Fluorescent probes for cancer detection

Abstract

Provided are fluorescent probes capable of detecting breast cancer, lung cancer, and squamous cell carcinoma. A fluorescent probe for detecting breast cancer and benign breast tumors comprises a compound represented by the following general formula (I) or a salt thereof.

Description

Technical Field

[0001] The present invention relates to a fluorescent probe capable of specifically detecting cancer, and more particularly to a fluorescent probe capable of detecting malignant and benign breast tumors, lung adenocarcinoma, or lung squamous cell carcinoma. Background Art

[0002] Methods for evaluating enzyme activity in malignant tissues can provide information on effective biomarkers for guiding cancer detection. Fluorescence-induced detection of cancer is one of the most promising methods for improving the efficiency of local excision surgery. The research team of the present inventors has so far developed an active aminopeptidase-responsive fluorescent probe (Non-Patent Document 1) and successfully detected human breast cancer and esophageal cancer within a few minutes by locally spraying the probe solution (Non-Patent Documents 2 and 3).

[0003] However, many malignancies cannot be detected with high sensitivity / specificity by these fluorescent probes due to the lack of obvious differences in aminopeptidase activity between cancer and normal tissues.

[0004] Prior art literature

[0005] Non-patent literature

[0006] Non-patent document 1: Urano, Y. et al. Science Transl Med, 3, 110 (2011)

[0007] Non-patent document 2: Onoyama, H. et al. Sci. Rep., 6, 26399 (2016)

[0008] Non-patent document 3: Ueo, H. et al. Sci. Rep., 5, 12080 (2015) Summary of the Invention

[0009] Problems to be solved by the invention

[0010] An object of the present invention is to provide a fluorescent probe capable of detecting malignant and benign breast tumors, lung adenocarcinoma, or lung squamous cell carcinoma.

[0011] Solutions for solving problems

[0012] The present inventors focused on glycosidase activity in cancer, prepared various fluorescent probes for detecting different glycosidase activities, and used these fluorescent probes to conduct comprehensive screening of glycosidase activity in human surgical specimens. They discovered that glycosidase activity varies depending on the type of tumor and that certain fluorescent probes are effective for specific imaging of tumors, which led to the completion of the present invention.

[0013] That is, the present invention provides:

[0014] [1] A fluorescent probe for detecting breast cancer and benign breast tumors, comprising a compound represented by the following general formula (I) or a salt thereof.

[0015]

[0016] In formula (I),

[0017] When R1 is present, R1 represents the same or different monovalent substituents present on the benzene ring;

[0018] R2 and R3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;

[0019] R4 and R5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;

[0020] R6 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluoroalkyl group having 1 to 5 carbon atoms;

[0021] When R7 and R8 are present, R7 and R8 each independently represent an alkyl group or an aryl group having 1 to 6 carbon atoms,

[0022] Here, when X is an oxygen atom, R7 and R8 do not exist;

[0023] X represents an oxygen atom, a silicon atom or a carbon atom;

[0024] n is an integer from 1 to 3;

[0025] L is selected from any of the following formulae (1) to (6),

[0026]

[0027] [2] The fluorescent probe according to [1], wherein X is an oxygen atom.

[0028] [3] The fluorescent probe according to [1] or [2], wherein R6 is a fluorinated alkyl group having 1 to 5 carbon atoms.

[0029] [4] A fluorescent probe for detecting breast cancer and benign breast tumors, comprising the fluorescent probe described in any one of [1] to [3] and a GGT activity detection probe.

[0030] [5] A fluorescent probe for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising a compound represented by the following general formula (I) or a salt thereof,

[0031]

[0032] In formula (I),

[0033] When R1 is present, R1 represents the same or different monovalent substituents present on the benzene ring;

[0034] R2 and R3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;

[0035] R4 and R5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;

[0036] R6 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluoroalkyl group having 1 to 5 carbon atoms;

[0037] When R7 and R8 are present, R7 and R8 each independently represent an alkyl group or an aryl group having 1 to 6 carbon atoms,

[0038] Here, when X is an oxygen atom, R7 and R8 do not exist;

[0039] X represents an oxygen atom, a silicon atom or a carbon atom;

[0040] n is an integer from 1 to 3;

[0041] L is selected from the following groups of formula (5) or (6),

[0042]

[0043] [6] The fluorescent probe according to [5], wherein X is an oxygen atom.

[0044] [7] The fluorescent probe according to [5] or [6], wherein R6 is a fluorinated alkyl group having 1 to 5 carbon atoms.

[0045] [8] A method for detecting breast cancer and benign breast tumors, comprising the following steps: (a) applying the fluorescent probe described in any one of [1] to [4] to a clinical specimen of the breast; and (b) measuring a fluorescent image of the clinical specimen of the breast to which the fluorescent probe is applied.

[0046] [9] A method for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising the following steps: (a) applying the fluorescent probe described in any one of [5] to [7] to a clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma; and

[0047] (b) Fluorescent images of clinical specimens of lung adenocarcinoma or lung squamous cell carcinoma to which the fluorescent probes are applied are measured.

[0048]

[10] A fluorescent probe for detecting breast cancer and benign breast tumors, comprising the fluorescent probe described in any one of [1] to [4] and a GGT activity detection probe.

[0049]

[11] The fluorescent probe for detecting breast cancer and benign breast tumors according to

[10] , wherein the GGT activity detection probe is a compound represented by the following general formula (II) or a salt thereof,

[0050]

[0051] In formula (II),

[0052] X represents Si(R a )(R b )、Ge(R a )(R b )、Sn(R a )(R b )、C(R a )(R b )、P(=O)(R a ) or O,

[0053] Here, R a and R b each independently represents a hydrogen atom, an alkyl group or an aryl group;

[0054] R 1 represents a hydrogen atom or 1 to 4 identical or different substituents independently selected from the group consisting of optionally substituted alkyl, carboxyl, ester, alkoxy, amide and azide groups;

[0055] R 2 represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, an aryl group or a heteroaryl group;

[0056] R 3 and R 4 Each independently represents: a hydrogen atom; 1 to 3 identical or different substituents independently selected from the group consisting of a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, and an azide group;

[0057] R 5 、R 6 and R 7 Each independently represents a hydrogen atom or an alkyl group,

[0058] Here, R 6 or R 7 Optionally, each together with R4 forms a ring structure including the nitrogen atom to which they are bonded;

[0059] R 8 represents an acyl residue derived from an amino acid.

[0060]

[12] A fluorescent probe for detecting breast cancer and benign breast tumors, comprising the fluorescent probe described in any one of [1] to [4] and a compound of the following formula (7) or a salt thereof,

[0061]

[0062]

[13] A kit for detecting breast cancer and benign breast tumors, comprising the fluorescent probe described in any one of [1] to [4].

[0063]

[14] A kit for detecting breast cancer and benign breast tumors, comprising the fluorescent probe described in any one of [1] to [4] and a GGT activity detection probe.

[0064]

[15] A kit for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising the fluorescent probe described in any one of [5] to [7].

[0065] Effects of the Invention

[0066] According to the present invention, a fluorescent probe capable of detecting malignant tumors and benign tumors of the breast can be provided.

[0067] Furthermore, the present invention can provide a fluorescent probe capable of detecting lung adenocarcinoma or lung squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 These are 12 glycosidase-responsive fluorescent probes synthesized in the examples.

[0069] Figure 2 Shown are the results of a screening assay for the detection of breast and benign glycosidase-responsive fluorescent probes.

[0070] Figure 3 These are the evaluation results of fluorescent probes in breast cancer specimens using glycosidase-responsive fluorescent probes (1, 2, 5, 7, 11, and 12).

[0071] Figure 4 The results are a comparison of the fluorescence increases of glycosidase-responsive fluorescent probes 5 and 11 in breast cancer and breast fibroadenoma (benign tumor).

[0072] Figure 5 Shown are the results of fluorescence imaging experiments on breast cancer (IDC) using 12 glycosidase-responsive fluorescent probes.

[0073] Figure 6 Shown are the results of fluorescence imaging experiments of benign tumors (FA) using 12 glycosidase-responsive fluorescent probes.

[0074] Figure 7Shown are the results of breast tumor imaging experiments using glycosidase-responsive fluorescent probe 5 (HMRef-aD-Man).

[0075] Figure 8 Shown are the results of breast tumor imaging experiments using glycosidase-responsive fluorescent probe 5 (HMRef-aD-Man).

[0076] Figure 9 This is the result of dual-color imaging using a combination of glycosidase-responsive fluorescent probe 5 and gGlu-2-OMeSiR600. DETAILED DESCRIPTION

[0077] Throughout this specification, unless otherwise specified, "alkyl" or the alkyl portion of a substituent containing an alkyl portion (e.g., alkoxy) refers to, for example, an alkyl group having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 3 carbon atoms, in a linear, branched, or cyclic form, or a combination thereof. More specifically, examples of the alkyl group include methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, cyclopropylmethyl, n-pentyl, and n-hexyl.

[0078] When referred to as a "halogen atom" in this specification, any of a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom may be mentioned, and a fluorine atom, a chlorine atom, or a bromine atom is preferred.

[0079] The present inventors focused on glycosidase activity in cancer and developed various fluorescent probes for detecting different glycosidase activities. Using these fluorescent probes, they successfully visualized and evaluated glycosidase activity in intact, surgically resected cancer tissue without homogenization.

[0080] Specifically, the present inventors conducted a comprehensive screening of glycosidase activity in human surgical specimens and determined that glycosidase activity varies depending on tumor type and that certain fluorescent probes are effective for cancer-specific imaging. Details are described below.

[0081] 1. Fluorescent probe for breast cancer detection

[0082] One embodiment of the present invention is a fluorescent probe for detecting breast cancer and benign breast tumors, comprising a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as "embodiment 1").

[0083]

[0084] In the general formula (I), when R1 is present, R1 represents the same or different monovalent substituents present on the benzene ring. Examples of the monovalent substituents include halogen and optionally substituted alkyl.

[0085] m is an integer from 0 to 4.

[0086] In a preferred embodiment of the present invention, m is 0 and R1 is absent. That is, the benzene ring bonded to the xanthene skeleton is an unsubstituted benzene ring.

[0087] In the general formula (I), R2 and R3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom.

[0088] When R2 and R3 represent an alkyl group, the alkyl group may contain one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. For example, the alkyl group represented by R2 or R3 may be a haloalkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc. R2 and R3 are each independently preferably a hydrogen atom or a halogen atom. When R2 and R3 are halogen atoms, preferably both R2 and R3 are fluorine atoms or chlorine atoms.

[0089] In a preferred embodiment of the present invention, R2 and R3 are both hydrogen atoms.

[0090] R4 and R5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom, and are the same as those described for R2 and R3. Preferably, both R4 and R5 are hydrogen atoms.

[0091] R6 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluoroalkyl group having 1 to 5 carbon atoms. Preferred alkyl groups for R6 include methyl and ethyl groups. Preferred fluoroalkyl groups for R6 include -CH2-CF3 and -CH2-CH2-CF3.

[0092] In a preferred embodiment of the present invention, R6 is -CH2-CF3.

[0093] In the general formula (I), when R7 and R8 are present, R7 and R8 each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group. However, R7 and R8 each independently represent an alkyl group having 1 to 3 carbon atoms, and more preferably, both R7 and R8 are methyl groups. The alkyl group represented by R7 and R8 may contain one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. For example, the alkyl group represented by R7 or R8 may be a haloalkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.

[0094] When R7 or R8 represents an aryl group, the aryl group can be any of a monocyclic aromatic group or a condensed aromatic group, and the aryl ring can contain one or more ring-forming heteroatoms (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom). As the aryl group, a phenyl group is preferred. The aryl ring can have one or more substituents. As substituents, for example, one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. can be present.

[0095] When X described below is an oxygen atom, R7 and R8 do not exist.

[0096] X represents an oxygen atom, a silicon atom or a carbon atom.

[0097] In a preferred embodiment of the present invention, X is an oxygen atom.

[0098] n is an integer of 1 to 3, and preferably n is 1.

[0099] In the general formula (I), L is selected from any of the following formulae (1) to (6).

[0100]

[0101] The fluorescent probe having a group represented by formula (1) as L is an α-mannosidase-responsive fluorescent probe, the fluorescent probe having a group represented by formula (2) as L is an α-L-fucosidase-responsive fluorescent probe, the fluorescent probe having a group represented by formula (3) as L is a β-hexosaminidase-responsive fluorescent probe, the fluorescent probe having a group represented by formula (4) as L is a β-N-acetylgalactosidase-responsive fluorescent probe, the fluorescent probe having a group represented by formula (5) as L is a β-glucosidase-responsive fluorescent probe, and the fluorescent probe having a group represented by formula (6) as L is a β-galactosidase-responsive fluorescent probe. According to the research results of the present inventors, these fluorescent probes are effective for imaging diagnosis of breast fibroadenoma, invasive ductal carcinoma, and non-invasive ductal carcinoma. Therefore, the fluorescent probe of the present invention having any of the groups represented by formulas (1) to (6) as L can detect both breast cancer and benign tumors.

[0102] A preferred embodiment of the first embodiment of the present invention is a fluorescent probe for detecting breast cancer and benign breast tumors, comprising a compound represented by the following formula (Ia) or a salt thereof.

[0103]

[0104] Here, L is selected from any of the groups in the following formulae (1) to (6).

[0105]

[0106] 2. Fluorescent probes for lung cancer or squamous cell carcinoma detection

[0107] Another embodiment of the present invention is a fluorescent probe for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising a compound represented by the following general formula (I) or a salt thereof (hereinafter also referred to as "embodiment 2").

[0108]

[0109] In the general formula (I), R1 to R8, X, and n are the same as those described in the first embodiment.

[0110] In Embodiment 2, L is selected from the groups represented by the following formula (5) or (6).

[0111]

[0112] The fluorescent probe having a group of formula (5) or (6) as L is a β-glucosidase- or β-galactosidase-responsive fluorescent probe. According to the research results of the present inventors, it has been found that the fluorescent probe is effective for specific imaging of not only lung squamous cell carcinoma, which cannot be detected by aminopeptidase-responsive fluorescent probes, but also major lung cancers including lung adenocarcinoma. Therefore, the fluorescent probe of the present invention having a group of formula (5) or (6) as L can specifically detect lung adenocarcinoma or lung squamous cell carcinoma.

[0113] A preferred aspect of the second embodiment of the present invention is a fluorescent probe for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising a compound represented by the following formula (Ia) or a salt thereof.

[0114]

[0115] Here, L is selected from the groups of the following formula (5) or (6).

[0116]

[0117] The compound represented by general formula (I) in embodiment 1 and 2 can exist in the form of acid addition salt or base addition salt. As acid addition salt, for example, inorganic acid salts such as hydrochloride, sulfate, nitrate or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, tartrate etc. can be listed, and as base addition salt, metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt or organic amine salt such as triethylamine salt etc. can be listed. In addition to these, salts with amino acids such as glycine are sometimes formed. The compound represented by general formula (I) or its salt sometimes exists in the form of hydrate or solvate, and these substances can also be used in the present invention.

[0118] The compound represented by general formula (I) sometimes has one or more asymmetric carbon atoms depending on the type of substituents. In the present invention, stereoisomers such as optically active forms based on one or more asymmetric carbon atoms, diastereomers based on two or more asymmetric carbon atoms, and any mixtures and racemic forms of stereoisomers can also be used.

[0119] The examples of this specification specifically illustrate methods for producing representative compounds of the compounds represented by the general formula (I). Therefore, those skilled in the art can, based on these descriptions and appropriately selecting reaction raw materials, reaction conditions, and reaction reagents, modify or alter these methods as needed to produce compounds represented by the general formula (I).

[0120] 3. Fluorescent probes used in combination with GGT activity detection probes

[0121] The fluorescent probe of embodiment 1 can be used in combination with GGT activity detection probe.For example, when detecting breast cancer, if the fluorescent probe of red GGT probe and embodiment 1 is used in combination, then breast cancer (malignant tumor) and benign tumor both emit red fluorescence in the same manner, and the glycosidase-responsive fluorescent probe used in the present invention can emit strong green fluorescence especially in benign tumor. When the images of the two are compounded, breast cancer tissue can be imaged as red and benign tumor tissue can be imaged as yellow. Therefore, by using glycosidase-responsive fluorescent probe and GGT probe in combination, it is possible to determine that the position where only glycosidase-responsive fluorescent probe emits fluorescence is benign tumor portion, glycosidase-responsive fluorescent probe and GGT probe both emit fluorescence as breast cancer (malignant tumor) portion, it is possible to distinguish benign tumor from malignant tumor. Thus, in surgery, benign tumor portion will not be excised, and the efficiency of local resection can be improved.

[0122] That is, another embodiment of the present invention is a fluorescent probe for detecting breast cancer and benign breast tumors, which comprises the fluorescent probe of embodiment 1 and a GGT activity detection probe.

[0123] Here, examples of GGT activity detection probes include compounds represented by the following general formula (II) or salts thereof.

[0124]

[0125] In the general formula (II), X represents Si(R a )(R b )、Ge(R a )(R b )、Sn(R a )(R b )、C(R a )(R b )、P(=O)(R a ) or O. Here, R a and R b Each independently represents a hydrogen atom, an alkyl group or an aryl group.

[0126] R 1represents a hydrogen atom or 1 to 4 identical or different substituents independently selected from the group consisting of an optionally substituted alkyl group, a carboxyl group, an ester group, an alkoxy group, an amide group, and an azide group.

[0127] R 2 represents a hydrogen atom, a hydroxyl group, a cyano group, an optionally substituted alkyl group, an alkoxy group, an aryl group or a heteroaryl group.

[0128] R 3 and R 4 Each independently represents: a hydrogen atom; and 1 to 3 identical or different substituents independently selected from the group consisting of a hydroxyl group, a halogen atom, an optionally substituted alkyl group, a sulfo group, a carboxyl group, an ester group, an amide group, and an azide group.

[0129] R 5 、R 6 and R 7 Each independently represents a hydrogen atom or an alkyl group.

[0130] Here, R 6 or R 7 Can be respectively with R 4 Together they form a ring structure including the nitrogen atom to which they are bonded.

[0131] R 8 represents an acyl residue derived from an amino acid. Here, the acyl residue refers to a residue that is a partial structure remaining after removing the OH group from the carboxyl group of the amino acid. That is, the carbonyl portion of the acyl residue derived from the amino acid and R 8 The adjacent NH groups form an amide bond, thereby connecting to the rhodamine skeleton.

[0132] "Amino acid" can be any compound having both an amino group and a carboxyl group, and any compound can be used, including natural and non-natural compounds. It can be any of neutral amino acids, basic amino acids or acidic amino acids. In addition to amino acids that function as transmitters such as neurotransmitters, amino acids that are constituents of polypeptide compounds such as physiologically active peptides (including dipeptides, tripeptides, tetrapeptides and oligopeptides) and proteins can also be used. For example, α-amino acids, β-amino acids, γ-amino acids, etc. can be used. As amino acids, optically active amino acids are preferably used. For example, for α-amino acids, any of D-amino acids or L-amino acids can be used, but sometimes it is preferred to select optically active amino acids that function in organisms.

[0133] R 8 The target peptidase may be γ-glutamyl transpeptidase (GGT), dipeptidyl peptidase IV (DPP-IV), or calpain. 8Preferably, γ-glutamyl. In addition, when the target peptidase is dipeptidyl peptidase IV, R 8 Preferably, it is an acyl group containing a proline residue. When the target peptidase is calpain, R 8 For example, it may be an acyl group containing a cysteine ​​residue, or Suc-Leu-Leu-Val-Tyr (Suc-LLVY) or AcLM, which are well-known in the art, may be used as a calpain substrate.

[0134] The principle of fluorescence emission by the compound represented by general formula (II) can be referred to Japanese Patent Application No. 2018-210101, which is an application currently under review.

[0135] A preferred embodiment of the present invention is a fluorescent probe for detecting breast cancer and benign breast tumors, which comprises the fluorescent probe of embodiment 1 and R 8 A compound of the general formula (II) or a salt thereof which is a γ-glutamyl group.

[0136] Among the compounds represented by the general formula (II), the following compounds can be preferably used.

[0137]

[0138] The compound represented by general formula (II) can exist in the form of acid addition salt or base addition salt. As acid addition salt, for example, inorganic acid salts such as hydrochloride, sulfate, nitrate, or organic acid salts such as methanesulfonate, p-toluenesulfonate, oxalate, citrate, tartrate, etc. can be listed. As base addition salt, metal salts such as sodium salt, potassium salt, calcium salt, magnesium salt, ammonium salt, or organic amine salts such as triethylamine salt can be listed. In addition to these, salts with amino acids such as glycine are sometimes formed. The compound represented by general formula (II) or its salt sometimes exists in the form of hydrate or solvate, and these substances can also be used in the present invention.

[0139] The compound represented by general formula (II) sometimes has one or two or more asymmetric carbon atoms depending on the type of substituents. In the present invention, stereoisomers such as optically active forms based on one or two or more asymmetric carbon atoms, diastereomers based on two or more asymmetric carbon atoms, and any mixtures and racemic forms of stereoisomers can also be used.

[0140] A preferred embodiment of the present invention is a fluorescent probe for detecting breast cancer and benign breast tumors, comprising the fluorescent probe of embodiment 1 and a compound of formula (7) or a salt thereof.

[0141] 4. Kit for detecting breast cancer and benign breast tumors using the fluorescent probe of the present invention

[0142] Another embodiment of the present invention is a kit for detecting breast cancer and benign breast tumors, comprising the fluorescent probe of embodiment 1.

[0143] Another embodiment of the present invention is a kit for detecting breast cancer and benign breast tumors, comprising the fluorescent probe of embodiment 1 and a GGT activity detection probe. The GGT activity detection probe is as described above.

[0144] Another embodiment of the present invention is a kit for detecting lung adenocarcinoma or lung squamous cell carcinoma, comprising the fluorescent probe of the second embodiment.

[0145] In this kit, the fluorescent probe of the present invention is usually prepared as a solution, but can be provided in the form of a composition in an appropriate form, such as a powder mixture, freeze-dried product, granules, tablets, liquid preparations, etc., or can be dissolved in distilled water for injection or an appropriate buffer solution for use.

[0146] In addition, the kit may contain other reagents as needed. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents may be used, and those skilled in the art can appropriately select the amounts of these additives.

[0147] Another embodiment of the present invention is a method for detecting breast cancer and benign breast tumors, comprising: (a) applying the fluorescent probe of embodiment 1 to a clinical breast specimen; and (b) measuring a fluorescent image of the clinical breast specimen to which the fluorescent probe is applied.

[0148] In the step (a), the application of the fluorescent probe to the clinical breast specimen can be performed by, for example, locally spraying a solution of the fluorescent probe onto the clinical breast specimen.

[0149] In addition, another embodiment of the present invention is a method for detecting lung adenocarcinoma or lung squamous cell carcinoma, which includes the following steps: (a) applying the fluorescent probe of embodiment 2 to a clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma; and (b) measuring the fluorescent image of the clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma to which the above-mentioned fluorescent probe is applied.

[0150] In step (a), the fluorescent probe can be applied to a clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma by, for example, locally spraying a solution of the fluorescent probe onto the clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma.

[0151] Example

[0152] Hereinafter, the present invention will be described with reference to Examples, but the present invention is not limited thereto.

[0153] [Synthesis Example 1]

[0154] Synthesis of probe 1 (β-glucosidase-reactive probe)

[0155] HMRef (0.426 g, 1.07 mmol), 2,3,4,6-tetra-O-acetyl-α-D-pyranose glucopyranose bromide (8.26 g, 20.1 mmol), Ag2O (4.71 g, 20.3 mmol), and Na2SO4 (720 mg, 2.00 mmol) were dissolved in 30 mL of acetonitrile (super dehydrated) and stirred at room temperature for 24 hours. The reaction solution was filtered through Celite, the filtrate was recovered, and the acetonitrile was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 95:5) to obtain the acetylated sugar adduct. This was dissolved in methanol, and a solution of NaOMe (520 mg, 9.63 mmol) dissolved in 5 mL of methanol was added, followed by stirring at room temperature for 5 hours. The reaction solution was neutralized with Amberlite IR 120, and the methanol was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH 2 Cl 2 :MeOH=90:10) to obtain the target compound (301 mg, 0.535 mmol) in a yield of 50.2%.

[0156] [Synthesis Example 2]

[0157] Synthesis of probe 5 (α-mannosidase reactive probe)

[0158] HMRef (96.0 mg, 0.24 mmol), penta-O-acetyl-D-mannopyranoside (3.33 g, 8.53 mmol), boron trifluoride-ether complex (8.0 mL, 63.4 mmol), and Na₂SO₄ (500 mg, 3.34 mmol) were dissolved in 15 mL of dichloromethane and stirred for 24 hours. The reaction solution was filtered through celite, and the dichloromethane layer was extracted using 1 M NaOH by separation and removed under reduced pressure. The residue was dissolved in 15 mL of methanol, and a solution of NaOMe (1.60 g, 29.6 mmol) dissolved in 5 mL of methanol was added, followed by stirring at room temperature for 5 hours. The reaction solution was neutralized with Amberlite IR 120, and the methanol was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH₂Cl₂:MeOH = 90:10) to obtain a crude product. This was further purified by HPLC (5-80% aqueous MeCN (0.1% TFA), over 90 minutes) to obtain the target compound (61.6 mg, 0.200 mmol) in a yield of 45.6%.

[0159] [Synthesis Example 3]

[0160] Synthesis of probe 7 (α-L-fucosidase-reactive probe)

[0161] HMRef (90.0 mg, 0.226 mmol), 2,3,4-tri-O-acetyl-L-fucopyranosyltrichloroacetimide (862 mg, 1.99 mmol), and TMSOTf (442 mg, 1.99 mmol) were dissolved in 10 mL of dichloromethane and stirred at -41°C for 12 hours. The reaction solution was diluted with dichloromethane and neutralized with saturated aqueous sodium bicarbonate. The organic layer was separated by separation and removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 95:5) to obtain an intermediate. This intermediate was dissolved in 5 mL of methanol, and a solution of NaOMe (1.33 g, 24.6 mmol) dissolved in 5 mL of methanol was added, followed by stirring at room temperature for 5 hours. The reaction solution was neutralized with Amberlite IR 120, and the methanol was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 95:5) to obtain a crude product. This was further purified by HPLC (5-80% aqueous MeCN (0.1% TFA), over 90 minutes) to obtain the target compound (14.4 mg, 0.0257 mmol) in a yield of 11.7%.

[0162] [Synthesis Example 4]

[0163] Synthesis of probe 12 (β-N-acetylgalactosidase-reactive probe)

[0164] HMRef (46.7 mg, 0.117 mmol), 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-aD-galactopyranosyl chloride (400 mg, 1.09 mmol), Ag2O (400 mg, 1.72 mmol), NaI (88.3 mg, 0.589 mmol), and Na2SO4 (500 mg, 3.34 mmol) were dissolved in 10 mL of acetonitrile (super dehydrated) and stirred at room temperature for 24 hours. The reaction solution was filtered through Celite, the filtrate was recovered, and the acetonitrile was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH = 95:5) to obtain the acetylated sugar adduct. This was dissolved in methanol, and a solution of NaOMe (0.50 g, 9.25 mmol) dissolved in 5 mL of methanol was added, followed by stirring at room temperature for 5 hours. The reaction solution was neutralized with Amberlite IR 120, and the methanol was removed under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2:MeOH=90:10) to obtain a crude product, which was further purified by HPLC (5-80% aqueous MeCN (0.1% TFA) over 90 minutes) to obtain the target compound (11.3 mg, 0.0216 mmol) in a yield of 28.2%.

[0165] Probe 2 was synthesized according to the description of Matsuzaki, H et al, Bioconju. Chem., 27(4), 973-981 (2016). Probe 11 was synthesized according to the description of Asanuma, D et al, Nat. Commun., 6, 6463 (2015).

[0166] Probes 3, 4, 6, 8, 9, and 10 were synthesized by the same method as the above-mentioned probes.

[0167] [Example 1]

[0168] Screening of glycosidase-responsive fluorescent probes for the detection of breast cancer and benign breast cancer

[0169] The 12 glycosidase-responsive fluorescent probes synthesized above were used (refer to Figure 1 ), screening for detecting breast cancer and benign cancer was performed according to the following steps.

[0170] 200 μL of a PBS solution containing each fluorescent probe at a 50 μM concentration was added to each well containing a breast clinical specimen. Fluorescence images at 540 nm were acquired using a Maestro in vivo imaging system (PerkinElmer) at 1, 3, 5, 10, 20, and 30 minutes. Regions of interest (ROIs) were generated for each fluorescence intensity value and quantified and compared using the Maestro software. The filter settings used were Ex / Em = 465-30 nm / 515 nm long-pass.

[0171] The results are shown in Figure 2 .

[0172] Figure 2 FIG. 1a shows the results of screening using fluorescent probes in surgically resected human breast specimens.

[0173] Figure 2 (b) shows the fluorescence increase in breast FA tissue at 30 minutes in the presence and absence of each inhibitor. Black bars indicate the fluorescence increase in the absence of inhibitors, while gray bars indicate the fluorescence increase in the presence of inhibitors. Fluorescent probe concentration = 50 μM, inhibitor concentration = 500 μM.

[0174] Figure 2 Figure c shows the results of a comprehensive analysis of non-invasive glycosidase activity in normal breast, IDC (breast cancer), and FA (benign tumor) tissues using 12 fluorescent probes. The increase in fluorescence represents the increase from 1 minute to 30 minutes after the addition of the fluorescent probe. Each point represents the increase in fluorescence in normal breast, IDC, DCIS, and FA tissues, starting from the left. Figure 3c, In both breast cancer and FA (benign tumor) tissues, glycosidase-responsive fluorescent probes 1, 2, 5, 7, 11, and 12 showed increased fluorescence.

[0175] Figure 2 d shows the results of immunohistochemical analysis of normal breast, FA, or IDC tissues for MAN2C1. Tissues were evaluated as negative when no stained cells were observed, and positive otherwise. Stronger staining was observed in FA and IDC tissues than in normal tissues, confirming overexpression of MAN2C1 in these tissues.

[0176] Figure 2 The result of the DEG method is shown in e. The DEG method was performed according to the procedure described in J. Am. Chem. Soc., 135, 6002-6005 (2013).

[0177] In FA tissue, DEG analysis using α-mannosidase-reactive probe 5 revealed only a single fluorescent spot by two-dimensional electrophoresis, from which MAN2C1 was identified using fluorescent spot peptide mass fingerprinting. Furthermore, only a single similar fluorescent spot was confirmed in IDC tissue by two-dimensional electrophoresis. These results indicate that MAN2C1 is the responsible enzyme for the increased fluorescence of α-mannosidase-reactive probe 5.

[0178] for Figure 2 Six glycosidase-responsive fluorescent probes (1, 2, 5, 7, 11, and 12) that showed increased fluorescence in c were evaluated in breast cancer specimens. Four breast cancer IDCs and one DCIS were used as specimens, and the fluorescent probe concentration was 50 μM.

[0179] The results are shown in Figure 3 .Depend on Figure 3 It was confirmed that the fluorescence increase of glycosidase-responsive fluorescent probes 5 and 11 was large, and the fluorescence intensity ratio between tumor and normal tissues was also large.

[0180] The results of comparing the fluorescence increase in breast cancer and benign tumors for glycosidase-responsive fluorescent probes 5 (HMRef-α-D-Man) and 11 (HMRef-β-D-GlcNAc) are shown in FIG. Figure 4 (Fluorescent probe concentration = 50 μM).

[0181] 200 μL of a PBS solution containing the fluorescent probe at the above concentration was added to each well containing a breast clinical specimen. Fluorescence images at 540 nm were acquired using a Maestro in vivo imaging system (PerkinElmer) at 1, 3, 5, 10, 20, and 30 minutes. Regions of interest (ROIs) were generated for each fluorescence intensity value and quantified and compared using the Maestro software. The filter settings used were Ex / Em = 465-30 nm / 515 nm long-pass.

[0182] The results showed that the glycosidase-responsive fluorescent probes 5 (HMRef-α-D-Man) and 11 (HMRef-β-D-GlcNAc) showed increased fluorescence in tumor tissue compared to normal breast tissue (Normal). Furthermore, they showed increased fluorescence in benign tumor tissue (FA) compared to cancerous tissue (IDC, DCIS). These results suggest that both probes may be effective for fluorescence imaging of breast cancer or for the specific detection of benign tumors.

[0183] In addition, the imaging images of the time changes of fluorescence rise for breast cancer (IDC) and benign tumor (FA) using 12 glycosidase-responsive fluorescent probes are shown in FIG. Figures 5-6 The experiment was carried out under the following conditions.

[0184] 200 mL of a PBS solution containing the fluorescent probe at the above concentration was added to each well containing a breast clinical specimen. Fluorescence images at 540 nm were acquired using the Maestro in vivo imaging system (PerkinElmer) at 1, 3, 5, 10, 20, and 30 minutes. Regions of interest (ROIs) were generated for each fluorescence intensity value and quantified and compared using the Maestro software. The filter settings used were Ex / Em = 465-30 nm / 515 nm long-pass.

[0185] Figure 5 This is the result of fluorescence imaging of breast cancer tissue (right image). Figure 6 The results of fluorescence imaging of a benign tumor (FA) are shown (right).

[0186] [Example 2]

[0187] Breast tumor imaging using HMRef-aD-Man

[0188] Imaging was performed using glycosidase-responsive fluorescent probe 5 (HMRef-aD-Man) (concentration: 50 mM) under the following conditions.

[0189] 3 mL of a PBS solution containing the fluorescent probe at the above concentration was added to a culture dish containing a clinical breast specimen (a mixed sample of normal and tumor areas), and fluorescence images were obtained at various times. Fluorescence images of clinical specimens with DCIS and FA were obtained using the Maestro in vivo imaging system (PerkinElmer). Each filter setting used Ex / Em = 465-30nm / 515nm long-pass. Fluorescence images of a clinical IDC specimen were obtained using a portable imaging device manufactured by Homme, equivalent to the Maestro in vivo imaging system.

[0190] The results are shown in Figures 7-8 .

[0191] Figure 7 a shows the imaging results of IDC (breast cancer) obtained by surgical resection.

[0192] After the probe was spread, only the IDC site was visualized with strong fluorescence for about 20 minutes.

[0193] Figure 7 b shows the results of histological analysis of the specimen and the results of immunostaining for MAN2C1. It was confirmed that the sites where increased fluorescence was observed coincided with the sites that histologically indicated IDC and the sites where MAN2C1 was highly expressed.

[0194] Figure 7 Figure c shows the imaging result of DCIS (breast cancer) obtained by surgical resection. Within 15 minutes after the probe was spread, only the DCIS site was visualized with strong fluorescence.

[0195] Figure 7 Figure d shows examples of regions of interest (ROIs) in the aforementioned specimens, their histological analysis results, and MAN2C1 immunostaining results. It was confirmed that the areas of increased fluorescence corresponded to histologically identified DCIS and areas with high MAN2C1 expression. Furthermore, very small DCIS tissue, less than 1 mm in size, was also detected.

[0196] Figure 7 Figure e shows the fluorescence increase value of the region of interest (ROIs) of the above-mentioned specimen at 15 minutes. No significant increase in fluorescence was observed in the histologically normal area, but a significant increase in fluorescence was observed in the area histologically diagnosed as DCIS.

[0197] Figure 8 Figure a shows the imaging result of FA (benign tumor) obtained by surgical resection. Within 10 minutes after the probe was spread, only the FA site was visualized with strong fluorescence.

[0198] Figure 8b shows the results of histological analysis of the specimen and the results of immunostaining for MAN2C1. It was confirmed that the sites where increased fluorescence was observed coincided with the sites histologically identified as FA and the sites with high MAN2C1 expression.

[0199] [Example 3]

[0200] Differentiation between benign tumors and breast cancer using a combination of HMRef-α-D-Man and GGT probes

[0201] Two-color imaging was performed under the following conditions using a combination of glycosidase-responsive fluorescent probe 5 (HMRef-α-D-Man) (concentration: 50 μM) and gGlu-2-OMeSiR600 (concentration: 50 μM) as a GGT probe.

[0202] 200 μL of PBS solution containing the fluorescent probe at the above concentration was added to each well where the breast clinical specimen was placed, and fluorescence images at 540 nm and 640 nm were obtained at 1, 3, 5, 10, 20, and 30 minutes using the Maestroin in vivo imaging system (PerkinElmer). For each fluorescence intensity value, regions of interest (ROIs) were obtained on the Maestro software and quantified and compared. For the glycosidase-responsive fluorescent probe 5, the filter setting used Ex / Em = 465-30 nm / 515 nm long-pass. For the GGT probe, Ex / Em = 570-40 nm / 610 nm long-pass was used.

[0203] The results are shown in Figure 9 .

[0204] Figure 9 a shows a fluorescence image at 540 nm and a pseudo real color image at 500 to 720 nm. Figure 9 b shows a fluorescence image at 640 nm and a pseudo-true color image at 600 to 820 nm. The exposure time (milliseconds) of the fluorescence image is shown at the bottom of each image.

[0205] Figure 9 c shows a composite image of green and red fluorescence images 30 minutes after the two probes were applied to breast cancer and benign tumor tissues, with exposure times of 40 milliseconds for the green image and 20 milliseconds for the red image. Figure 9 d shows the comparison of the composite images at each time point.

[0206] according to Figure 9c, glycosidase-responsive fluorescent probe 5 emits green fluorescence in the case of benign tumors, and red GGT probe gGlu-2OME-SiR600 emits red fluorescence in the case of both breast cancer (malignant tumors) and benign tumors. When the images of the two are compounded, breast cancer tissue can be imaged as red, and benign tumor tissue can be imaged as yellow. Therefore, by combining the use of glycosidase-responsive fluorescent probe and GGT probe, it can be determined that only the part where the glycosidase-responsive fluorescent probe emits fluorescence is the benign tumor part, and the part where the glycosidase-responsive fluorescent probe and GGT probe emit fluorescence is the breast cancer (malignant tumor) part, which can distinguish benign tumors from malignant tumors. Thus, the benign tumor part will not be removed during surgery, and the efficiency of local resection surgery can be improved.

Claims

1. A fluorescent probe for detecting breast cancer and benign breast tumors, comprising a compound represented by the following general formula (I) or a salt thereof, wherein: Benign breast tumors are fibroadenomas. In formula (I), m is 0, R1 does not exist; R2 and R3 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R4 and R5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom; R6 represents a fluoroalkyl group having 1 to 5 carbon atoms; R7 and R8 do not exist; X represents an oxygen atom; n is 1; L is a group of the following formula (1), 2. A fluorescent probe for detecting breast cancer and benign breast tumors, comprising the fluorescent probe according to claim 1 and a compound of the following formula (7) or a salt thereof, wherein: Benign breast tumors are fibroadenomas.

3. A kit for detecting breast cancer and benign breast tumors, comprising the fluorescent probe according to claim 1, wherein: Benign breast tumors are fibroadenomas.

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