Fluorescent probe solution

Abstract

Provided is a fluorescent probe solution in which a decrease in the purity of a fluorescent probe is suppressed. A fluorescent probe solution according to the present invention comprises: a compound represented by formula (I) or a salt thereof; at least one organic solvent selected from the group consisting of dimethyl sulfoxide, an alcohol having 1-3 carbon atoms, and N, N-dimethylformamide; and water, the content ratio of the organic solvent being 5% by volume to 95% by volume, and the content ratio of the water being 5% by volume to 95% by volume. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a fluorescent probe solution. Background Art

[0002] In recent years, the development of fluorescent probes that can specifically detect cancer by evaluating the enzyme activity of the test body has been continuously developed. For example, Patent Document 1 discloses a fluorescent probe in which the enzyme activity as the evaluation object focuses on glycosidase activity, and can detect malignant and benign tumors of the breast, lung adenocarcinoma, or lung squamous cell carcinoma.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent document 1: WO2020 / 175688 Summary of the invention

[0006] Technical problem to be solved by the invention

[0007] The inventors of the present invention have studied the practical application of the fluorescent probe of Patent Document 1 and found that the purity of the fluorescent probe may decrease during storage.

[0008] The present invention is used to solve the above-mentioned technical problems, and its main purpose is to provide a fluorescent probe solution that suppresses the reduction in purity of the fluorescent probe.

[0009] Technical solutions for solving technical problems

[0010] According to one aspect of the present invention, there is provided a fluorescent probe solution comprising: a compound represented by the following formula (I) or a salt thereof; at least one organic solvent selected from dimethyl sulfoxide, alcohols having 1 to 3 carbon atoms and N,N-dimethylformamide; and water, wherein the organic solvent is contained in an amount of 5% to 95% by volume, and the water is contained in an amount of 5% to 95% by volume.

[0011]

[0012] In formula (I), when R1 exists, it represents the same or different monovalent substituents present on the benzene ring; 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 hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluoroalkyl group having 1 to 5 carbon atoms; when R7 and R8 exist, they each independently represent an alkyl group having 1 to 6 carbon atoms or an aryl group; X represents an oxygen atom, a silicon atom, or a carbon atom; wherein, when X is an oxygen atom, R7 and R8 do not exist; n is an integer from 1 to 3; and L is any group selected from the following formulas (1) to (6).

[0013]

[0014] Effects of the Invention

[0015] According to an embodiment of the present invention, by allowing water to exist in the solvent, a fluorescent probe solution capable of suppressing the purity reduction of the compound represented by formula (I) and capable of long-term storage is obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a graph showing the amount of purity reduction (converted value) of the fluorescent probe solutions of Test Examples 1 to 11 when stored at room temperature for 3 years.

[0017] Figure 2 This is a graph showing the amount of purity reduction (converted value) of the fluorescent probe solutions of Test Examples 1 to 11 when stored at 50° C. for one year.

[0018] Figure 3 This is a graph showing the increase in the amount of oxides (converted value) when the fluorescent probe solutions of Test Examples 1 to 11 were stored at 50° C. for one year.

[0019] Figure 4 This is a graph showing the amount of purity reduction (converted value) of the fluorescent probe solutions of Test Examples 13 to 15 when stored at room temperature for 3 years.

[0020] Figure 5 This is a graph showing the amount of oxide increase (converted value) when the fluorescent probe solutions of Test Examples 13 to 15 were stored at room temperature for 3 years.

[0021] Figure 6 These are fluorescent images of breast clinical specimens obtained using the fluorescent probe solutions for detection of Test Examples 12, 16, and 17. DETAILED DESCRIPTION

[0022] Hereinafter, preferred embodiments of the present invention will be described, but the present invention is not limited to these embodiments. In addition, in this specification, "to" indicating a numerical range includes its upper and lower limits. Moreover, each embodiment can be combined unless otherwise specified.

[0023] In the present specification, "alkyl" or the alkyl part of a substituent (e.g., alkoxy group) containing the alkyl part, unless otherwise specified, means, for example, an alkyl group consisting of a straight chain, branched chain, cyclic group, or a combination thereof having 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms, and more preferably 1 to 3 carbon atoms. 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.

[0024] When a "halogen atom" is mentioned in the present specification, any of a fluorine atom, a chlorine atom, a bromine atom or an iodine atom may be mentioned, and a fluorine atom, a chlorine atom or a bromine atom is preferable.

[0025] A. Fluorescent Probe Solution

[0026] The fluorescent probe solution according to an embodiment of the present invention comprises: a compound represented by formula (I) (hereinafter sometimes referred to as "compound (I)") or a salt thereof; at least one organic solvent selected from dimethyl sulfoxide, an alcohol having 1 to 3 carbon atoms and N,N-dimethylformamide; and water. The content ratio of the organic solvent in the fluorescent probe solution is 5% to 95% by volume, and the content ratio of water is 5% to 95% by volume. The fluorescent probe solution may further comprise any appropriate additive as required. In addition, in this specification, "the compound represented by formula (I) or a salt thereof" is sometimes referred to as "compound (I)". In addition, in this specification, the content ratio (volume %) of the organic solvent, water, and the additive that is liquid at 25°C in the above-mentioned fluorescent probe solution is each synonymous with the ratio (volume %) of the volume of each component before mixing to the total volume of these components before mixing, and the value measured at 25°C and 1013.25hPa can be applied. The compound represented by formula (I) and other additives are small in amount, and their influence on the volume of the fluorescent probe solution can be ignored. According to another aspect of the present invention, a method for producing a fluorescent probe solution can be provided, which comprises the steps of mixing compound (I) or a salt thereof, at least one organic solvent selected from dimethyl sulfoxide, alcohols having 1 to 3 carbon atoms and N,N-dimethylformamide, water, and an additive as an arbitrary component, wherein before mixing, the ratio of the organic solvent to the total volume of the organic solvent, the water and the additive which is liquid at 25°C is 5% by volume to 95% by volume, and the mixing ratio of the water is 5% by volume to 95% by volume.

[0027] A-1. Compound represented by formula (I)

[0028]

[0029] In the general formula (I), when R1 exists, it represents the same or different monovalent substituents present on the benzene ring. Examples of the monovalent substituents include halogen, alkyl which may be substituted, and the like.

[0030] m is an integer of 0 to 4. In one embodiment, m is 0 and R1 does not exist. That is, the benzene ring bonded to the xanthene skeleton is an unsubstituted benzene ring.

[0031] 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.

[0032] When R2 and R3 represent an alkyl group, the alkyl group may be substituted. Specifically, 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.

[0033] R2 and R3 are preferably independently hydrogen atoms or halogen atoms. When R2 and R3 are halogen atoms, R2 and R3 are preferably both fluorine atoms or chlorine atoms. In one embodiment, R2 and R3 are both hydrogen atoms.

[0034] R4 and R5 each independently represent a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom, which is the same as the description of R2 and R3. In one embodiment, R4 and R5 are both hydrogen atoms.

[0035] R6 represents a hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluoroalkyl group having 1 to 5 carbon atoms. The alkyl group represented by R6 is preferably a methyl group or an ethyl group. The fluoroalkyl group represented by R6 is preferably -CH2-CF3 or -CH2-CH2-CF3. In one embodiment, R6 is -CH2-CF3.

[0036] In the general formula (I), when R7 and R8 exist, they each independently represent an alkyl group or an aryl group having 1 to 6 carbon atoms. Preferably, R7 and R8 each independently represent an alkyl group having 1 to 3 carbon atoms, and more preferably, both R7 and R8 represent a methyl group.

[0037] The alkyl group represented by R7 or R8 may be substituted. Specifically, 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 R7 or R8 may be a haloalkyl group, a hydroxyalkyl group, a carboxyalkyl group, etc.

[0038] When R7 or R8 represents an aryl group, the aryl group may be any of a monocyclic aromatic group or a condensed aromatic group, and the aromatic ring may also contain one or more heteroatoms (e.g., a nitrogen atom, an oxygen atom, or a sulfur atom, etc.) constituting the ring. As the aryl group, a phenyl group is preferred. One or more substituents may be present on the aromatic ring. As the substituent, for example, one or more halogen atoms, carboxyl groups, sulfonyl groups, hydroxyl groups, amino groups, alkoxy groups, etc. may be present.

[0039] When X described below is an oxygen atom, R7 and R8 do not exist.

[0040] X represents an oxygen atom, a silicon atom or a carbon atom. In one embodiment, X is an oxygen atom.

[0041] n is an integer of 1 to 3, and is preferably 1.

[0042] In the general formula (I), L is any group selected from the following formulae (1) to (6).

[0043]

[0044] In one embodiment, compound (I) is a compound represented by the following formula (Ia) (wherein L is the same as L in formula (I) and is selected from any group of the above formulae (1) to (6)).

[0045]

[0046] Compound (I) may have one or more asymmetric carbon atoms depending on the type of substituents. In the present invention, in addition to stereoisomers such as optically active forms based on one or more asymmetric carbon atoms and diastereomers based on two or more asymmetric carbon atoms, any mixture of stereoisomers, racemic forms, etc. can also be used.

[0047] The salt of compound (I) can be an acid addition salt or a base addition salt. As an 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 cited, and as a 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 cited. In addition to these, there are also cases where salts are formed with amino acids such as glycine. Compound (I) or its salt also exists in the form of hydrates or solvates, and these substances can also be used in the present invention.

[0048] Compound (I) and a salt thereof can be synthesized by any appropriate method. Examples of the method for synthesizing Compound (I) and a salt thereof include the method described in Patent Document 1.

[0049] In one embodiment, the content ratio of compound (I) in the fluorescent probe solution can be, for example, 0.1mM to 100mM, for example, 0.5mM to 100mM, preferably 1mM to 60mM, and more preferably 3mM to 20mM. Such a fluorescent probe solution containing compound (I) at a high concentration can be diluted with a diluent such as PBS or physiological saline before use.

[0050] A-2. Organic solvents

[0051] The organic solvent is selected from the group consisting of dimethyl sulfoxide (DMSO), alcohols having 1 to 3 carbon atoms, and N,N-dimethylformamide (DMF). As the alcohol having 1 to 3 carbon atoms, ethanol or isopropanol is preferably exemplified. These organic solvents have good miscibility with water and can dissolve compound (I) well. The organic solvent may be used alone or in combination of two or more.

[0052] The ratio of the organic solvent in the fluorescent probe solution is typically 5% to 95% by volume. If the ratio is less than 5% by volume, the solubility of compound (I) may be reduced. On the other hand, if the ratio exceeds 95% by volume, the effect of improving the stability of compound (I) may be reduced. The ratio is preferably more than 10% by volume, more preferably more than 20% by volume, for example, it can be set to more than 40% by volume. In addition, the ratio is preferably less than 90% by volume, more preferably less than 80% by volume, and more preferably less than 75% by volume.

[0053] In one embodiment, the content ratio of the organic solvent in the fluorescent probe solution is, for example, 40% by volume or more, preferably 45% by volume or more, more preferably 50% by volume or more, for example, 55% by volume or more or 60% by volume or more, preferably 90% by volume or less, more preferably 80% by volume or less, and further preferably 75% by volume or less. According to this embodiment, even without using a solubilizing agent described later, the solubility of compound (I) can be ensured, and the effect of improving stability due to the addition of water can be well obtained.

[0054] In one embodiment, the content ratio of the organic solvent in the fluorescent probe solution is, for example, 10% to 90% by volume, preferably 20% to 80% by volume, more preferably 25% to 70% by volume, further preferably 30% to 60% by volume, and further preferably 35% to 45% by volume. By reducing the content ratio of the organic solvent, the effect of improving the stability of compound (I) (e.g., the effect of suppressing the reduction of purity) can be better obtained. In addition, by using a solubilizing agent described later, even when the content ratio of the organic solvent is small, the solubility of compound (I) can be ensured, and the effect of improving stability due to the addition of water can be well obtained.

[0055] In one embodiment, the organic solvent includes DMSO. In this embodiment, the amount of DMSO used may be 50% by volume or more, preferably 75% by volume or more, for example 100% by volume, of the total organic solvent.

[0056] A-3.Water

[0057] By adding water to the fluorescent probe solution, the stability of compound (I) in the fluorescent probe solution can be improved. As water, purified water such as RO water, ion exchange water, and distilled water is preferably used.

[0058] The ratio of water contained in the fluorescent probe solution is typically 5% to 95% by volume. If the contained ratio is less than 5% by volume, the effect of improving the stability of compound (I) may be reduced. On the other hand, if the contained ratio is greater than 95% by volume, the contained ratio of the organic solvent will be reduced, and as a result, the solubility of compound (I) may be reduced. The contained ratio is preferably more than 10% by volume, more preferably more than 20% by volume, and further preferably more than 25% by volume. The contained ratio is preferably less than 90% by volume, more preferably less than 80% by volume, for example, can be set to less than 60% by volume.

[0059] In one embodiment, the content ratio of water in the fluorescent probe solution is, for example, 10% by volume or more, preferably 20% by volume or more, more preferably 25% by volume or more, preferably 60% by volume or less, more preferably 55% by volume or less, more preferably 50% by volume or less, for example, 45% by volume or less or 40% by volume or less. According to this embodiment, even without using a solubilizing agent described later, the solubility of compound (I) can be ensured, and the effect of improving stability can be obtained.

[0060] In one embodiment, the content ratio of water in the fluorescent probe solution is, for example, 10% to 90% by volume, preferably 20% to 80% by volume, more preferably 30% to 75% by volume, further preferably 40% to 70% by volume, and further preferably 55% to 65% by volume. By increasing the content ratio of water, the effect of improving the stability of compound (I) (e.g., the effect of suppressing the reduction of purity) can be better obtained. In addition, by using a solubilizing agent described later, even when the content ratio of the organic solvent is small, the solubility of compound (I) can be ensured, and the effect of improving stability due to the addition of water can be well obtained.

[0061] A-4. Additives

[0062] The additive is an arbitrary component used as needed. Therefore, the fluorescent probe solution may be composed of only the above three components, that is, only compound (I), an organic solvent and water, or may contain additives in addition to these components. In the fluorescent probe solution of an embodiment of the present invention, the total content of the organic solvent and water is, for example, 95% to 100% by volume (for example, 95% to 99.9% by volume), preferably 99% to 100% by volume (for example, 99% to 99.8% by volume).

[0063] The additives can be appropriately selected according to the purpose. Specific examples of additives include buffers, isotonic agents, solubilizers, and the like. Buffering agents include phosphate buffers, TRIS buffers, citric acid buffers, boric acid buffers, and the like. Isotonic agents include sodium chloride, potassium chloride, and the like. Solubilizers include cationic surfactants, anionic surfactants, amphoteric surfactants, nonionic surfactants, and other surfactants, among which nonionic surfactants are preferred. Examples of nonionic surfactants include polyethylene glycol, glycerol fatty acid esters, polysorbates (e.g., Tween (R) series), sorbitan fatty acid esters (e.g., RHEODOL (R) series), polyoxyethylene alkyl ethers (e.g., EMULGEN (R) series), polyoxyethylene alkylphenyl ethers (e.g., Triton-X (R) series), polyoxyethylene aryl ether, polyoxyalkylene derivatives, alkyl alkanolamides, polyoxyethylene alkylamines, alkyl imidazolines, polyoxyethylene hydrogenated castor oil, aliphatic alcohol ethoxylates, polyoxyethylene polyoxypropylene glycol (e.g., Poloxamer), etc. The additives may be used alone or in combination of two or more.

[0064] Those skilled in the art can appropriately select the amount of additives. For example, the content ratio of the buffer in the fluorescent probe solution can be, for example, 1mM to 100mM, preferably 5mM to 20mM. The content ratio of the solubilizing agent in the fluorescent probe solution can be, for example, 0.05w / v% to 5.0w / v%, preferably 0.5w / v% to 3.0w / v%. In addition, when the additive is a liquid (for example, when it is a liquid at 25°C), its content ratio is, for example, 0.01% by volume to 5.0% by volume, preferably 0.02% by volume to 1.0% by volume.

[0065] A-5. Preparation method

[0066] The fluorescent probe solution can be prepared by mixing the above-mentioned components at the above-mentioned content ratio (volume ratio before mixing) and dissolving compound (I). In one embodiment, the fluorescent probe solution can be prepared by dissolving compound (I) in a mixed solution comprising an organic solvent and water. In another embodiment, the fluorescent probe solution can be prepared by dissolving compound (I) in an organic solvent and mixing the resulting solution with water and any additives. The pH of the fluorescent probe solution is, for example, 5 to 9, preferably 6 to 8, and more preferably 6.5 to 7.8.

[0067] The fluorescent probe solution can be used directly or diluted with water, buffer, saline, etc. The concentration of the fluorescent probe solution during use, calculated based on the concentration of compound (I), can be, for example, 5 μM to 200 μM, preferably 10 μM to 100 μM, and more preferably 40 μM to 60 μM. Those skilled in the art can appropriately select the buffer for dilution. For example, PBS, TRIS hydrochloric acid buffer, etc. can be used. The concentration of the buffer can be, for example, 1 mM to 100 mM, preferably 5 mM to 20 mM. The pH of the buffer can be, for example, 6 to 8, more preferably 7.2 to 7.6, and further preferably 7.4.

[0068] In one embodiment, the fluorescent probe solution can be manufactured into a vial, which is contained in a vial with a rubber stopper sealing the opening. Specifically, the fluorescent probe solution can be filled in a vial, its opening is sealed with a rubber stopper, and a vial is made by tightening a lid such as aluminum. In the case where the fluorescent probe solution containing compound (I) is made into a vial, the problem of reduced purity during storage may occur, but according to the fluorescent probe solution of an embodiment of the present invention, by making water present in the solvent, it is possible to have both the solubility and stability of compound (I).

[0069] As a material for forming the vial, a glass material such as borosilicate glass or a resin material such as polypropylene can be used. Glass material is preferred. The vial may be colorless or colored.

[0070] As materials for forming the rubber stopper, there can be cited: butyl rubber (IIR), isoprene rubber (IR), butadiene rubber (BR), styrene-butadiene rubber (SBR), fluororubber (FKM), ethylene-propylene rubber (EPDM), etc. When a rubber stopper formed of butyl rubber is used, the effect of the present invention can be better obtained. The rubber stopper can also be subjected to lamination processing. When a rubber stopper subjected to lamination processing is used, the effect of the present invention can also be well obtained.

[0071] The purity of compound (I) in the fluorescent probe solution just prepared (e.g., within 12 hours after preparation) is, for example, 95% or more, preferably 98% or more, and more preferably 99% or more. The purity of compound (I) in the fluorescent probe solution can be measured, for example, by the method described in the Examples.

[0072] The amount of purity reduction of compound (I) when the fluorescent probe solution is stored at room temperature (representatively 25° C.) for 3 years (purity immediately after preparation - purity after storage for 3 years) is, for example, 3.0% or less, preferably 2.0% or less, more preferably 1.5% or less, and further preferably 1.0% or less. In addition, the amount of purity reduction of compound (I) when the fluorescent probe solution is stored at room temperature for 3 years may be a value calculated by converting the amount of purity reduction when stored at room temperature for about 1 month to 3 years.

[0073] In one embodiment, the purity reduction amount of compound (I) when the fluorescent probe solution is stored at 50°C for 1 year (purity immediately after preparation - purity after storage for 1 year) is, for example, 7.0% or less, preferably 5.0% or less, more preferably 4.0% or less, and further preferably 3.0% or less. In addition, the purity reduction amount of compound (I) when the fluorescent probe solution is stored at 50°C for 1 year may be a value calculated by converting the purity reduction amount when stored at 50°C for about 1 month to 1 year.

[0074] B. Uses of fluorescent probe solutions

[0075] The above compound (I) is a glycosidase-responsive fluorescent probe. Specifically, in formula (I), the compound (I) having a group of formula (1) as L is an α-mannosidase-responsive fluorescent probe; the compound (I) having a group of formula (2) is an α-L-fucosidase-responsive fluorescent probe; the compound (I) having a group of formula (3) is a β-hexosaminidase-responsive fluorescent probe; the compound (I) having a group of formula (4) is a β-N-acetylgalactosaminidase-responsive fluorescent probe; the compound (I) having a group of formula (5) is a β-glucosidase; and the compound (I) having a group of formula (6) is a β-galactosidase-responsive fluorescent probe.

[0076] By using compound (I), the glycosidase activity of surgically excised tissue specimens can be visualized and evaluated without homogenization.

[0077] In one embodiment, in formula (I), compound (I) having any group of formula (1) to (6) as L can detect both breast cancer and benign tumors. Therefore, the fluorescent probe solution containing compound (I) is effective in imaging diagnosis of breast cancer and benign breast tumors, such as breast fibroadenoma, invasive ductal carcinoma, and non-invasive ductal carcinoma.

[0078] The fluorescent probe solution described in item A can be used in combination with a γ-glutamyltranspeptidase (GGT) activity detection probe. For example, when a red GGT activity detection probe is used in combination with compound (I) in the detection of breast cancer, the GGT activity detection probe emits red fluorescence for both breast cancer (malignant tumor) and benign tumor, whereas compound (I) emits strong green fluorescence particularly for benign tumor. Therefore, if the images of the two are superimposed, the breast cancer tissue can be imaged in red and the benign tumor tissue can be imaged in yellow. Therefore, by using compound (I) and a red GGT activity detection probe in combination, a site where both compound (I) and the GGT activity detection probe fluoresce can be determined as a benign tumor site, while a site where only the GGT activity detection probe fluoresces can be determined as a breast cancer (malignant tumor) site, thereby enabling discrimination between benign tumors and malignant tumors. Therefore, the efficiency of partial resection can be improved by not removing the benign tumor site during surgery.

[0079] Examples of the GGT activity detection probe include compounds represented by the general formula (II) described in Patent Document 1 or salts thereof.

[0080] Detection of breast cancer and benign breast tumors can be carried out, for example, by a method comprising (step a1) and (step b1), wherein (step a1) is: applying a fluorescent probe solution containing a compound (I) having any group of formula (1) to (6) as L to a clinical specimen of the breast; and (step b1) is: measuring the fluorescent image of the clinical specimen of the breast to which the fluorescent probe solution is applied.

[0081] (Step a1) applying the fluorescent probe solution to the clinical specimen of the breast can be performed, for example, by spraying the fluorescent probe solution locally on the clinical specimen of the breast, immersing the clinical specimen of the breast (excision specimen) in the probe solution, etc. (Step b1) measuring the fluorescent image can be performed, for example, by using a fluorescent observation device to obtain a fluorescent image of the clinical specimen to which the fluorescent probe solution has been applied.

[0082] In one embodiment, the compound (I) having a group of formula (5) or (6) as L in formula (I) is effective for specific imaging of major lung cancers including lung adenocarcinoma, in addition to lung squamous cell carcinoma that cannot be detected using an aminopeptidase-responsive fluorescent probe. Therefore, a fluorescent probe solution containing such a compound (I) can specifically detect lung adenocarcinoma or lung squamous cell carcinoma.

[0083] The detection of lung adenocarcinoma or lung squamous cell carcinoma can be carried out, for example, by a method comprising (step a2) and (step b2), wherein (step a2) is: applying a fluorescent probe solution of a compound (I) having a group having formula (5) or (6) as L to a clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma; and (step b2) is: measuring the fluorescent image of the clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma to which the fluorescent probe is applied.

[0084] (Step a2) applying the fluorescent probe solution to a clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma, for example, can be performed by locally spraying the fluorescent probe solution on the clinical specimen of lung adenocarcinoma or lung squamous cell carcinoma, immersing the excised specimen in the probe solution, etc. (Step b2) measuring the fluorescent image can be performed by using a fluorescent observation device to obtain a fluorescent image of the clinical specimen to which the fluorescent probe solution is applied.

[0085] Example

[0086] Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples in any way.

[0087] [Synthesis Example 1] Synthesis of HMRef-αMan

[0088]

[0089] Dichloromethane (7.2 mL) was added to HMRef (109 mg, 0.273 mmol), penta-O-acetyl-D-mannopyranoside (1.07 g, 2.73 mmol), and MS4A (200 mg), and the mixture was stirred at room temperature for 1 hour. Boron trifluoride diethylether complex (2.4 mL, 19.1 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 16 hours. After cooling to 0°C, a saturated aqueous sodium bicarbonate solution was added, and the reaction solution was filtered through celite. The mixture was extracted with dichloromethane, and the organic phase was washed with saturated brine, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in methanol (4 mL), and a 28% sodium methoxide methanol solution (1 mL) was added, and the mixture was stirred at room temperature for 1.5 hours. The mixture was neutralized with a 10% aqueous phosphoric acid solution and extracted with dichloromethane, and the organic phase was washed with saturated brine and dried with sodium sulfate, and then the solvent was distilled off under reduced pressure. The residue was purified by column chromatography to obtain the target compound (18.5 mg, 12%).

[0090] [Test Examples 1 to 12]

[0091] The components were mixed to obtain a fluorescent probe solution as shown in Table 1. Specifically, HMRef-αMan obtained in Synthesis Example 1 was dissolved in DMSO to a concentration of 10 mM, and 125 μL of the resulting solution was mixed with a required amount of water or phosphate buffer (pH 7.4) to obtain a fluorescent probe solution.

[0092] [Table 1]

[0093]

[0094] 0.125 mL to 0.312 mL of the fluorescent probe solution obtained in Test Examples 1 to 11 were respectively filled into a 25 mL glass vial (manufactured by Yamato Glass Co., Ltd.), the opening was plugged with a rubber stopper (manufactured by MARUEMU Co., Ltd., No. 20-T), and the vial was tightly sealed from the top with an aluminum cap. The vials filled with the fluorescent probe solution were stored at 25°C or 50°C in a light-shielded place for 25 to 32 days.

[0095] The purity of HMRef-αMan in the fluorescent probe solution within 12 hours after preparation and in the fluorescent probe solution after storage in the vial for 25 to 32 days was measured as follows, and the difference (former - latter) was divided by the number of storage days to calculate the purity reduction per day. Furthermore, the purity reduction (converted value) when stored at room temperature (3 years) or 50°C (1 year) was calculated from the purity reduction per day. The results are shown in Figure 1 and Figure 2 In addition, the purity of HMRef-αMan in the fluorescent probe solutions obtained in Experimental Examples 1 to 11 was above 99% within 12 hours after preparation.

[0096] <Measurement method of HMRef-αMan solution>

[0097] HPLC analysis was performed using a liquid chromatography system (UltiMate 3000, Thermo Fisher Scientific) under the following conditions, and the ratio of the peak area of ​​HMRef-αMan to the total peak area was calculated as the purity.

[0098] Column: Meteoric core C8, 5μm, 4.6×150mm (YMC Corporation)

[0099] Column temperature: 25°C

[0100] Mobile phase A: water / TFA (1000:1)

[0101] Mobile phase B: acetonitrile / TFA (1000:1)

[0102] Liquid delivery volume: 1.0mL / min.

[0103] Wavelength: 254nm

[0104] Liquid delivery of mobile phase: Liquid delivery was carried out under the conditions shown in the table below.

[0105] [Table 2]

[0106] Time after injection (min) Mobile phase A (vol%) Mobile phase B (vol%) 0～20 84～59 16～41

[0107] After the fluorescent probe solution was stored in the vial for 25 to 32 days, the separated peaks were mass analyzed under the same conditions as the above-mentioned liquid chromatography method, and a product (oxide) in which two hydrogen atoms had been lost from HMRef-αMan was observed as the main product.

[0108] In the same manner as above, the oxide content of HMRef-αMan in the fluorescent probe solution within 12 hours after preparation and in the fluorescent probe solution after being stored in a vial at 50°C for 25 to 32 days (ratio of oxide peak area to total peak area: %) was measured, and the difference (latter - former) was divided by the number of storage days to calculate the oxide increase per day. Furthermore, the oxide increase per day was converted to a 1-year value and calculated as the oxide increase after 1-year storage. The results are shown in Figure 3 .

[0109] like Figure 1 and Figure 2 As shown in FIG. 1 , the presence of water in the solvent suppressed the decrease in purity of HMRef-αMan and improved its stability during storage. This effect was also confirmed in the presence of phosphate. Figure 2 and Figure 3 It is known that the generation of HMRef-αMan oxides causes a decrease in purity. Since the oxidation of HMRef-αMan is expected to occur on the fluorescent skeleton of formula (I), it is speculated that the effects of the present invention can be obtained regardless of the type of sugar in the L residue.

[0110] [Test Examples 13 to 15]

[0111] The components were mixed to form the composition shown in Table 3 to obtain a fluorescent probe solution. Specifically, HMRef-αMan obtained in Synthesis Example 1 was dissolved in DMSO in a concentration of 10 mM, and 100 μL of the obtained DMSO solution, 100 μL of a Tween 80 aqueous solution containing 2 w / v% Tween 80 (manufactured by TCI), and a required amount of water were mixed to obtain a fluorescent probe solution. By adding Tween 80 as a solubilizing agent, the fluorescent probe can be dissolved even if the amount of the organic solvent is small.

[0112] [Table 3]

[0113]

[0114] The fluorescent probe solutions obtained in Test Examples 13 to 15 were filled into glass vials, respectively, and sealed with rubber stoppers. The vials filled with the fluorescent probe solutions were stored at 25° C. in a light-shielded place for 85 days and analyzed by liquid chromatography.

[0115] As in Experimental Examples 1 to 11, the purity of HMRef-αMan and the content of its oxide in the fluorescent probe solution within 12 hours after preparation and in the fluorescent probe solution after 85 days of storage in the vial were measured, and the purity decrease and oxide increase over 3 years were calculated as conversion values. The results are shown in Figure 4 and Figure 5 In addition, the purity of HMRef-αMan in the fluorescent probe solutions obtained in Experimental Examples 13 to 15 was above 99% within 12 hours after preparation.

[0116] like Figure 4 and Figure 5 As shown in the figure, even when a solubilizing agent is added to reduce the content ratio of the organic solvent, the purity of HMRef-αMan can be suppressed from decreasing. In addition, it is known that the formation of HMRef-αMan oxides causes a decrease in purity. From the obtained results, it is known that the greater the water content ratio, the more effective the stabilization is, and by adding a solubilizing agent, a probe solution with a higher water content ratio can be prepared.

[0117] [Test Example 16]

[0118] The HMRef-αMan obtained in Synthesis Example 1 was dissolved in an aqueous solution containing 6.5 w / v% mannitol, 10 mM phosphate, and 0.33 w / v% Tween 80 (manufactured by TCI) to a concentration of 1.67 mM to obtain a fluorescent probe solution. The obtained fluorescent probe solution was freeze-dried using a freeze dryer (Tokyo Rikaki Co., Ltd., FDU-1200) to obtain a white powder.

[0119] [Test Example 17]

[0120] HMRef-αMan obtained in Synthesis Example 1 was dissolved in an aqueous solution containing 6.5 w / v% mannitol, 10 mM phosphate, and 1.67 w / v% polyoxyethylene polyoxypropylene glycol (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 1.67 mM to obtain a fluorescent probe solution. The obtained fluorescent probe solution was freeze-dried using a freeze dryer (Tokyo Rikaki Co., Ltd., FDU-1200) to obtain a white powder.

[0121] MCF-7 cells were inoculated in a glass-bottomed culture dish and cultured for 1 to 2 days (D-MEM high glucose + 10% FBS). The culture medium was removed and a fluorescent probe solution for detection was added. The fluorescent probe solution for detection was prepared by diluting or dissolving the fluorescent probe solution of Test Example 12 or the white powder obtained in Test Examples 16 and 17 with PBS so that the HMRef-αMan concentration was 10 μM. After 20 minutes, the images were taken using a fluorescence microscope (Leica DMI 6000CS). The results are shown in Figure 6 .

[0122] like Figure 6 As shown, it can be seen that the fluorescence intensity of the fluorescent probe solution obtained by dissolving after freeze-drying is weaker than that of the fluorescent probe solution of Test Example 12, and the detection ability is poor.

[0123] Industrial Applicability

[0124] The fluorescent probe solution according to the embodiment of the present invention can be suitably used for detecting malignant tumors and benign tumors of the breast, lung adenocarcinoma, lung squamous cell carcinoma, and the like.

Claims

1. A fluorescent probe solution, characterized in that: The invention comprises: a compound represented by the following formula (I) or a salt thereof; at least one organic solvent selected from dimethyl sulfoxide, an alcohol having 1 to 3 carbon atoms and N,N-dimethylformamide; and water. The content ratio of the organic solvent is 5 volume % to 95 volume %, The water content is 5 volume % to 95 volume %; In formula (I), When R1 exists, it represents the same or different monovalent substituents present on the benzene ring; 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 hydrogen atom, an alkyl group having 1 to 5 carbon atoms, or a fluorinated alkyl group having 1 to 5 carbon atoms; When R7 and R8 exist, they each independently represent an alkyl group or an aryl group having 1 to 6 carbon atoms, X represents an oxygen atom, a silicon atom or a carbon atom; Wherein, when X is an oxygen atom, R7 and R8 do not exist; n is an integer from 1 to 3; L is any group selected from the following formulae (1) to (6), 2. The fluorescent probe solution according to claim 1, characterized in that: A solubilizing agent is also included.

3. The fluorescent probe solution according to claim 1 or 2, characterized in that: The content ratio of the organic solvent is 40 volume % to 95 volume %.

4. The fluorescent probe solution according to claim 1 or 2, characterized in that: The organic solvent is dimethyl sulfoxide.

5. The fluorescent probe solution according to claim 1 or 2, characterized in that: The content ratio of the water is 20 volume % to 60 volume %.

6. The fluorescent probe solution according to claim 1 or 2, characterized in that: The content ratio of the compound represented by the formula (I) is 0.5 mM to 100 mM.

7. The fluorescent probe solution according to claim 1 or 2, characterized in that: Also contains a buffer.

8. The fluorescent probe solution according to claim 1 or 2, characterized in that: It is housed in a vial with an opening sealed with a rubber stopper.

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