidase specifically expressed in the target cell and the fluorescent probe, The method of claim 12.   14. The method of claim 13, comprising observing the fluorescence response using a fluorescence imaging means.   The method according to claim 12, wherein the target cell is a cancer cell.   An apparatus comprising fluorescence imaging means for observing a fluorescence response due to a reaction between a peptidase specifically expressed in a target cell and the fluorescent probe for detecting a peptidase activity according to any one of claims 1 to 8.   The apparatus of claim 16, wherein the apparatus is an endoscope or an in vivo fluorescence imaging apparatus.   A kit comprising the fluorescent probe for detecting peptidase activity according to any one of claims 1 to 8.

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