阅读说明：本技术 用于检测脑肿瘤的荧光探针 (Fluorescent probe for detecting brain tumor ) 是由 北川阳介 田中将太 齐藤延人 栗木优五 神谷真子 浦野泰照 于 2020-05-19 设计创作，主要内容包括：【问题】提供一种能够用作喷雾剂、灵敏度特异性优异并具有即时性的能够检测脑肿瘤的新型荧光探针。【解决手段】一种用于检测脑肿瘤的荧光探针,包含下式(I)所示的化合物或其盐,其中,P1表示精氨酸残基、组氨酸残基、或酪氨酸残基,P2表示脯氨酸残基或甘氨酸残基,其中,P1与相邻的N原子形成酰胺键并连接,P2与P1形成酰胺键并连接；R～(1)表示1-4个相同或不同的取代基,独立地选自由氢原子、或可分别被取代的烷基、羧基、酯基、烷氧基、酰胺基、及叠氮基组成的组；R～(2)、R～(3)、R～(4)、R～(5)、R～(6)及R～(7)各自独立地表示氢原子、羟基、可被取代的烷基、或卤原子；R～(8)及R～(9)各自独立地表示氢原子或烷基；X表示O、Si(R～(a))(R～(b))、Ge(R～(a))(R～(b))、Sn(R～(a))(R～(b))、C(R～(a))(R～(b))、或P(＝O)(R～(a))；R～(a)及R～(b)各自独立地表示氢原子、烷基、或芳基；Y表示C1-C3亚烷基。([ problem ] to provide aThe novel fluorescent probe can be used as a spray, has excellent sensitivity and specificity, and has instantaneity and can detect the brain tumor. [ MEANS FOR solving PROBLEMS ] A fluorescent probe for detecting brain tumor, comprising a compound represented by the following formula (I) or a salt thereof, wherein P1 represents an arginine residue, a histidine residue, or a tyrosine residue, and P2 represents a proline residue or a glycine residue, wherein P1 is linked to an adjacent N atom through an amide bond, and P2 is linked to P1 through an amide bond; r 1 Represents 1 to 4 same or different substituents independently selected from the group consisting of a hydrogen atom, or an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group, each of which may be substituted; r 2 、R 3 、R 4 、R 5 、R 6 And R 7 Each independently represents a hydrogen atom, a hydroxyl group, an alkyl group which may be substituted, or a halogen atom; r 8 And R 9 Each independently represents a hydrogen atom or an alkyl group; x represents O, Si (R) a )(R b )、Ge(R a )(R b )、Sn(R a )(R b )、C(R a )(R b ) Or P (═ O) (R) a )；R a And R b Each independently represents a hydrogen atom, an alkyl group, or an aryl group; y represents a C1-C3 alkylene group.)

1. A fluorescent probe for detecting brain tumors,

comprising a compound represented by the following formula (I) or a salt thereof,

(wherein,

p1 represents an arginine residue, a histidine residue, or a tyrosine residue, and P2 represents a proline residue or a glycine residue, wherein P1 is amide-bonded to the adjacent N atom, and P2 is amide-bonded to P1;

R¹represents 1 to 4 same or different substituents independently selected from the group consisting of a hydrogen atom, or an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group which may be substituted;

R²、R³、R⁴、R⁵、R⁶and R⁷Each independently represents a hydrogen atom, a hydroxyl group, an alkyl group which may be substituted, or a halogen atom;

R⁸and R⁹Each independently represents a hydrogen atom or an alkyl group;

x represents O, Si (R)^a)(R^b)、Ge(R^a)(R^b)、Sn(R^a)(R^b)、C(R^a)(R^b) Or P (═ O) (R)^a)；

R^aAnd R^bEach independently represents a hydrogen atom, an alkyl group, or an aryl group;

y represents a C1-C3 alkylene group).

2. The fluorescent probe according to claim 1, wherein the brain tumor is a glioblastoma or glioma.

3. A fluorescent probe according to claim 1 or 2, for identifying brain tumors or gliomas by detecting the presence of proteases expressed in tumor cells.

4. The fluorescent probe according to claim 3, wherein the protease is calpain 1 or cathepsin D.

5. The fluorescent probe according to any one of claims 1 to 4, wherein X is O (oxygen atom) and Y is methylene.

6. The fluorescent probe of any one of claims 1-5, wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹Are all hydrogen atoms.

7. A coating or topical spray composition for detecting tumors, comprising a solution of the fluorescent probe of any one of claims 1-6.

8. A method of detecting or visualizing a brain tumor, comprising:

a step of bringing the fluorescent probe according to any one of claims 1 to 6 into contact with a target tissue; and

and observing a fluorescent response of a reaction between the protease expressed in the target tissue and the fluorescent probe.

9. The method according to claim 8, wherein the step of bringing the fluorescent probe into contact with the target tissue is performed by locally applying or spraying a solution containing the fluorescent probe to the target tissue.

10. A method according to claim 8 or 9, comprising the step of visualising said fluorescent response using a fluorescence imaging device.

11. The method according to any one of claims 8 to 10, wherein the brain tumor is a glioblastoma or a glioma.

12. The method according to any one of claims 8 to 11, wherein the protease expressed in the target tissue is calpain 1 or cathepsin D.

Technical Field

The invention relates to a fluorescent probe for detecting brain tumors. More particularly, the present invention relates to a fluorescent probe capable of specifically detecting and labeling a brain tumor by coating or spraying a tissue sample, and a detection method using the same.

Background

Gliomas (gliomas) account for approximately 30% of primary brain tumors, with the median survival of the most malignant glioblastoma tumors being approximately 1.5 years. Even low-grade malignant gliomas rarely heal completely, mostly worsen within a few years and die within 5-10 years. In surgery for glioma, although total resection is a prognostic factor, due to functional limitations, extensive resection of the surrounding brain is not possible and visualization of the tumor has been attempted to achieve safer and maximized resection.

To date, 5-aminolevulinic acid (5-ALA) is the only safe-record used as a fluorescent agent for relevant tumor visualization. However, the method using 5-ALA has a problem of sensitivity specificity, the necessity of oral administration before operation, the duration of metabolism, and difficulty in re-administration, and further, the method has a problem that fluorescent labeling cannot be performed in cases of oncomelania, and the like (for example, non-patent documents 1 and 2). Moreover, the traditional tumor visualization method is difficult to combine the enhancement of the resection rate and the preservation of the cranial nerve function in a trade-off relationship.

Documents of the prior art

Non-patent document

Non-patent document 1: cobuiler (cobarger), et al, Neurosurgical focus (Neurosurgical focus), 36.2 (2014): e3

Non-patent document 2: felarro (FerRaro), et al, Neurosurgical review (Neurosurgical review), 39.4 (2016): 545-555

Disclosure of Invention

Problems to be solved by the invention

Therefore, it is required to develop a highly safe fluorescent probe that can be used in place of conventional 5-ALA to diagnose or visualize brain tumors by coating or local spraying. Accordingly, an object of the present invention is to provide a novel fluorescent probe that can be used as an aerosol, has excellent sensitivity and specificity, and can detect a brain tumor with immediacy.

Means for solving the problems

The present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have found that a fluorescent probe having a structure in which a specific amino acid residue is introduced as a side chain into a xanthene-like skeleton exhibits a specific fluorescent response to tumor cells or tissues of a brain tumor, and is useful for labeling and diagnosis of a brain tumor. Moreover, the fluorescent probe is found to be used for solving a plurality of problems of the traditional fluorescent labeling method, and a novel brain tumor operation supporting technology capable of improving the problems can be realized. Based on these findings, the present invention has been completed.

That is, in one aspect of the present invention, there is provided:

<1> a fluorescent probe for detecting brain tumor, which is characterized by comprising a compound represented by the following formula (I) or a salt thereof,

formula 1:

wherein P1 represents an arginine residue, a histidine residue, or a tyrosine residue, and P2 represents a proline residue or a glycine residue, wherein P1 is amide-bonded to the adjacent N atom, and P2 is amide-bonded to P1; r¹Represents 1 to 4 same or different substituents independently selected from the group consisting of a hydrogen atom, or an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group, each of which may be substituted; r²、R³、R⁴、R⁵、R⁶And R⁷Each independently represents a hydrogen atom, a hydroxyl group, an alkyl group which may be substituted, or a halogen atom; r⁸And R⁹Each independently represents a hydrogen atom or an alkyl group;

x represents O, Si (R)^a)(R^b)、Ge(R^a)(R^b)、Sn(R^a)(R^b)、C(R^a)(R^b) Or P (═ O) (R)^a)；

R^aAnd R^bEach independently represents a hydrogen atom, an alkyl group, or an aryl group; y represents a C1-C3 alkylene group;

<2> the fluorescent probe according to <1>, wherein the brain tumor is glioblastoma or glioma;

<3> the fluorescent probe according to <1> or <2> above, for identifying a brain tumor or glioma by detecting the presence of a protease expressed in a tumor cell;

<4> the fluorescent probe according to <3>, wherein the protease is calpain 1 or cathepsin D;

<5> the fluorescent probe according to any one of <1> to <4> above, wherein X is O (oxygen atom) and Y is methylene;

<6>according to the above<1>-<5>The fluorescent probe of any one of the above, wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹Are each a hydrogen atom; and

<7> a coating type or local spray type composition for detecting tumor, which comprises the solution of the fluorescent probe of any one of <1> to <6 >.

And, in another aspect, the invention relates to a method of detecting or visualizing a brain tumor or glioma,

<8> a method for detecting or visualizing a brain tumor, which comprises the step of contacting the fluorescent probe of any one of <1> to <6> above with a target tissue; and observing a fluorescent response of a reaction between the protease expressed in the target tissue and the fluorescent probe;

<9> the method according to <8>, wherein the step of bringing the fluorescent probe into contact with the target tissue is performed by locally applying or spraying a solution containing the fluorescent probe to the target tissue;

<10> the method according to <8> or <9> above, characterized by comprising a step of visualizing the fluorescence response using a fluorescence imaging device;

<11> the method according to any one of <8> and <10>, wherein the brain tumor is glioblastoma or glioma; and

<12> the method according to any one of <8> to <11>, wherein the protease expressed in the target tissue is calpain 1 or cathepsin D.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the fluorescent probe of the invention, the brain tumor can be detected and marked specifically, with high sensitivity and in real time. Since the fluorescent probe of the present invention can be directly and locally coated or sprayed on a brain tumor, the presence of a brain tumor or the like can be accurately, rapidly and highly sensitively identified and imaged by a simple method regardless of the formulation, and an excellent effect is obtained.

Thus, by marking the affected part of a brain tumor or the like in the operation, it is expected to improve the resection rate and the overall survival rate. In addition, the problem that no fluorescent probe can be labeled for low-malignant glioma at present can be solved.

The detection method using the fluorescent probe of the present invention can perform detection by visible light safe to living bodies, and has extremely excellent practical applicability in that the amount of the fluorescent probe used is extremely small, in addition to the simple detection procedure.

Drawings

FIG. 1 is a fluorescence image obtained when a fluorescent probe compound group was added to a fresh raw specimen of a tumor and a tumor periphery.

FIG. 2 is a graph showing the arrangement of changes in fluorescence intensity obtained when a fluorescent probe compound group is added to a fresh raw specimen of a tumor and a tumor periphery.

FIG. 3 is a graph showing the presence or absence of a fluorescent response in a fluorescent probe compound group.

FIG. 4 is a graph showing the change in fluorescence intensity of calpain 1 and cathepsin D due to the reaction with fluorescent probe YK 190.

FIG. 5 shows fluorescence images of SiRNA transfected with liposomes (Lipofectamine) using a U87 glioblastoma cell line, and observed after incubation in the reaction with a fluorescent probe.

FIG. 6 is a graph showing the enzyme expression levels by real-time PCR.

Detailed Description

Hereinafter, embodiments of the present invention will be described. The scope of the present invention is not limited to these descriptions, and can be modified and implemented as appropriate within a range not to impair the gist of the present invention, except for the following examples.

1. Definition of

Herein, "amino acid residue" refers to a structure corresponding to the remaining part of the structure (acyl group) from which a hydroxyl group is removed from the carboxyl group of an amino acid. Also, any compound may be used for the "amino acid" as long as the compound has both an amino group and a carboxyl group, including natural and non-natural compounds. Any of neutral amino acids, basic amino acids, or acidic amino acids may be used, and in addition to amino acids that act as transmitters themselves such as neurotransmitters, amino acids that are components of polypeptide compounds such as bioactive peptides (including oligopeptides in addition to dipeptides, tripeptides, and tetrapeptides) and proteins, for example, α amino acids, β amino acids, γ amino acids, and the like may also be used. As the amino acid, an optically active amino acid is preferably used. For example, any of D-and L-amino acids can be used as the alpha amino acid, but it is preferable to select an optically active amino acid that functions in an organism.

Herein, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

As used herein, an "alkyl group" can be any of aliphatic hydrocarbon groups consisting of straight chain, branched chain, cyclic, or combinations thereof. The number of carbon atoms of the alkyl group is not particularly limited, but for example, the number of carbon atoms is 1 to 20 (C)_1-20) The number of carbon atoms is 3-15 (C)_3-15) The number of carbon atoms is 5-10 (C)_5-10). When the number of carbon atoms is specified, it refers to "alkyl" groups having a number of carbon atoms within the range of the number. E.g. C_1-8Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, n-heptyl, and n-octyl, and the like. Herein, the alkyl group may have one or more of any substituent. Examples of such substituents includeIncluding but not limited to alkoxy, halogen, amino, mono-or di-substituted amino, substituted silyl, or acyl groups, and the like. When the alkyl group has two or more substituents, they may be the same or different. The same applies to alkyl moieties containing other substituents of the alkyl moiety (e.g., alkanyl, aralkyl, etc.).

Herein, when it is defined that a specific functional group "may have a substituent", the type of the substituent, the substitution position, and the number of the substituent are not particularly limited, and when two or more substituents are present, they may be the same or different. Examples of substituents include, but are not limited to, alkyl, alkoxy, hydroxy, carboxy, halogen, sulfo, amino, alkoxycarbonyl, oxo, and the like. Among these substituents, a substituent may also be present. Examples include, but are not limited to, halogenated alkyls, dialkylaminos, and the like.

As used herein, "alkylene" is a divalent group consisting of straight or branched chain saturated hydrocarbons, and includes, for example, methylene, 1-methylmethylene, 1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene, 1-dimethylethylene, 1, 2-dimethylethylene, 1-diethylethylene, 1, 2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltrimethylene, 1-dimethyltrimethylene, 1, 2-dimethyltrimethylene, 2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1-diethyltrimethylene, 1, 2-diethyltrimethylene, 2, 2-diethyltrimethylene group, 2-ethyl-2-methyltrimethylene group, tetramethylene group, 1-methyltetramethylene group, 2-methyltetramethylene group, 1-dimethyltetramethylene group, 1, 2-dimethyltetramethylene group, 2-di-n-propyltrimethylene group and the like.

As used herein, an "alkoxy group" is a structure in which the above alkyl group is bonded to an oxygen atom, and for example, a saturated alkoxy group that may include a straight chain, a branched chain, a cyclic group, or a combination thereof. For example, as preferable examples, methoxy, ethoxy, n-propoxy, isopropoxy, cyclopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, cyclobutoxy, cyclopropylmethoxy, n-pentoxy, cyclopentoxy, cyclopropylethoxy, cyclobutylmethoxy, n-hexoxy, cyclohexoxy, cyclopropylpropoxy, cyclobutylethoxy, cyclopentylmethoxy and the like can be mentioned.

As used herein, the "aryl group" may be either a monocyclic or fused polycyclic aromatic hydrocarbon group, or an aromatic heterocyclic group containing one or more hetero atoms (for example, oxygen atom, nitrogen atom, sulfur atom, or the like) as ring-constituting atoms. In this case, this may be referred to as "heteroaryl" or "heteroaromatic". Aryl groups, whether monocyclic or fused, can be bonded at all possible positions. Non-limiting examples of monocyclic aryl groups can include phenyl (Ph), thienyl (2-or 3-thienyl), pyridyl, furyl, thiazolyl, oxazolyl, pyrazolyl, 2-pyrazinyl, pyrimidinyl, pyrrolyl, imidazolyl, pyridazinyl, 3-isothiazolyl, 3-isoxazolyl, 1, 2, 4-oxadiazol-5-yl or 1, 2, 4-oxadiazol-3-yl, and the like. Non-limiting examples of the fused polycyclic aryl group may include 1-naphthyl group, 2-naphthyl group, 1-indenyl group, 2, 3-indan-1-yl group, 2, 3-indan-2-yl group, 2-anthryl group, indazolyl group, quinolyl group, isoquinolyl group, 1, 2-dihydroisoquinolyl group, 1, 2, 3, 4-tetrahydroisoquinolyl group, indolyl, isoindolyl, phthalazinyl, quinoxalinyl, benzofuranyl, 2, 3-dihydrobenzofuran-1-yl, 2, 3-dihydrobenzofuran-2-yl, 2, 3-dihydrobenzothien-1-yl, 2, 3-dihydrobenzothien-2-yl, benzothiazolyl, benzimidazolyl, fluorenyl, or thioxanthyl, and the like. In this context, an aryl group may have one or more arbitrary substituents on its ring. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono-or di-substituted amino groups, substituted silyl groups, or acyl groups, and the like. When the aryl group has two or more substituents, they may be the same or different. The same applies to aryl moieties that contain another substituent of the aryl moiety (e.g., aryloxy or aralkyl, etc.).

As used herein, "alkylamino" and "arylamino" refer to-NH₂An amino group in which a hydrogen atom of the group is substituted with one or two of the above-mentioned alkyl groups or aryl groups. For example, methylamino, dimethylamino, ethylamino, diethylamino, ethyl group may be includedMethylamino, benzylamino, and the like. Likewise, "alkylthio" and "arylthio" refer to groups in which the hydrogen atom of the-SH group is replaced by an alkyl or aryl group as described above. For example, methylthio, ethylthio, benzylthio and the like can be included.

As used herein, "amide" includes both RNR 'CO- (alkylaminocarbonyl-, in the case of R ═ alkyl) and RCONR' - (alkylcarbonylamino-, in the case of R ═ alkyl).

As used herein, "ester" includes both ROCO- (alkoxycarbonyl-, in the case of R ═ alkyl) and RCOO- (alkylcarbonyloxy-, in the case of R ═ alkyl).

As used herein, the term "ring structure" when formed by a combination of two substituents refers to a heterocyclic or carbocyclic group, which may be saturated, unsaturated, or aromatic. Thus, cycloalkyl, cycloalkenyl, aryl, and heteroaryl groups as defined above are included. For example, cycloalkyl, phenyl, naphthyl, morpholinyl, piperidinyl, imidazolyl, pyrrolidinyl, pyridyl, and the like can be included. In this context, a substituent may form a ring structure with another substituent, and when such substituents are bonded to each other, those skilled in the art will appreciate that certain substitutions are formed, for example, a bond to hydrogen. Thus, when particular substituents are described together to form a ring structure, it will be understood by those skilled in the art that the ring structure may be formed or readily formed by conventional chemical reactions. The relevant ring structures and their formation are within the purview of those skilled in the art.

2. Fluorescent probe molecules

The fluorescent probe for detecting brain tumor of the present invention has a structure in which a dipeptide site, which is a substrate for a specific protease, is introduced into a xanthene-like skeleton as a side chain, and in one embodiment, the fluorescent probe comprises a compound having a structure represented by the following formula (I) or a salt thereof.

Chemical formula 2:

in the general formula (I), P1 represents an arginine residue, a histidine residue, or a tyrosine residue, and P2 represents a proline residue or a glycine residue. As demonstrated in the examples below, in glioblastoma, a type of localized brain tumor, the dipeptide site consisting of P1 and P2 is selected from the point of view of the combination of amino acid residues specifically expressed by certain types of proteases (peptidases) and susceptible to selective hydrolysis by the relevant proteases. The protease that cleaves the dipeptide site consisting of P1 and P2 is preferably calpain 1 or cathepsin D. Thus, in the combination of P1 and P2, preferably P1 is an arginine residue, a histidine residue, or a tyrosine residue, P2 is a proline residue or a glycine residue, more preferably P1 is an arginine residue, and P2 is a proline residue.

Wherein P1 is linked by amide bond formation with the adjacent NH in formula (la), i.e. the xanthene backbone is linked by amide bond formation of the carbonyl moiety of amino acid residue P1 with the NH in formula (I). In addition, P1 can be linked to P2 in the same manner as a normal peptide chain, and as a result, P2 forms an amide bond with P1 and is linked. As described above, the "amino acid residue" refers to a structure corresponding to the remaining part of the structure in which a hydroxyl group is removed from a carboxyl group of an amino acid. Thus, P2 has the same structure as the so-called N-terminal residue, and P1 as the intermediate amino acid residue can be linked to P2 in the same manner as a normal peptide chain.

In the above general formula (I), R¹Represents 1 to 4 same or different substituents independently selected from the group consisting of a hydrogen atom, or an alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group, each of which may be substituted. In the case of having two or more substituents on the benzene ring, they may be the same or different from each other. Preferably a hydrogen atom as R¹。

R²、R³、R⁴、R⁵、R⁶And R⁷Each independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom. Preferably, R³And R⁴Is a hydrogen atom. And, also preferably, R²、R⁵、R⁶And R⁷Is a hydrogen atom. More preferably, R²、R³、R⁴、R⁵、R⁶And R⁷Are all hydrogen atoms。

R⁸And R⁹Each independently represents a hydrogen atom or an alkyl group. At R⁸And R⁹In the case where they all represent an alkyl group, they may be the same or different. For example, at R⁸And R⁹In the case of both being hydrogen atoms, preferably R⁸Is alkyl and R⁹Is a hydrogen atom. More preferably, R⁸And R⁹Are all hydrogen atoms.

In the above formula (I), X represents O, Si (R)^a)(R^b)、Ge(R^a)(R^b)、Sn(R^a)(R^b)、C(R^a)(R^b) Or P (═ O) (R)^a). Wherein R is^aAnd R^bEach independently represents a hydrogen atom, an alkyl group, or an aryl group. At R^aAnd R^bIn the case of alkyl groups, they may have one or more substituents, and as such substituents, there may be mentioned, for example, one or more alkyl groups, alkoxy groups, halogen atoms, hydroacid groups, carboxyl groups, amino groups, sulfo groups and the like. R^aAnd R^bThere may be alkyl groups having 1 to 6 carbon atoms each of which may be substituted, both of which are preferably methyl groups. And, in some cases, R^aAnd R^bMay be bonded to each other to form ring structures. For example, at R^aAnd R^bIn the case of both alkyl radicals, R^aAnd R^bMay be bonded to each other to form a spiro carbocyclic ring. The ring formed is preferably, for example, an approximately 5 to 8 membered ring. X is preferably O (oxygen atom).

Y represents C₁-C₃. The alkylene group may be one of a linear alkylene group or a branched alkylene group. For example, in addition to methylene (-CH)₂-) ethylene (-CH₂-CH₂-) propylene (-CH)₂-CH₂-CH₂-) outside of the group, -CH (CH)₃)-、-CH₂-CH(CH₃)-、-CH(CH₂CH₃) And the like may also be used as the branched alkylene group. Among them, a methylene group or an ethylene group is preferable, and a methylene group is more preferable.

The compounds of the general formula (I) can be present in the form of salts. Such salts may include base addition salts, acid addition salts, amino acid salts, and the like. Base addition salts may include metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, etc., ammonium salt, or organic amine salts such as triethylamine salt, piperidine salt, morpholine salt, etc., and acid addition salts may include inorganic acid salts such as hydrochloride, sulfate, nitrate, etc., organic acid salts such as carboxylate, methanesulfonate, p-toluenesulfonate, citrate, oxalate, etc., and the like. The amino acid salt may include a glycine salt and the like. However, these salts are not limited.

The compound represented by the formula (I) may have 1 or 2 or more asymmetric carbons depending on the kind of the substituent, and stereoisomers such as optical isomers or diastereomers may be present. Stereoisomers in pure form, any mixture of stereoisomers, racemates and the like are included within the scope of the present invention.

The compound represented by the formula (I) or a salt thereof may exist in the form of a hydrate or a solvate, and these substances are included in the scope of the present invention. The type of the solvent for forming the solvate is not particularly limited, and examples thereof include solvents such as ethanol, acetone, and isopropyl alcohol.

The compounds of formula (I) can be readily prepared by the following method: for example, a xanthene compound having amino groups at the 3-and 6-positions and a 2-carboxytolyl group or 2-alkoxycarbonylphenyl group at the 9-position is used as a starting material, and the 2-carboxytolyl group or 2-alkoxycarbonylphenyl group at the 9-position is converted into a hydroxyalkyl group to aminoacylate the 3-position. The 3, 6-diaminoxanthene compound that can be used as a raw material may include, for example, Rhodamine (Rhodamine)110 or Rhodamine 123 and the like that are commercially available, but is not limited thereto, and an appropriate xanthene compound may be selected depending on the structure of the target compound. Further, a fluorescent probe having the same function as that of the general formula (I) in the present invention can also be produced by using a compound having a skeleton in which an oxygen atom of a xanthene skeleton portion in the compound represented by the general formula (I) is substituted with a C atom or an Si atom having a specific substituent, or a Ge atom or a Pb atom.

Also, since the preparation methods of representative compounds included in the compounds of the present invention represented by the general formula (I) are specifically shown in the examples herein, one skilled in the art can easily prepare any compound included in the general formula (I) by referring to the disclosure herein and appropriately selecting starting materials, reagents, reaction conditions, and the like as needed.

The fluorescent probe can be used as a composition by mixing with an additive for preparing a reagent in a sugar refinery as required. For example, additives such as a dissolution assistant, a pH adjuster, a buffer, a tonicity agent (tonicity agent), and the like may be used as additives for physiological environments, and the mixing thereof may be appropriately selected by those skilled in the art. These compositions may be provided as a composition in a suitable form such as a mixture in powder form, a lyophilizate, a granule, a tablet, a liquid, and the like.

3. Mechanism of luminescence of fluorescent probe molecules

The mechanism of fluorescence emission of the fluorescent probe of the present invention is explained below.

The compound represented by the above formula (I) is a skeleton of a xanthene-based fluorescent dye which is widely used for biological imaging because of its high water solubility, high fluorescence quantum yield, and the like, but when the upper part of the xanthene skeleton is in a closed loop state, the fluorescent probe itself is substantially not absorbed and does not fluoresce (the fluorescence response is in an Off state) in a neutral region (for example, a range of pH5 to 9). In response to these, as shown in the following figure, the dipeptide site in P2-P1-NH is hydrolyzed by protease and, when cleaved from the xanthene skeleton, rapidly changes to a ring-opened tautomer to produce a strongly fluorescent compound (II).

And (3) conversion:

that is, the fluorescent probe of the present invention containing the compound represented by the general formula (I) or a salt thereof as an active ingredient has a property that the ring-opened compound (II) is hydrolyzed by a protease expressed in a brain tumor tissue and emits strong fluorescence. Thus, by using the fluorescent probes of the present invention, the presence of brain tumors expressing the protease can be detected or labeled. The relevant proteases may include calpain 1 or cathepsin D as described above.

In more detail, for example, the compound represented by the general formula (I) wherein X is an O atom or a salt thereof hardly emits light when irradiated with, for example, excitation light of about 440-510nm in the neutral region, but the above ring-opened compound has a property of emitting strong fluorescence (for example, emission (emission): 524nm) under the same conditions. Therefore, when the fluorescent probe of the present invention is used for detection, it is generally sufficient to irradiate visible light of about 440-510 nm. The wavelength of fluorescence to be observed is generally about 510-800nm, for example, it is preferable to observe fluorescence at about 516-556 nm.

4. Detection/visualization method using fluorescent probes

Brain tumors expressing specific proteases can also be specifically detected or visualized by using the above-mentioned fluorescent probes, according to the relevant luminescence mechanism.

Specifically, the method of the present invention is characterized by comprising:

A) bringing the fluorescent probe into contact with a target tissue; and

B) and a step of observing a fluorescent response of a reaction between the protease expressed in the target tissue and the fluorescent probe. Only the target tissue (or target cells) expressing a particular protease can be specifically detected or visualized as a fluorescent signal. Furthermore, the term "detecting" herein should be interpreted in its broadest sense, including detecting for various purposes, e.g., quantitatively, qualitatively, etc.

As mentioned above, the specific protease may preferably be calpain 1 or cathepsin D. However, it is not limited thereto. The target tissue is a brain tumor tissue. Brain tumors may include glioblastomas and gliomas. Tumor cells present in brain tumor tissue may also be targeted.

Also, the method of the present invention may further comprise the step of observing the above-described fluorescent response using a fluorescence imaging device. In the above step of observing the fluorescence response, a fluorescence photometer having a wide measurement wavelength may be used, but the fluorescence response may also be visualized by using a fluorescence imaging device capable of displaying the above fluorescence response as a two-dimensional image. By using the step of fluorescence imaging, the fluorescence response can be visualized in two dimensions, so that the target tissue (or cell) expressing the above protease can be instantaneously recognized. As the fluorescence imaging device, a device known in the art may be used. Further, if necessary, the reaction of the protease with 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 step a), as a method of bringing the sample to be measured into contact with the fluorescent probe, typically, a step of spraying, adding, or applying a solution containing the fluorescent probe to the sample may be included, but may be appropriately selected depending on the form of the sample, the measurement environment, and the like. In the case of spraying a solution containing a fluorescent probe onto a sample, for example, a solution including: a liquid medicine storage section for storing a liquid medicine containing a fluorescent probe; and a chemical liquid spraying section configured to be capable of spraying the chemical liquid onto a sample to be measured. The associated device may be an endoscope. Endoscopes having such a function may include those having the structures disclosed in japanese patent laid-open nos. 2010-240188 and 2015-23904. Also, since the apparatus observes a fluorescent response through the fluorescent probe, the fluorescent imaging method as described above may be further included.

The concentration of the fluorescent probe of the present invention to be used is not particularly limited, but, for example, a solution having a concentration of about 0.1 to 10. mu.M can be suitably used.

The compound represented by the above formula (I) or a salt thereof can be used as it is as the fluorescent probe of the present invention, but an additive usually used for preparing a reagent may be added as a composition as needed. For example, as an additive for using the agent in a physiological environment, additives such as a dissolution assistant, a pH adjuster, a buffer, a tonicity agent, and the like may be applied, and the mixing amount thereof may be appropriately selected by those skilled in the art. These compositions are generally provided in the form of a composition in an appropriate form, such as a mixture in the form of powder, a lyophilizate, a granule, a tablet, a liquid, etc., but when used, it can be suitably dissolved in distilled water for injection or an appropriate buffer.

The detection or visualization of brain tumors according to the method of the invention can generally be performed under neutral conditions, e.g., in the range of pH5.0-9.0, preferably in the range of pH6.0-8.0, more preferably in the range of pH 6.8-7.6. As a method of adjusting pH, for example, any pH adjusting agent or buffer solution known in the art, for example, phosphate buffer, may be applied.

Brain tumors detected or visualized by the methods of the invention can be used to diagnose these tumors. The term "diagnosis" herein is to be interpreted in its broadest sense, including the visual or microscopic confirmation of the presence of tumor tissue in any biological site.

The detection method of the present invention can be used further, for example, in surgery or examination. The term "surgery" herein is any application for diagnosing and treating tumors, including endoscopic procedures, such as neuroendoscopy, gastroscopy, colonoscopy, laparoscopy or thoracoscopy, in addition to procedures such as open wound craniotomy, puncture surgery, thoracotomy, or laparotomy, or skin surgery, and the like. Also, the term "examination" includes an examination using an endoscope, a treatment such as excision and collection of a tissue related to the examination, and an extracorporeal examination of a tissue separated/collected from a cargo lift, and the like.

As one of preferable modes, for example, the fluorescent probe of the present invention is applied to a part or all of the surgical field by an appropriate method such as spraying, coating or injecting under the naked eye or microscope, and after several tens of seconds to several minutes, emits light having a wavelength of about 50nm, and can irradiate the applied site. In the case where the site to which it is applied contains tumor tissue, since the tissue fluoresces, the tissue is recognized as tumor tissue and excised together with surrounding tissue including the tumor tissue. For example, in the case of surgical treatment of tumors, not only can a confirmed diagnosis be visually confirmed for tumor tissues, but also infiltration and metastasis of lymph tissues such as lymph nodes and peripheral organ tissues can be diagnosed, and the range of resection can be determined by performing intraoperative rapid diagnosis.

In another preferred embodiment, the fluorescent probe of the present invention is applied to an examination site by an appropriate method such as spraying, coating, or injection, for example, in an endoscopic examination, and after several tens of seconds to several minutes, the site to which light is applied is irradiated with light having a wavelength of about 500nm, and when a tissue that fluoresces is confirmed, the tissue can be recognized as a tumor tissue. When the tumor tissue can be confirmed in the endoscopic examination, the tissue can be subjected to an examination resection or a therapeutic resection.

[ examples ]

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[ example 1]

1. Synthesis of fluorescent probes

Hydroxymethyl rhodamine green (HRMG) having various dipeptide sites was synthesized according to the reaction scheme described in example 1 of International publication WO 2016/006678. By making various changes to the Fmoc-amino acid used in the synthesis of compound a7 in this example 1, compounds having different dipeptide sites were obtained.

In the synthesized compound, R in the above general formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸And R⁹Each hydrogen atom, X ═ O, Y ═ methylene, and P1 and P2 are the following combinations.

TABLE 1

Compound (I)	P1	P2
			YK190	Arginine residue	Proline
YK213	Histidine	Glycine
			YK19	L-alanine	Glycine
YK281	Methionine alum	Acetylleucine

Example 2

2. Screening by fluorescent probes

The fluorescent probe compound groups containing YK190, YK213, YK19, and YK281 synthesized in example 1 were added to fresh specimens of tumors and tumor peripheries, and the fluorescence intensities were compared. Glioblastoma was used for the samples. As a comparative example, hydroxymethylrhodamine green (HRMG) without a dipeptide site was used.

After dissolving 0.5. mu.L of each fluorescent probe In 10mM of 0.5. mu.L of RPMI1640 (phenol-free) (final probe concentration: 50. mu.M) and dropping 200. mu.L of each sample, the fluorescence intensity was measured with time using a Maestro In Vivo Imaging System Ex.

Excitation wavelength: 465nm

Emission wavelength: 515nm

As shown in fig. 1 and 2, it was found that (1) YK190 and (2) YK213 were labeled with tumor-specific fluorescence compared to the peripheral tissues, while (3) YK218 was not much different from the peripheral side tissues. (5) HRMG (b) in (b) is a comparative example having no dipeptide site in the side chain. As shown in fig. 2, it was found that YK190 and YK213 both obtained higher fluorescence intensity in tumors, but YK190 can be said to be a better fluorescent probe because it showed almost 2-fold fluorescence intensity. And (4) YK19 also obtained excellent tumor-specific fluorescent response.

FIG. 3 is a graph summarizing the results of screening for fluorescence responses of these fluorescent probe compounds (the horizontal axis represents the sample, the vertical axis represents the probe name, and gray color in which no fluorescence response is observed).

Example 3

3. Identification of proteases

Next, proteases associated with the fluorescence response obtained in example 2 were identified.

Cathepsin D, cytosol non-specific dipeptidase, calpain 1, cytosol aminopeptidase were identified as candidate enzymes using DEG (electrophoretic gel) assay and LC MS/MS for protease analysis by peptide mass fingerprinting.

As a result of measuring changes in fluorescence intensity using inhibitors (SNJ-1945 and Amastatin) for these candidate enzymes, it was found that the fluorescence of calpain 1 and cathepsin D was inhibited by SNJ-1945.

When changes in fluorescence intensity due to reaction with the fluorescent probe YK190 were measured for these calpain 1 and cathepsin D, a significant increase in fluorescence intensity was observed as shown in fig. 4, indicating that the fluorescent response of glioblastoma in example 2 is caused by the reaction of these enzymes with the fluorescent probe.

Further, in searching for the expressed enzyme by real-time PCR, it was found that the gene expression of calpain 1 and cathepsin D was increased in the tumor lysate as compared with that in the tumor periphery, which is consistent with the above results.

Example 4

4. Validation Using the U87 cell line by SiRNA

Calpain 1 and cathepsin D were knocked out by SiRNA in cell lines, and reaction with fluorescent probe YK190 was confirmed. The experiment was carried out in the following procedure.

The reaction with the fluorescent probe was confirmed after transfection of SiRNA with liposome using U87 glioblastoma cell line and 48 hours of culture.

As a result, both calpain 1 and cathepsin D expression were reduced in the U87 cell line compared to the control SiRNA cell line (fig. 5). The results also indicate that the fluorescent response to glioblastoma in example 2 is caused by the reaction of these enzymes with the fluorescent probe. In addition, as an experiment supporting this, the enzyme expression level was confirmed by real-time PCR, and the results were consistent (fig. 6).