WO2021177060A1 - Fluorescent probe which becomes substrate of lat1 - Google Patents

Abstract

[Problem] To provide a novel fluorescent substrate compound which becomes a substrate of LAT1 and is introduced into cells. [Solution] A compound represented by general formula (I) or a salt thereof.

Description

Fluorescent probe that serves as an LTA1 substrate

The present invention relates to a novel fluorescent probe that serves as an LTA1 substrate. More specifically, the present invention relates to a fluorescent substrate compound that is selectively incorporated in LAT1 and a method for visualizing cancer cells via LTA1 using the same.

LAT1 (Large amino acid transporter 1) is one of the isoforms of the transporter LAT that is driven independently of sodium, and has large side chains such as Ph, His, Ile, Leu, Met, Trp, and Tyr. Sexual amino acids and amino acid derivatives such as L-DOPA are antiported inside and outside the cell. In recent years, the three-dimensional structure of the protein has been clarified by crystal structure analysis, and LAT1 transports the substrate by forming a dimer with the transmembrane protein 4F2hc on the cell membrane.

Expression of LAT1 in normal tissues is limited to many normal tissues that require an intermittent supply of amino acids, such as nerve cells, glial cells, activated T cells, bone marrow, testis, placenta barrier, and blood-brain barrier. Membrane transport of large neutral amino acids in tissues is mainly carried out by LAT2, which is one of the isoforms of LAT.

In addition, it has been reported that LAT1 is highly expressed in tumor tissues, and its upregulation in a wide range of cancer types and cancer-specific expression distribution have attracted attention as a new cancer biomarker in recent years. ing. Lung cancer, prostate cancer, ovarian cancer, breast cancer, bladder cancer, brain cancer, esophageal cancer, gastric cancer, bile duct cancer, liver cancer, pancreatic cancer, head and neck cancer, kidney cancer, leukemia, skin cancer In a large number of cancer types such as thyroid cancer, increased LAT1 expression has been confirmed at the tissue level derived from human patients (Non-Patent Documents 1 and 2), especially non-small cell lung cancer, brain tumor, and prostate. In cancer and breast cancer, the high expression group of LAT1 has a poor prognosis, suggesting that the malignancy of cancer is related to the expression of LAT1.
In this way, LAT1 has been clarified to be closely related to the ecology of cancer, and is the subject of basic research aimed at elucidating the ecology of cancer, and is attracting attention as a new drug discovery target for cancer. Is collecting.

On the other hand, in recent years, the fluorescence imaging method has been attracting attention as a method for realizing simple and highly sensitive cancer diagnosis, especially intraoperative diagnosis of cancer. In our laboratory, an active-table fluorescent probe that emits fluorescence only after reacting with an enzyme whose expression is upregulated in cancer, such as γ-glutamyl transpeptidase (GGT) and dipeptidyl peptidase IV (DPPIV). We have developed a plurality of these and have shown that cancer in fresh human surgical specimens can be detected quickly and with high sensitivity (Non-Patent Documents 3 and 4).

However, as mentioned above, although LAT1 has been verified to be useful as a cancer marker and is attracting attention as a drug discovery target, most of the fluorescence imaging techniques for visualizing cancer tissues via LAT1 have been reported. No.
In addition, if the fluorescence imaging method that can be used with high sensitivity and safety can be applied to cancer detection, it is expected to become an epoch-making medical technology that can diagnose a wide range of cancer types intraoperatively. Little has been done to develop a fluorescent substrate that can be incorporated into cancer tissue through it.

An object of the present invention is to provide a novel fluorescent substrate compound that serves as a substrate for LAT1 and is taken up into cells.
Another object of the present invention is to provide a new cancer diagnostic technique that enables fluorescence imaging of cancer using LAT1 using such a fluorescent substrate compound.

In solving the above problems, the present inventors focused on the substrate requirements required by LAT1 and investigated and developed the structure of a compound in which a fluorescent group was bound to the amino acid side chain site. Specifically, NBD (nitrobenzoxaziazole, molecular weight: 164) having a small molecular weight was selected as the fluorescent group, and a compound in which the side chain sites of various amino acids were bound to NBD and a derivative of the compound were synthesized, and these were synthesized. The present invention was completed as a result of diligent studies, considering that the compound may be efficiently taken up into cultured cells of cancer with high expression of LAT1.

That is, the present invention
[1] A compound represented by the following general formula (I) or a salt thereof.

(During the ceremony,
R ¹ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms:
X represents ^{NR 2}
Here, R ² is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms:
Y is a nitro group (-NO ₂ ), a sulfonic acid group (-SO ₃ H) or a sulfonamide group (-SO ₂ NR'R''), where R'and R'' are independent of each other. In addition, it is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms;
L is selected from the group consisting of alkylene, arylene, and a group composed of an arbitrary bond of alkylene and arylene. )
[2] The compound according to [1] or a salt thereof, wherein L is selected from the following.
-(CR ^a R ^b ) _n- , ^{-Ar-, *} -Ar- (CR ^a R ^b ) _m- , ^* -(CR ^a R ^b ) _s -Ar-, ^* -(CR ^a R ^b ) _s- Ar- (CR ^a R ^b ) _m-
(Ar represents an arylene, ^Ra and R ^b are each independently and independently at each appearance, a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, n is an integer of 1 to 8, and m is an integer of 1 to 8. It is an integer of 1 to 5, s is an integer of 1 to 8, and * indicates the side to be combined with X.)
[3] The compound according to [1] or [2] or a salt thereof, wherein L is selected from the following.
_{^{-CH 2 -, * -Bz-CH}} 2 -, - (CH 2) 4 -, * -CH 2 -Bz-CH 2 -
(Bz represents a benzene ring, and * indicates the side bonded to X.)
[4] The compound according to [3] or a salt thereof, wherein L is ^* -Bz-CH _2-.
[5] The compound according to [3] or a salt thereof, wherein L is -CH _2-.
[6] L is, - _{(CH 2) 4} - The compound or a salt thereof according to [3].
[7] The compound according to [3] or a salt thereof, wherein L is ^* -CH _2- Bz-CH _2-.
[8] The compound according to any one of [1] to [7] or a salt thereof, wherein Y is a nitro group (-NO _2).
[9] A fluorescent probe containing the compound according to any one of [1] to [8] or a salt thereof.
[10] A method for detecting intracellular uptake by LAT1.
(A) The step of introducing a fluorescent probe containing the compound according to any one of [1] to [8] or a salt thereof into the cell, and (b) the fluorescence emitted by the compound or the salt thereof in the cell. A method that includes the step of measuring.
[11] A method for visualizing cancer cells or tissues.
(A) A step of applying a fluorescent probe containing the compound according to any one of [1] to [8] or a salt thereof to a cell or tissue, and (b) fluorescence of the cell or tissue to which the fluorescent probe is applied. A method comprising irradiating with an appropriate wavelength at which the cell or tissue is measured, thereby observing or measuring the fluorescence emitted within the cell or tissue.
Is to provide.

It was confirmed that the compound of the present invention is efficiently taken up into A549 cells, which are cultured cells with high LAT1 expression, while the uptake into cells is significantly inhibited in the presence of a LAT1 inhibitor such as BHC. Was done. Therefore, the compound of the present invention can be used as a fluorescent substrate to be incorporated into cancer tissue via LAT1.
In addition, the compound of the present invention can be applied to a fluorescence imaging technique for visualizing cancer tissue via LAT1.

The optical properties of compound 2 (NBD-pAP) are shown. It is a fluorescence image of A549 cells treated with NBD-pAP. The time-lapse fluorescence image of HEK293 cells treated with NBD-pAP is shown. Fluorescent images of A549, HeLa and HEK293 cells treated with NBD-pAP are shown. The pH dependence of the absorption spectrum and the fluorescence spectrum of NBD-pAP is shown. The absorption and emission spectra of NBD-pMAP in 100 mM NaPi buffer with various pH values containing 0.1% DMSO as a co-solvent are shown. Fluorescent images of A549 and HeLa cells treated with NBD-mAP are shown. A comparison of the uptake of NBD-pAP and NBD-mAP into A549 cells and HeLa cells is shown. The optical characteristics of NBD-pAMP are shown. Fluorescent images of A549 cells treated with 5 μM NBD-pAMP are shown. FIG. 11A shows a time-lapse fluorescence image of A549 cells treated with NBD-pAMP after washing out of extracellular buffer. FIG. 11 (b) shows the quantification of the fluorescence intensity in the ROI of each cell. FIG. 12 (a) shows a fluorescence confocal image of a 3D spheroid prepared by A549 or HEK293. FIG. 12B shows the quantification of the fluorescence intensity of each spheroid at ROI. FIG. 13 (a) shows a fluorescence image of a fresh brain tumor sample incubated with 50 μM NBD-pAP for 30 minutes in the presence or absence of a LAT1 inhibitor (20 μM JPH203). FIG. 13 (b) shows the time-dependent fluorescence changes of a fresh brain tumor sample after the addition of the NBD-pAP solution (in the presence or absence of JPH203). The photophysical characteristics of NBD-lys (upper) and NBD-amino-ala (lower) are shown. FIG. 15 shows a fluorescence image of A549 cells treated with 5 μM NBD-lys and NBD-amino-ala.

In the present specification, "alkyl" may be any of an aliphatic hydrocarbon group consisting of a linear chain, a branched chain chain, a cyclic chain, or a combination 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 6 (C _{1 to 6} ), the number of carbon atoms is 1 to 10 (C _{1 to 10} ), and the number of carbon atoms is 1 to 15 (C _{1 to 15).} ), The number of carbon atoms is 1 to 20 (C _{1 to 20} ). When the number of carbon atoms is specified, it means "alkyl" having the number of carbon atoms in the range of the number of carbon atoms. For _example, the _{C 1 ~ 8} alkyl, methyl, ethyl, n- propyl, isopropyl, n- butyl, isobutyl, sec- butyl, tert- butyl, n- pentyl, isopentyl, neo-pentyl, n- hexyl, isohexyl, Includes n-heptyl, n-octyl and the like. As used herein, the alkyl group may have one or more arbitrary substituents. Examples of such a substituent include, but are not limited to, an alkoxy group, a halogen atom, an amino group, a mono or di-substituted amino group, a substituted silyl group, or an acyl. If the alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl moiety of other substituents containing the alkyl moiety (eg, alkane group, arylalkyl group, etc.).

In the present specification, the term "halogen atom" may be any of a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom, and is preferably a fluorine atom, a chlorine atom, or a bromine atom.

In the present specification, when a functional group is defined as "may be substituted", the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and two or more substitutions are made. If they have groups, they may be the same or different. Examples of the substituent include, but are not limited to, an alkyl group, an alkoxy group, a hydroxyl group, a carboxyl group, a halogen atom, a sulfo group, an amino group, an alkoxycarbonyl group, an oxo group and the like. Further substituents may be present in these substituents. Examples of such include, but are not limited to, alkyl halide groups, dialkylamino groups, and the like.

1. 1. A compound represented by the general formula (I) or a salt thereof One embodiment of the present invention is a compound represented by the following general formula (I) or a salt thereof (hereinafter, also referred to as “compound of the present invention”). ..

In the present invention, in developing a fluorescent substrate to be incorporated into cancer tissues via LAT1, the present inventors focused on the substrate requirements required by LAT1 and examined binding a fluorescent group to the amino acid side chain site. bottom. Although not intended to be bound by theory, LAT1 is considered to be preferable as a fluorescent group having a small molecular weight like other transporters. Among them, among them, although it is compact in molecular size, it is used in clinical practice. NBD (nitrobenzoxaziazole, molecular weight: 164), which can be used at the fluorescence wavelength used, was selected. Therefore, it is important that the compound of the present invention has a structure in which NBD and side chain sites of various amino acids are bonded.

In the general formula (I), R ¹ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. The alkyl group is preferably a methyl group.

In the general formula (I), L is a divalent group selected from the group consisting of alkylene, arylene, and a group composed of an arbitrary bond of alkylene and arylene.

The alkylene is a divalent group consisting of linear or branched saturated hydrocarbons, for example, methylene, 1-methylmethylene, 1,1-dimethylmethylene, ethylene, 1-methylethylene, 1-ethylethylene. , 1,1-dimethylethylene, 1,2-dimethylethylene, 1,1-diethylethylene, 1,2-diethylethylene, 1-ethyl-2-methylethylene, trimethylene, 1-methyltrimethylene, 2-methyltri Methylene, 1,1-dimethyltrimethylene, 1,2-dimethyltrimethylene, 2,2-dimethyltrimethylene, 1-ethyltrimethylene, 2-ethyltrimethylene, 1,1-diethyltrimethylene, 1,2- Diethyltrimethylene, 2,2-diethyltrimethylene, 2-ethyl-2-methyltrimethylene, tetramethylene, 1-methyltetramethylene, 2-methyltetramethylene, 1,1-dimethyltetramethylene, 1,2-dimethyl Examples thereof include tetramethylene, 2,2-dimethyltetramethylene and 2,2-di-n-propyltrimethylene.

The arylene is a divalent group consisting of a monocyclic or condensed polycyclic aromatic hydrocarbon, and contains one or more heteroatoms (for example, oxygen atom, nitrogen atom, sulfur atom, etc.) as a ring-constituting atom. It may be an aromatic heterocycle. Whether the arylene is a monocyclic ring or a condensed ring, it can be bonded at all possible positions. Non-limiting examples of monocyclic aryl include a phenylene group (Ph) and the like. Non-limiting examples of condensed polycyclic arylenes include naphthylene and the like.

In one preferred embodiment of the invention, L is selected from:
-(CR ^a R ^b ) _n- , ^{-Ar-, *} -Ar- (CR ^a R ^b ) _m- , ^* -(CR ^a R ^b ) _s -Ar-, ^* -(CR ^a R ^b ) _s- Ar- (CR ^a R ^b ) _m-
Here, Ar represents an arylene, ^Ra and R ^b are independent hydrogen atoms or alkyl groups having 1 to 3 carbon atoms at each appearance, and n is an integer of 1 to 8. m is an integer of 1 to 5, s is an integer of 1 to 8, and * indicates the side to be combined with X.

Further, in one preferred embodiment of the present invention, L is selected from the following.
^{_{- (CH 2) n1 -,}} - Ph-, * -Ph- (CH 2) m1 -, * - (CH 2) s1 -Ph-, * - (CH 2) s1 -Ph- (CH 2) m1 -
Here, Ph represents a phenylene group, n1 is an integer of 1 to 8, m1 is an integer of 1 to 5, s1 is an integer of 1 to 8, and * indicates a side to be bonded to X. ..

Since the group in which L or L and X (NR ² ) are bound corresponds to the side chain site of the amino acid that is the substrate of LAT1, the hydrogen atom is removed from the group at the end of the side chain of the amino acid that is the substrate target of LAT1. A group from which one is removed (for example, if the amino acid of the substrate target of LAT1 is alanine, a methylene group (-CH ^2- )) is preferable, but the present invention is not limited to this, and can be selected depending on the type of amino acid. ..
For example, if the amino acid of the substrate target of LAT1 is phenylalanine, L is ^{preferably *} -Bz-CH _2- (Bz represents a benzene ring) and ^* -CH _2- Bz-CH _2- .

In a more preferred embodiment of the invention, L is selected from:
^{_{-CH 2 -, * -Bz-CH}} 2 -, - (CH 2) 4 -, * -CH 2 -Bz-CH 2 -
Here, Bz represents a benzene ring, and * represents a side bonded to X.

In the general formula (I), X represents ^{NR 2.} Here, R ² is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms. When R ² is an alkyl group, the pH dependence of the absorption spectrum and fluorescence spectrum of the compound of the present invention can be suppressed. The alkyl group is preferably a methyl group.

In one preferred aspect of the invention, L is ^* -Bz-CH _2- .

In one preferred aspect of the present invention, L ^{_{is, * -CH 2 -Bz-CH 2}} - it is.

In one preferred aspect of the present invention, L is, -CH ² - is.

In another preferred aspect of the present invention, L is, - _{(CH 2) 4} - a.

In general formula (I), Y is a nitro group (-NO ₂ ), a sulfonic acid group (-SO ₃ H) or a sulfonamide group (-SO ₂ NR'R''), where R'and R ″ is independently a hydrogen atom or an alkyl group having 1 to 4 carbon atoms.
Y is preferably a nitro group (-NO ₂ ).

In the general formula (I), when L is ^* -Bz-CH _2- , X can be introduced at the para-position or meta-position of Bz (benzene ring) with respect to _{two -CH groups.}

The compound represented by the general formula (I) can exist as an acid addition salt or a base addition salt. Examples of the acid addition salt include mineral salts such as hydrochloride, sulfate and nitrate, methanesulfonate, p-toluenesulfonate, oxalate, citrate, tartrate, trifluoroacetate and the like. Examples of the base addition salt include metal salts such as sodium salt, potassium salt, calcium salt and magnesium salt, ammonium salt, and organic amine salt such as triethylamine salt. .. In addition to these, it may form a salt with an amino acid such as glycine. The compound represented by the general formula (I) or a salt thereof may exist as a hydrate or a solvate, but these substances can also be used in the present invention.

The compound represented by the general formula (I) may have one or two or more asymmetric carbons depending on the type of the substituent, but in the present invention, one or two or more asymmetric carbons. In addition to stereoisomers such as optically active compounds based on the above and diastereoisomers based on two or more asymmetric carbons, any mixture of stereoisomers, racemates and the like can also be used.

A method for producing a typical compound of the compound represented by the general formula (I) is specifically shown in the examples of the present specification. Therefore, those skilled in the art can appropriately select reaction raw materials, reaction conditions, reaction reagents, etc. based on these explanations, and modify or modify these methods as necessary to obtain the general formula ( The compound represented by I) can be produced.

The compound represented by the general formula (I) can be used as a substrate for LAT1.

2. Fluorescent probe of the present invention Another aspect of the present invention is a fluorescent probe containing the compound of the general formula (I) or a salt thereof (hereinafter, also referred to as "fluorescent probe of the present invention").

Another aspect of the present invention is a method for detecting intracellular uptake by LAT1.
It is a method including (a) a step of introducing a fluorescent probe containing the compound of the general formula (I) or a salt thereof into a cell, and (b) a step of measuring the fluorescence emitted by the compound or a salt thereof in the cell (). Hereinafter, it is also referred to as "detection method of the present invention").
Here, examples of cells include normal cells, cancer cells, nerve cells and the like.

Another aspect of the present invention is a method of visualizing cancer cells or tissues.
(A) A step of introducing a fluorescent probe containing the compound of the general formula (I) or a salt thereof into a cell or tissue, and (b) using an appropriate wavelength at which fluorescence is measured in the cell or tissue into which the fluorescent probe has been introduced. It is a method including the step of observing or measuring the fluorescence emitted in the cell or tissue by irradiating the cell or tissue (hereinafter, also referred to as “visualization method of the present invention”).

Another embodiment of the present invention is a method for detecting cancer, wherein (a) a step of applying a fluorescent probe containing a compound of the general formula (I) or a salt thereof to a clinical sample of a subject, and ( b) A method including measuring a fluorescent image of a clinical sample to which the fluorescent probe is applied (hereinafter, also referred to as "detection method of the present invention").
The application of the fluorescent probe to the clinical sample in the step (a) can be performed, for example, by locally spraying the solution of the fluorescent probe onto the clinical sample.
Clinical specimens include, for example, lung cancer, prostate cancer, ovarian cancer, breast cancer, bladder cancer, brain cancer, esophageal cancer, gastric cancer, bile duct cancer, liver cancer, pancreatic cancer, head and neck cancer, and kidney. Hmm, leukemia, skin cancer, thyroid cancer, etc.

Another aspect of the present invention is (a) a step of administering a fluorescent probe containing a compound of the general formula (I) or a salt thereof to a subject, and (b) fluorescence of cells, tissues or organs of the subject. Is a method for diagnosing cancer, which comprises a step of irradiating with an appropriate wavelength to be measured and thereby observing or measuring fluorescence inside the cell, tissue or organ (hereinafter, "diagnosis method of the present invention"). Also called. "
Examples of the administration method include local administration, oral administration, intravenous administration and the like.
The types of cancers subject to the diagnostic method of the present invention include lung cancer, prostate cancer, ovarian cancer, breast cancer, bladder cancer, brain tumor, esophageal cancer, gastric cancer, bile duct cancer, liver cancer, and pancreatic cancer. , Head and neck cancer, kidney cancer, leukemia, skin cancer, thyroid cancer.

In the present specification, the term "cancer tissue" means any tissue including cancer cells. The term "tissue" should be interpreted in the broadest sense, including part or all of the organ, and should not be construed in a limited way in any sense. As the cancer tissue, a tissue highly expressing LAT1 is preferable. In addition, the term "diagnosis" in the present specification needs to be interpreted in the broadest sense, including confirming the presence of cancerous tissue at any biological site with the naked eye or under a microscope.

In the above-mentioned method for visualizing cancer cells or tissues and the method for diagnosing cancer, the compound of the present invention or a salt thereof is taken up into cells, tissues or organs via LAT1, and the compound is incorporated into cells, tissues or organs. This is preferably performed by observing or measuring the fluorescence emitted inside the organ.

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

The method of using the fluorescent probe of the present invention is not particularly limited, and it can be used in the same manner as a conventionally known fluorescent probe. Usually, the compound of the present invention or a salt thereof is added to an aqueous medium such as physiological saline or a buffer solution, or a mixture of an aqueous medium and a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide. The solution may be added to a suitable buffer containing cells or tissues and the fluorescence spectrum may be measured. The fluorescent probe of the present invention may be used in the form of a composition in combination with a suitable additive. For example, it can be combined with additives such as buffers, solubilizers and pH regulators.
In addition, the concentration of the compound of the present invention in the fluorescent probe of the present invention can be appropriately determined according to the type of cells to be measured, measurement conditions, and the like.

Another embodiment of the present invention is a kit for detecting cancer cells or tissues, which comprises the fluorescent probe of the present invention.

In the kit, the fluorescent probe of the present invention is usually prepared as a solution, but is provided as a composition in an appropriate form such as a mixture in powder form, a lyophilized product, a granule, a tablet, or a liquid preparation, and is provided at the time of use. It can also be applied by dissolving it in distilled water for injection or an appropriate buffer solution.

In addition, the kit may appropriately contain other reagents and the like, if necessary. For example, as the additive, additives such as a solubilizing agent, a pH adjusting agent, a buffering agent, and an isotonicizing agent can be used, and the blending amount thereof can be appropriately selected by those skilled in the art.

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

1. 1. Abbreviation Description Boc: tert-butoxycarbonyl DCM: dichloromethane DMEM: Drubecco's improved eagle medium DMSO: dimethylsulfoxide ESI: electrospray ionization FLIM: fluorescence lifetime imaging microscope _Φfl : fluorescence quantum yield HBSS: Hank's equilibrium salt solution HEPES: 4- (2-Hydroxyethyl) -1-piperazine ethanesulfonic acid HPLC: High Performance Liquid Chromatography HRMS: High Resolution Mass Spectrometry MeOH: Methanol MeCN: Acetonitrile MPLC: Medium Pressure Liquid Chromatography MS: Mass Spectrometry NaPi: Sodium Phosphate PBS : Phosphoric acid buffered physiological saline ROI: Region of interest RT-qPCR: Real-time quantitative polymerase chain reaction TFA: Trifluoroacetonitrile TLC: Thin layer chromatography UPLC-MS: Ultra-high performance liquid chromatography Mass spectrometry

2. Materials and methods
Materials and general procedures Reagents and solvents are Aldrich Chemical Co., Ltd., Tokyo Chemical Industry Co., Ltd. and Wako Pure Chemical Industries, Ltd., Kanto Chemical Co., Ltd., Gibco Co., Ltd., Invitrogen, Toyobo and Thermo Scientific. It was of the available grade grade supplied by and used without further purification. The reaction was observed by TLC and ESI mass spectrometry, or UPLC-MS.

The instrumental NMR spectrum was measured at 400 MHz for ^{1 1 HNMR and 101 MHz for 13} CNMR by a JEOL JNM-LA400 apparatus.
The mass spectrum (MS) was measured using JEOL JMS-T100LC AccuToF (ESI). Preparative HPLC uses an HPLC system consisting of a pump (PU-2080, JASCO) and a detector (MD-2025 or FP-2025, JASCO) and contains eluent A (0.1% TFA (v / v)). ₂ O) and eluent B (20% H ₂ O and CH ₃ CN containing 0.1% TFA (v / v)) at a flow rate of 5 mL / min, Inertsil ODS-3 (10.0 × 250 mm) column (GL). This was performed using Sciences Inc.).
The preparative MPLC is a silica gel column (silica gel 40 μm, Yamazen) using an MPLC system consisting of a pump and a detector (EPCLC AI-580S, Yamazen), or a SNAP Ultra C18 30 g (Biotage) using ^{Isolera TM One (Biotage).} I went there.
LC-MS analysis includes pumps (LC-30AD, Shimadzu), PDA detectors (SPD-M30A, Shimadzu), FP detectors (RF-20Axs, Shimadzu) and MS detectors (LCMS-2020, Shimadzu). ) Was used on an Agilent Poroshell 120 (10 cm × 2.1 mm, EC-C18 1.9 μm) column (Agilent).
qPCR was performed on the Light Cycler® 480 system (Roche).

Measurement of optical characteristics The absorption spectrum was obtained with Shimadzu UV-1850 (Tokyo, Japan). The fluorescence spectrum was performed using Hitachi F7100 (Tokyo, Japan). The slit width was 5 nm for both excitation and emission. The photomultiplier tube voltage was 400V or 700V. Absolute fluorescence quantum yields were determined using Hamamatsu Photonics Quantaurus QY. The area under the emission spectrum of the test sample was compared with the standard sample to obtain the relative fluorescence quantum yield, which was calculated by the following formula.

In the formula, st = standard; x = sample; A = absorbance at excitation wavelength; n = refractive index; D = area under the fluorescence spectrum on the energy scale.
The optical properties of these probes were examined in PBS (−) or Na-Pi buffer containing 0.1% DMSO as a co-solvent. Fluorescence lifetime was measured by Quantaurus-Tau C 11367 (HAMAMATSU).

Cell Culture A549 cells, HEK293 cells and HeLa cells were cultured in DMEM (Gibco) containing 10% fetal bovine serum (Gibco) and 1% penicillin streptomycin (Gibco).
Caco-2 cells were cultured in DMEM containing 20% fetal bovine serum, 1% penicillin streptomycin and 1% MEM non-essential amino acid solution (100x, Gibco).
All cells were cultured in a humidified incubator in 95% air under _{5% CO 2.}

Fluorescence confocal microscopy imaging with LAT inhibitors A549, HEK293, HeLa and Caco-2 cells were ^{seeded at 4.0 × 10 4} cells / well in an 8-well chamber (ibidi) and cultured in the appropriate medium. Cells were washed once with Na ⁺ Free buffer (pH 5.3), were preincubated for 30 minutes at Na ⁺ Free buffer (pH 5.3). The buffer is a probe in 200 μL of Na ⁺ free buffer (pH 5.3) in the presence or absence of a LAT inhibitor (5 mM BCH or 5 μM HCl 203) containing less than 0.5% DMSO as a co-solvent. It was replaced with a solution (when JPH203 was used as an inhibitor, the buffer contained 100 μM HCl as a co-solvent) and the chamber was incubated for 30 minutes. Fluorescence images were acquired using a confocal fluorescence microscope (TCS SP8, Leica) equipped with a 40x objective lens (HC PL APO 40x / 1.30 OIL, Leica) and an Ar laser.
The excitation wavelength and the emission wavelength were 488 nm and 534 to 634 nm, respectively.

Fluorescence imaging of probe efflux A549 cells on an 8-well chamber are incubated with probe solution for 30 minutes according to the same protocol as above, and then buffer solution is Na ^{Replaced with +} free buffer (pH 5.3). Next, a 50-minute time-lapse fluorescence image was acquired using a confocal microscope under the same conditions as described above.

Comparison of probe uptake rates Following the same protocol as above, A549 and HeLa cells in an 8-well chamber were pre-incubated and then the chamber was placed on a confocal microscope. The buffer was then rapidly replaced with a probe solution of ^{200 μL Na +} free buffer (pH 5.3) containing less than 0.25% DMSO as a co-solvent. After application of the probe, images were recorded by Leica TCS SP8 every minute for a total of 10 or 20 minutes under the following conditions: Using a 63x objective lens and an Ar laser, the excitation wavelength and emission wavelength were 488 nm and 534 to 634 nm (A549) or 519 to 590 nm (HeLa), respectively.

A 96-well round bottom plate (Corning) of spheroid culture was coated with 30 mg / mL poly-HEMA (Sigma-Aldrich) 95% EtOH solution and then dried overnight on a clean bench. ^{A549 cells and HEK293 cells were seeded with 2.0 × 10 3} cells containing 2.5% Matrigel (Corning) on each well (seeding procedure on ice to prevent Matrigel polymerization). rice field). The plates were centrifuged (1000 G x 10 minutes, 4 ° C.) and incubated in DMEM for 3 days to prepare spheroids with a radius of about 400 μm.

These spheroids cultured by fluorescence imaging <br/> above protocol spheroids were washed once with Na ⁺ Free buffer (pH 5.3), were preincubated for 30 minutes at Na ⁺ Free buffer (pH 5.3). The spheroids are then transferred to an 8-well chamber and the buffer is in ^{the presence or absence of a 200 μL Na +} free buffer LAT1 inhibitor (5 μM JPH203) containing less than 0.25% DMSO and 100 μM HCl as co-solvent. It was replaced with the probe solution in the presence and the chamber was incubated for 30 minutes. Fluorescence images were obtained with a confocal fluorescence microscope (TCS SP8, Leica) equipped with a 10x objective lens (HC PL APO CS 10x / 0.40 DRY, Leica) and an Ar laser. The excitation wavelength and the emission wavelength were 488 nm and 534 to 634 nm, respectively. Images were taken at 10 μm each in the z direction, for a total of 150 μM. After overlaying the z-direction image with LASX software, the fluorescence intensity of the spheroid was quantified with ImageJ software.

Fresh brain tumor specimens used in the fluorescence imaging experiments of fresh specimens were resected during surgery and provided unfrozen. ^{NBD-pAP in Na +} free buffer (pH 5.3) in the presence or absence of a LAT1 inhibitor (20 μM JPH203) containing 0.25% DMSO as a co-solvent, fresh brain tumors on an 8-well chamber. Applied to the sample. ^{Then, using the Maestro TM} In-Vivo Imaging System (PerkinElmer) before and 2 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes and 30 minutes after application of the probe under the following conditions. , Fluorescent images were obtained. Excitation filter: 490 nm, emission filter: 530 long pass. The fluorescence intensity was quantified according to the following procedure. The region of interest (ROI) was set on the captured fluorescence image, the fluorescence intensity of each ROI was calculated and graphed using Maestro software.

3. 3. Synthesis and characterization of compounds [Synthesis Example 1]
Synthesis of Compound 2 (NBD-pAP) (1) Synthesis of Compound 1 Compound 1 was synthesized by the following synthesis scheme.

4-Amino-N-Boc-L-phenylalanine (101 mg, 0.36 mmol) and sodium bicarbonate (26.1 mg, 0.31 mmol) were dissolved in water (1.3 mL). A methanol solution (6.1 mL) of 4-chloro-7-nitrobenzoflazan (NBD-Cl, 87.6 mg, 0.44 mmol) was added dropwise, and the mixture was stirred at 55 ° C. for 2.25 hours. After cooling to room temperature, methanol was evaporated. The aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The combined organic layers were washed with brine and dried over Na ₂ SO ₄ to evaporate the solvent to give a bright orange solid (151.7 mg, 95% yield).
¹ H-NMR (400 MHz, CD ₂ Cl ₂ ) δ 8.42 (d, J = 8.7 Hz, 1H), 7.37-7.32 (m, 4H), 6.70 (d, J = 8.7 Hz, 1H), 5.03 (d , J = 4.6 Hz, 1H), 3.04-3.27 (m, 2H), 1.39 (s, 9H)

(2) Synthesis of Compound 2 (NBD-pAP) Compound 2 was synthesized by the following synthesis scheme.

Compound 1 (152 mg, 0.34 mmol) was dissolved in dichloromethane (15 mL) and cooled at 0 ° C. TFA (5.6 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 2.25 hours, the solvent was evaporated to dryness. The residue is HPLC (eluent: A: H2O, 0.1% TFA (v / v), B: CH3CN / H2O = 80/20, 0.1% TFA (v / v); gradient: A / B = 70. Purification by 30/30/70, 45 minutes) gave NBD-pAP (Compound 2, 36.3 mg, yield 31%).
^{1 1} H-NMR (400MHz, CD ₃ OD) δ8.48 (d, J = 8.7 Hz, 1H), 7.44 (dd, J = 19.0, 8.5 Hz, 4H), 6.75 (d, J = 8.7 Hz, 1H) , 4.16 (dd, J = 7.8, 5.5 Hz, 1H), 3.36-3.14 (m, 2H).
¹³ CNMR (101 MHz, CDCl ₃ ): δ 170.10, 145.18, 144.22, 142.32, 137.59, 136.43, 132.92, 130.60, 124.32, 124.11, 100.78, 53.94, 35.65.
^{HRMS (ESI -):. Calcd} For [MH] -, 342.08384, Found, 310.11301 (-0.85 mDa)

[Synthesis Example 2]
Synthesis of Compound 5 (NBD-pMAP) (1) Synthesis of Compound 3 Compound 3 was synthesized by the following synthesis scheme.

4-Amino-N-Boc-L-Phenylalanine (430 mg, 1.53 mmol) was dissolved in a mixed solution of methanol (2 mL) and acetic acid (0.4 mL). 37% formaldehyde · _{H 2} O solution (113μL) was added dropwise, then borane-2-methylpyridine complex _{(pic · BH 3, 164mg,} 1.53mmol) in methanol (2.3 mL) was added and 3 at room temperature Stirred for hours. The MeOH was evaporated and 1N HClaq was added to the residue. The aqueous solution was stirred at room temperature for 5 minutes and saturated NaHCO ₃ aq was added to make the solution alkaline. The aqueous layer was extracted with ethyl acetate, the combined organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The residue was purified by a preparative MPLC system (AcOEt / n-hexane, Rf = 0.30) to obtain compound 3 (43.1 mg, yield 9.6%) as a pale yellow solid.
¹ H-NMR (400 MHz, CD ₃ CN) δ 6.94 (d, J = 8.2 Hz, 2H), 6.49 (d, J = 8.2 Hz, 2H), 5.33 (d, J = 6.4 Hz, 1H), 4.19 (dd, J = 13.3, 7.8 Hz, 1H), 2.95-2.72 (m, 2H), 2.70 (s, 3H), 1.34 (s, 9H)
¹³ CNMR (101 MHz, CD ₃ CN): δ 172.95, 155.31, 148.86, 129.93, 124.49, 112.06, 79.00, 55.10, 36.24, 29.77, 27.56.

(2) Synthesis of Compound 5 (NBD-pMAP) Compound 5 was synthesized by the following synthesis scheme.

Compound 3 (43.1 mg, 0.15 mmol) and sodium bicarbonate (11.1 mg, 0.13 mmol) were dissolved in water (2.0 mL). A methanol solution (2.0 mL) of NBD-Cl (26.6 mg, 0.13 mmol) was added dropwise, and the mixture was stirred at room temperature for 3.5 hours. The methanol was evaporated and the aqueous mixture was acidified with 2N HCl and extracted with dichloromethane. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane (10 mL) and cooled at 0 ° C. without further purification. TFA (2 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 2 hours, the solvent was evaporated to dryness. HPLC the residue (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient : A / B = 90/10 to 50/50, 45 minutes) to obtain NBD-pMAP (Compound 5, 12.9 mg, yield 25% in two steps).
^{1 1} H-NMR (400 MHz, CD ₃ OD) δ 8.40 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.7 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 6.26 (d, J = 9.1 Hz, 1H), 4.22 (dd, J = 7.8, 5.5 Hz, 1H), 3.89 (s, 3H), 3.38 (dd, J = 14.6, 5.5 Hz, 1H), 3.21 (q, J = 7.5 Hz, 1H)
HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ , 344.09949, Found, 344.10190 (+2.41 mDa)

[Synthesis Example 3]
Synthesis of Compound 8 (NBD-mAP) (1) Synthesis of Compound 6 Compound 6 was synthesized by the following synthesis scheme.

3-Amino-N-Boc-L-Phenylalanine (100 mg, 0.32 mmol) was dissolved in 10 mL methanol, followed by the addition of Pd / C (10%, 12 mg). Then, the mixture was stirred at _{room temperature under H 2 for 2 hours.} The reaction mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure to give compound 6 (88.6 mg, 98% yield).
^{1 1} H-NMR (400 MHz, CDCl ₃ ) δ 7.95 (d, J = 7.8 Hz, 1H), 7.66- 7.48 (m, 1H), 7.42 (d, J = 7.8 Hz, 2H), 5.31 (d, J = 5.9 Hz, 1H), 4.66 (s, 1H), 3.59-3.24 (m, 2H), 1.34 (s, 9H)

(2) Synthesis of Compound 8 (NBD-mAP) Compound 8 was synthesized by the following synthesis scheme.

Compound 6 (49.9 mg, 0.18 mmol) and sodium bicarbonate (12.9 mg, 0.15 mmol) were dissolved in water (4.0 mL). A methanol solution (2.0 mL) of NBD-Cl (42.7 mg, 0.21 mmol) was added dropwise, and the mixture was stirred at room temperature for 7 hours. The methanol was evaporated and the aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The mixed organic layer was washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane (10 mL) and cooled at 0 ° C. without further purification. TFA (2 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 6 hours, the solvent was evaporated. HPLC the residue (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient : A / B = 70/30 to 30/70, 45 minutes) to obtain NBD-mAP (Compound 8, 22.3 mg, yield 37% in two steps).
¹ H-NMR (400 MHz, CD ₃ OD) δ 8.51 (d, J = 8.7 Hz, 1H), 7.50 (t, J = 7.5 Hz, 1H), 7.41 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 7.3 Hz, 1H), 6.79 (d, J = 8.7 Hz, 1H), 4.29 (t, J = 6.6 Hz, 1H), 3.21-3.36 (m, 2H)
HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ , 358.11514, Found, 358.11873 (+3.58 mDa)

[Synthesis Example 4]
Synthesis of Compound 11 (NBD-pAMP) (1) Synthesis of Compound 9 Compound 9 was synthesized by the following synthesis scheme.

4-Cyano-N-Boc-L-Phenylalanine (216.7 mg, 0.77 mmol) was dissolved in a mixed solution of 30 mL methanol and 10 mL acetic acid, then Pd / C (10%, 36 mg) was added. Then, the mixture was stirred at _{room temperature under H 2 for 2 hours.} The reaction mixture was filtered through Celite® and the filtrate was concentrated under reduced pressure. The residue was purified by reverse phase column chromatography SNAP Ultra C18 30g (Biotage, _{eluent: A: H 2 O, 0.1} % TFA (v / v), B: CH 3 CN, 0.1% TFA (v / v) ) To give compound 9 (199.5 mg, yield 91%).
^{1 1} H-NMR (400 MHz, CD ₃ OD) δ 7.32 (dd, J = 17.6, 8.0 Hz, 4H), 4.27 (q, J = 4.6 Hz, 1H), 4.05 (s, 2H), 3.16 (dd, dd, J = 13.7, 5.0 Hz, 1H), 2.91 (dd, J = 13.7, 8.7 Hz, 1H), 1.36 (s, 9H)

(2) Synthesis of Compound 11 (NBD-pAMP) Compound 11 was synthesized by the following synthesis scheme.

Compound 9 (164 mg, 0.56 mmol) and sodium bicarbonate (281 mg, 3.3 mmol) were dissolved in water (15 mL). A methanol solution (15 mL) of NBD-Cl (134 mg, 0.67 mmol) was added dropwise, and the mixture was stirred at room temperature for 10 hours. The methanol was evaporated and the aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The mixed organic layer was washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane (47 mL) and cooled at 0 ° C. without further purification. TFA (13 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 2.5 hours, the solvent was evaporated. Residues HPLC (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient : A / B = 90/10 to 50/50, 45 minutes) to obtain NBD-pAMP (Compound 11, 69.8 mg, yield 31% in two steps).
¹ H-NMR (400 MHz, CD ₃ OD) δ 9.49 (d, J = 8.7 Hz, 1H), 8.48 (d, J = 8.2 Hz, 2H), 8.35 (d, J = 8.2 Hz, 2H), 7.33 (d, J = 8.7 Hz, 1H), 5.79 (s, 2H), 5.24 (dd, J = 7.8, 5.5 Hz, 1H), 4.31 (d, J = 5.5 Hz, 1H), 4.16 (dd, J = 14.4, 8.0 Hz, 1H)
HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ , 358.11514, Found, 358.12008 (+4.93 mDa)

[Synthesis Example 5]
Synthesis of Compound 12 (NBD-lys) Compound 12 was synthesized by the following synthesis scheme.

N-Boc-L-lysine (216 mg, 0.88 mmol) and sodium bicarbonate (86.6 mg, 0.65 mmol) were dissolved in water (4.0 mL). A solution of NBD-Cl (178 mg, 0.88 mmol) in methanol (10 mL) was added dropwise, and the mixture was stirred at room temperature for 3 hours. The methanol was evaporated and the aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane (20 mL) and cooled at 0 ° C. without further purification. TFA (7 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 3.5 hours, the solvent was evaporated. Residues HPLC (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient : A / B = 80/20 to 20/80, 45 minutes) to obtain NBD-Lys (compound 12,211 mg, yield 78% in two steps).
^{1 1} H-NMR (400MHz, CD ₃ OD) δ 8.50 (d, J = 8.7 Hz, 1H), 6.33 (d, J = 8.7 Hz, 1H), 3.93-3.95 (m, 1H), 3.55 (brs, 2H) ), 1.79-2.01 (m, 4H), 1.56-1.61 (m, 2H)
HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ , 310.11514, Found, 310.11301 (-2.14 mDa).

[Synthesis Example 6]
Synthesis of Compound 14 (NBD-amino-ala) Compound 14 was synthesized by the following synthesis scheme.

3-Amino-N- (Boc) -L-alanine (168 mg, 0.82 mmol) and sodium bicarbonate (61.0 mg, 0.61 mmol) were dissolved in water (4.0 mL). A solution of NBD-Cl (165 mg, 0.82 mmol) in methanol (15 mL) was added dropwise, and the mixture was stirred at room temperature for 3 hours. The methanol was evaporated and the aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The crude product was dissolved in dichloromethane (35 mL) and cooled at 0 ° C. without further purification. TFA (10 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 3.5 hours, the solvent was evaporated. HPLC the residue (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient : A / B = 80/20 to 20/80, 45 minutes) to obtain NBD-amino-ala (Compound 14, 99.4 mg, yield 45% in two steps).
^{1 1} H-NMR (400MHz, CD ₃ OD) δ8.54 (d, J = 8.7 Hz, 1H), 6.52 (d, J = 8.7 Hz, 1H), 4.04- 3.87 (m, 3H). HRMS (ESI ⁺ ): Calcd. For [M + H] ⁺ , 268.06819, Found, 268.06572 (-2.47 mDa).

[Synthesis Example 7]
Synthesis of Compound 16 (DBD-lys) Compound 16 was synthesized by the following synthesis scheme.

N-Boc-L-lysine (19.6 mg, 0.080 mmol) and sodium bicarbonate (8.70 mg, 0.10 mmol) were dissolved in water (0.4 mL). A solution of DBD-Cl (22.2 mg, 0.091 mmol) in methanol (2.0 mL) was added dropwise, and the mixture was stirred at room temperature for 5 hours. The methanol was evaporated and the aqueous mixture was acidified with 1N HCl and extracted with dichloromethane. The combined organic layers were washed with brine, dried over Na ₂ SO ₄ , and the solvent was evaporated to dryness. The residue was purified by a preparative MPLC system (silica gel, AcOEt + AcOH / n-hexane, Rf = 0.20). The crude product was then dissolved in dichloromethane (5 mL) and cooled at 0 ° C. TFA (1.5 mL) was added dropwise and the reaction mixture was warmed to room temperature. After stirring for 1 hour, the solvent was evaporated. HPLC the residue (eluent: A: H ₂ O, 0.1% TFA (v / v), B: CH ₃ CN / H ₂ O = 80/20, 0.1% TFA (v / v); gradient Purification with A / B = 70/30 to 30/70, 30 minutes) gave DBD-lys (Compound 16, 5.15 mg, yield 17% in two steps).
¹ H-NMR (400 MHz, CD ₃ OD) δ 7.86 (d, J = 8.2 Hz, 1H), 6.27 (d, J = 8.2 Hz, 1H), 3.94 (t, J = 6.2 Hz, 1H), 3.46 (t, J = 7.1 Hz, 2H), 2.77 (s, 6H), 1.90-2.01 (m, 2H), 1.77-1.84 (m, 2H), 1.54-1.65 (m, 2H). HRMS (ESI ⁺ ) : Calcd. For [M + H] ⁺ , 372.13416, Found, 372.13600 (+1.84 mDa).

4. Evaluation of Compound [Example 1]
Evaluation of Optical Properties of Compound 2 (NBD-pAP) The optical properties of Compound 2 (NBD-pAP) obtained in Synthesis Example 1 were obtained. The results are shown in FIG. 1 and Table 1 below.

FIG. 1 shows the optical characteristics of compound 2 (NBD-pAP), and the absorption spectrum and fluorescence spectrum were measured in a NaPi buffer solution (pH 7.5) containing 0.1% DMSO as a co-solvent.

^[A] Absorption maximum (Abs _max ) and release maximum (Em _max ) were measured in NaPi buffer (pH 7.5).^[B] Absolute fluorescence quantum yield ([Phi _fl) using Hamamatsu Photonics Quantaurus-QY PBS - measured in ().

The fluorescence quantum yield of NBD-pAP in the buffer solution was as low as 0.003, showing almost no fluorescence.

[Example 2]
Cell imaging with compound 2 (NBD-pAP)
(1) Evaluation using a LAT1 high-expressing cell line (A549) As examined in Example 1, it was revealed that the fluorescence of NBD-pAP was very weak, but the high sensitivity of fluorescence imaging was taken into consideration. Then, it was considered that it was possible to evaluate whether NBD-pAP could be a substrate for LAT1 by fluorescence imaging using cultured cells.
Therefore, first, A549 cells in which LAT1 expression has been reported are selected (Cell atlas --SLC7A5-The Human Protein Atlas.), And the presence or absence of BCH (structural formula is shown below), which is an inhibitor of LAT, is present. The intracellular uptake of NBD-pAP by LAT was evaluated from the fluorescence intensity below.

DMEM contains an amino acid that competes with NBD-pAP as a LAT1 substrate, and HBSS does not contain an amino acid ^{but contains Na +} ions, so it is Na ⁺ dependent. Since it is affected by the transport of amino acids by amino acid transporters, previous studies (Haefliger, P. et al. The LAT1 inhibitor JPH203 reduces growth of thyroid carcinoma in a fully immunocompetent mouse) ^{Na +} amino acid-free buffer (Na ⁺ free buffer: 125 mM Chocolate-Cl, 25 mM HEEPS,) prepared with reference to model. J. Exp. Clin. Cancer Res. 37, 1-15 (2018). Using 4.8 mM KCl, 1.2 mM sulfonyl ₄ , 1.2 mM KH ₂ PO ₄ , 1.3 mM CaCl ₂ , 5.6 mM D-glucose), it was evaluated whether NBD-pAP could be a LAT1 substrate.
In addition, since the pH of the buffer solution may affect the uptake of NBD-pAP into A549, evaluation was performed in four types of buffer solutions having different pH (Fig. 2). Since the Na ⁺ free buffer solution showed weak acidity (pH 5.3) before pH adjustment, the pH was adjusted to neutral by adding Tris base. The imaging protocol is as follows, and the parts not specifically mentioned in the subsequent experiments were evaluated by the same procedure.

<Protocol>
A549 cells are seeded in an Ibidi 8-well imaging chamber at ^{4.0 × 10 4 cells / well.}
_{↓ 37 ° C, CO 2 5} % under 1 wash ↓ Na ⁺ Free buffer for 30 minutes at 1-2 days of culture ↓ Na ⁺ Free buffer, 37 ° C., were pre-incubated with _CO 2 5% under.
↓ The buffer was replaced with a probe solution in ^{Na +} free buffer in the presence or absence of a LAT inhibitor.
↓ Na ⁺ Free buffer at 30 min, 37 ° C., and incubated _CO 2 5% under.
↓ The image was taken with a confocal microscope.

The results are shown in FIG. FIG. 2 is a fluorescence image of A549 cells treated with NBD-pAP. Cells were preincubated for 30 minutes at Na ⁺ free buffer pH 5.3 ~ 7.4, then pH 5.3 was incubated with Na ⁺ free buffer 50 [mu] M NBD-pAP of ~ 7.4 (5mM BCH (LAT In the presence or absence of an inhibitor)). Images were obtained with the Leica SP8. The imaging conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 2% Em. HyD3 530-630 nm, gain 500% offset-0.01.

As a result, it was observed that NBD-pAP had a difference in intracellular fluorescence intensity depending on the presence or absence of BCH under acidic conditions, suggesting that it is a substrate for LAT1 (Fig. 2). The intracellular fluorescence intensity decreased as the neutral condition was approached, suggesting that the uptake of NBD-pAP into A549 cells is affected by pH.

(2) Evaluation using a LAT1 low expression cell line (HEK293) Next, regarding HEK293 cells in which the expression level of LAT1 is reported to be low, NBD-pAP in a ^{Na + free buffer solution having a pH of 5.3 was also used.} Uptake was evaluated.

The results are shown in FIG. FIG. 3 shows a time-lapse fluorescence image of HEK293 cells treated with NBD-pAP. Cells were pre-incubated in pH 5.3 Na ⁺ free buffer for 30 minutes and then incubated with pH 5.3 Na ⁺ free buffer containing 50 μM NBD-pAP (in the presence of 5 mM BCH (LAT inhibitor) or In the absence). Images were obtained with the Leica SP8. The imaging conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 2% Em. HyD3 530-630 nm, gain 500% offset-0.01. Scale bar: 50 μm.

As a result, the intracellular fluorescence intensity in HEK293 cells was lower than that in A549 cells, suggesting that the uptake of NBD-pAP was low in HEK293 cells (Fig. 3).

(3) Evaluation Using LAT1 Selective Inhibitor JPH203 There are four types of isoforms of LAT1 to LAT4 in LAT. Of these, LAT1 is mainly highly expressed in tumor cells such as A549, but since BCH non-selectively inhibits all isoforms of LAT, NBD-pAP is LAT from previous studies. It was unclear which isoform of the throat was transported. Therefore, in order to evaluate whether NBD-pAP is a selective substrate for LAT1, JPH203 (structural formula is shown below), which exhibits a LAT1 selective inhibitory effect among the four isoforms of LAT, is used. , It was examined whether the uptake of NBD-pAP into A549 cells was inhibited. In addition, in order to investigate whether NBD-pAP is taken up via LAT1 regardless of cell type, a similar evaluation was performed using another cancer cultured cell line HeLa cell that highly expresses LAT1 (). FIG. 4).

Chemical structure of JPH203 (LAT1 selective inhibitor)

FIG. 4 shows fluorescence images of A549, HeLa and HEK293 cells treated with NBD-pAP. Cells were pre-incubated in pH 5.3 Na ⁺ free buffer for 30 minutes and then in pH 5.3 Na ⁺ free buffer containing 50 μM NBD-pAP for 30 minutes (5 μM JPH203 (LAT1)). Selective inhibitor) in the presence or absence). Images were obtained with the Leica SP8. The imaging conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 2% Em. HyD3 530-630 nm, gain 500% offset-0.01. Scale bar: 50 μm.

When JPH203 was used as an inhibitor, a difference in intracellular fluorescence intensity was observed in both A549 cells and HeLa cells depending on the presence or absence of JPH203. Further, for the LAT1 low expression cell HEK293, the intracellular fluorescence intensity was low as in FIG. 3 in which BCH was used as an inhibitor. Therefore, the results suggest that NBD-pAP is taken up via LAT1 among the four isoforms of LAT. In addition, from the studies so far, NBD-pAP can selectively observe the fluorescence signal from the inside of the cell without washing operation, and is high due to some mechanism such as intracellular concentration and activation of fluorescence inside the cell. It was suggested that the cells could be fluorescently visualized by the fluorescence intensity ratio inside and outside the cells.

[Example 3]
Development of an improved NBD fluorescent mother nucleus probe From the results of the study in Example 2, it was clarified that the compound NBD-pAP in which p-aminophenylalanine was introduced into NBD, which is a compact fluorescent group, serves as the LAT1 substrate. On the other hand, NBD-pAP has (1) intracellular fluorescence intensity changes depending on pH, (2) slow uptake into cells, and (3) low fluorescence quantum yield ( _Φfl <0). Since the point of 0.01) was recognized, next, structural development was carried out from NBD-pAP, and it was further examined whether these points could be improved.

(1) Examination of pH dependence First, the pH dependence of NBD-pAP was examined. NBD-pAP shows a pH-dependent absorption / fluorescence spectrum between pH 3.0 and 10.0, and the pKa of the nitrogen atom is about 7. It is known that it is around 5, and this data also agrees with this (Fig. 5).
FIG. 5 shows the pH dependence of the absorption spectrum and the fluorescence spectrum of NBD-pAP. (A, b) are absorption and emission spectra of NBD-pAP in 100 mM NaPi buffer of various pH values containing 0.1% DMSO as a co-solvent. The excitation wavelength was 470 nm. (C) is a plot of absorbance ratio vs. pH at 447 nm and 493 nm.

Therefore, in order to improve the pH dependence of NBD-pAP, a compound NBD-methylamino-phe (NBD-pMAP) in which a hydrogen atom on an NBD-bonded nitrogen atom was replaced with a methyl group was designed and synthesized (Synthesis Example 2). .. As a result of acquiring the absorption / fluorescence spectrum of NBD-pMAP, it was confirmed that the pH dependence of the absorption spectrum disappeared and that the deprotonation of the 4-position NH of NBD-pAP was the cause of the pH dependence of the absorption spectrum (Fig.). 6).
FIG. 6 shows the absorption and emission spectra of NBD-pMAP in 100 mM NaPi buffer with various pH values containing 0.1% DMSO as a co-solvent. The excitation wavelength was 490 nm.

(2) Examination of improvement of uptake rate into cells Next, it was examined whether the uptake rate of NBD-pAP into cells could be improved. When considering the intraoperative detection of cancer, the faster the rate of increase of the fluorescent signal is, the more suitable for rapid detection. Therefore, the substitution position of the substituent of phenylalanine to the phenyl group is a compound in which m-amino-phenylalanine is introduced into NBD with reference to the previous research that the transport rate of the LAT1 substrate is higher at the m-position than at the p-position. NBD-m-amino-phe (NBD-mAP) was designed and synthesized (Synthesis Example 3).

The result of applying NBD-mAP to A549 cells is shown in FIG.
FIG. 7 shows fluorescence images of A549 cells and HeLa cells treated with NBD-mAP. Cells were pre-incubated in pH 5.3 Na ⁺ free buffer for 30 minutes and then in pH 5.3 Na ⁺ free buffer containing 50 μM NBD-mAP for 40 minutes (in the presence or absence of 5 μM JPH203). In existence). Images were obtained with the Leica SP8. The imaging conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 2% Em. HyD3 530-630 nm, gain 500% offset-0.01. Scale bar: 50 μm.

From FIG. 7, an increase in fluorescence intensity was observed in the cells, and it was suppressed by the addition of JPH203, suggesting that NBD-mAP is also a substrate for LAT1. Therefore, in the substrate recognition of LAT1, it was shown that even if NBD is introduced at the m-position of the phenyl group of phenylalanine, it is acceptable as a LAT1 substrate.

Next, the uptake speed was compared according to the replacement position. In a confocal fluorescence microscope, a probe solution was added with the focal plane aligned with the cells (final 50 μM), and imaging was performed every minute for 10 minutes to track changes in intracellular fluorescence intensity for 10 to 20 minutes. As a result of measurement for two types of LAT1 high-expressing cells, A549 and HeLa, the uptake rate of the p-substituent was faster than that of the m-substitute in any of the cells, and the uptake was performed by changing the substitution position to the m-position. The speed did not improve (Fig. 8).

FIG. 8 shows a comparison of the uptake of NBD-pAP and NBD-mAP into A549 cells and HeLa cells. (A) is a time-dependent fluorescence image of A549 and HeLa cells treated with NBD-pAP and NBD-mAP. Cells were incubated 30 minutes prior to ^{Na +} free buffer at pH 5.3. Images were obtained with the Leica SP8. The image conditions were as follows: Ar 20%, HC PL APO CS2 63 × / 1.40 OIL, Ex. 488 nm 1% Em. HyD3 534-634 nm (A549) or 519-590 nm (HeLa), gain 500% offset-0.01. Scale bar: 50 μm. (B) is the quantification of the fluorescence intensity in the ROI of each cell. Error bars are represented by SD (n = 10).

(3) Examination of Improvement of Fluorescence Quantum Yield Next, a compound NBD-p-aminomethyl-phe (NBD) in which a methylene group was introduced between the amine at the NBD4 position and the aromatic ring of phenylalanine with the aim of improving the fluorescence quantum yield. -PAMP) was designed and synthesized (Synthesis Example 4), and the optical characteristics were obtained (Fig. 9).
FIG. 9 shows the optical characteristics of NBD-pAMP. The figure on the left is the absorption spectrum, and the figure on the right is the fluorescence spectrum. The entire spectrum was measured in PBS containing 0.1% DMSO as a co-solvent.
The fluorescence quantum yield of NBD-pAMP in PBS (−) was 5.9%, which was more than 10 times higher than that of NBD-pAP containing no methylene group (Table 2).

All properties were measured in PBS (−). Hamamatsu Photonics Quantaurus-QY to determine the absolute fluorescence quantum yield ([Phi _fl) used.

NBD-pAMP was applied to A549 cells and evaluated for LAT1 substrate (Fig. 10).
FIG. 10 shows a fluorescence image of A549 cells treated with 5 μM NBD-pAMP. Cells were pre-incubated in pH 5.3 Na ⁺ free buffer for 30 minutes and then incubated with pH 5.3 Na ⁺ free buffer containing 5 μM NBD-pAMP for 30 minutes (in the presence or absence of 5 μM JPH203). under). Images were obtained with the Leica SP8. The image conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1, 30 OIL, Ex. 488 nm 2% Em. HyD 3 530-630 nm, gain 500% offset-0.01. Scale bar: 50 μm.

From the results shown in FIG. 10, when NBD-pAMP was incubated at 5 μM, an increase in intracellular fluorescence intensity was observed, and since this increase was significantly reduced by JPH203, NBD-pAMP became a LAT1 substrate. It was suggested that Therefore, it was shown that the introduction of a methylene group between the NBD4-position amino group of NBD-pAP and the phenylalanine structure is acceptable for LAT1 substrate recognition.
Also, in NBD-pAMP, as in NBD-pAP, the fluorescence signal from the inside of the cell can be selectively observed without a washing operation, and some mechanism such as intracellular concentration and activation of fluorescence in the cell can be observed. It was suggested that the cells could be fluorescently visualized with a high intracellular and extracellular fluorescence intensity ratio.

Next, NBD-pAMP was incubated at 50 μM, washed, and then the intracellular fluorescence intensity was observed over time. As a result, it was observed that NBD-pAMP was excreted from the inside of the cell and stayed inside the cell. It was suggested that the sex was not high. Here, since LAT1 carries out the opposite transport of the substrate, considering the possibility that the transport of NBD-pAMP from the inside of the cell to the outside of the cell is carried out by LAT1, the excretion to the outside of the cell after the washing operation is added with JPH203. It was examined whether it was suppressed by (Fig. 11).

FIG. 11A shows a time-lapse fluorescence image of A549 cells treated with NBD-pAMP after washing out of extracellular buffer. Cells were pre-incubated in pH 5.3 Na ⁺ free buffer for 30 minutes and incubated with 5 μM NBD-pAMP for 30 minutes in the presence or absence of 5 μM JPH203 (LAT1 selective inhibitor). It is then washed with a pH 5.3 Na ⁺ free buffer (in the presence or absence of 5 μM JPH203). Images were obtained with the Leica SP8. The image conditions were as follows. Ar 20%, line average 16, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 0.5% Em. HyD3 530-630 nm, gain 500%. Scale bar: 50 μm.
FIG. 11 (b) shows the quantification of the fluorescence intensity in the ROI of each cell.

As shown in FIG. 11, the leakage of the probe from the cells after the washing operation was not inhibited by the addition of JPH203. It is considered possible that the probe is excreted from the intracellular to the extracellular by an amino acid transporter other than LAT1 or various drug excretion transporters whose expression is enhanced in cancer cells.

[Example 4]
Application to spheroid In Example 2, the fluorescent substrate NBD-pAP of LAT1 which was found to be taken up via LAT1 in two-dimensional cultured cells was applied to a 3D spheroid close to an actual tissue. It was verified whether the developed probe was taken up via LAT1 also in three-dimensional cultured cells. Specifically, a 3D spheroid having a diameter of about 400 mm was prepared from LAT1 high-expressing cell A549 and low-expressing cell HEK293 by the following protocol, and NBD-pAP was applied to this to perform fluorescence imaging with a confocal microscope (Fig.). 12).

<Protocol>
^{A549 cells containing 2.5% matrigel at 2.0 × 10 3} cells / well were seeded in a 96-well circular bottom plate coated with poly-HEMA (poly 2-hydroxyethyl methacrylate).
↓ 37 ° C., in _CO 2 5%, 30 min in a single washing ↓ Na ⁺ Free buffer (pH 5.3) at 3 days of culture ↓ Na ⁺ Free buffer _{(pH5.3), 37 ℃, CO} 2 5% Pre-incubation with ↓ The buffer ^{was replaced with a probe solution in Na +} free buffer (pH 5.3) in the ^{presence or absence of JPH203 ↓ Na +} free buffer (pH 5.3) for 30 minutes. 37 ° C., photographed images with incubation ↓ confocal microscopy _CO 2 5%

In imaging with a confocal microscope, a total of 16 stack images (total: 150 mm) are acquired at 10 mm intervals in the z-axis direction, and the maximum value of fluorescence intensity at each pixel is projected using the projection function of LAS X software. I created the image. The results are shown in FIG.

FIG. 12 (a) shows a fluorescence confocal image of a 3D spheroid prepared by A549 or HEK293. Pre-incubated at pH 5.3 in Na ⁺ free buffer for 30 minutes, then incubated in pH 5.3 Na ⁺ free buffer containing 50 μM NBD-pAP for 30 minutes (in the presence or absence of 5 μM JPH203). ). Images were obtained with a Leica SP8. The shooting conditions were Ar 20%, line average 5, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 2% Em. HyD 3 534-634 nm, Gain 500% Offset-0.01.
FIG. 12B shows the quantification of the fluorescence intensity of each spheroid at ROI. The data are shown as mean ± standard deviation (n = 4). ^* : P <0.05, ^** : p <0.001.

From the results shown in FIG. 12, when NBD-pAP was added to the spheroids of A549 cells, there was a difference in the fluorescence intensity with and without JPH203, and the spheroids of HEK293 cells were comparable to the A549-derived spheroids to which JPH203 was added. It was suggested that the probe was taken up via LAT1 even in the spheroids of the three-dimensional culture.

[Example 5]
Application to patient-derived fresh brain tumor specimens
It was examined whether NBD-pAP, which was suggested to be incorporated into three-dimensional spheroids via LAT1 in Example 4, is also incorporated into actual human fresh cancer tissues via LAT1. Brain tumors, which have been reported to have increased LAT1 expression and have been suggested to be associated with cancer malignancy and LAT1 expression, were selected as the cancer type to be evaluated. As an existing technique for fluorescently labeling brain tumors, there is 5-aminolevulinic acid (5-ALA), which is the only fluorescent reagent covered by insurance in the operation of malignant glioma. 5-ALA, which was orally administered 3 hours before the start of surgery, was synthesized intracellularly into protoporphyrin IX (PpIX) and selectively over-accumulated in tumor cells, to which blue visible light (375-445 nm) was emitted. Upon irradiation, it emits red fluorescence (600-740 nm). However, 5-ALA has problems such as low sensitivity specificity, poor resistance to photobleaching, and difficulty in additional administration during surgery.

If the NBD-pAP developed by the present invention is incorporated into brain tumor tissue via LAT1, it can be expected to be used as a fluorescence imaging tool for brain tumors that can be administered intraoperatively. By applying NBD-pAP to fresh brain tumor specimens derived from patients, it was evaluated whether NBD-pAP was also incorporated into human brain tumor tissues via LAT1 (FIG. 13).

<Protocol>
Fresh brain tumor specimens were placed in the Ibidi 8-well imaging chamber.
↓ Add 50 mM NBD-pAP to pH 5.3 Na + free buffer (in the presence or absence of 20 μM JPH203).
↓ Time-lapse image by Maestro ™ In-Vivo Imaging System (Parkin Elmer Inc.)

FIG. 13 (a) shows a fluorescent image of a fresh brain tumor sample incubated with 50 μM NBD-pAP for 30 minutes in the presence or absence of a LAT1 inhibitor (20 μM JPH203). Ex / Em = 488nm / 530nm long pass.
FIG. 13 (b) shows the time-dependent fluorescence changes of a fresh brain tumor sample after the addition of the NBD-pAP solution (in the presence or absence of JPH203).

As shown in FIG. 13, an increase in fluorescence intensity over time was confirmed in a brain tumor sample to which NBD-pAP was added, and the increase in fluorescence was suppressed by the LAT1 inhibitor JPH203. That is, it was suggested that NBD-pAP is also taken up via LAT1 in brain tumor tissues.

[Example 6]
Evaluation using NBD-Lys and NBD-amino-ala
(1) Optical characteristics Since the aromatic ring structure of the side chain was considered to be important for substrate recognition of LAT1, various structural developments have been carried out so far centering on phenylalanine as the amino acid structure. Next, considering the possibility that NBD can have a structure for new substrate recognition, NBD-Lys in which lysine was introduced as an amino acid structure in NBD and NBD-amino-ala in which 3-amino-alanine was introduced. (Synthesis Examples 5 and 6) were synthesized to obtain optical characteristics. The results are shown in FIG. 14 and Table 3.

FIG. 14 shows the photophysical characteristics of NBD-lys (upper row) and NBD-amino-ala (lower row). The figure on the left shows the absorption spectrum, and the figure on the right shows the fluorescence spectrum. All spectra were measured in 100 mM NaPi buffer containing 0.1% DMSO as a co-solvent.

^{[A] The} maximum absorption value (Abs _max ) and the maximum emission value (Em _max ) were measured in 100 mM NaPi buffer (pH 7.5). ^[B] Absolute fluorescence quantum yield ([Phi _fl) using Hamamatsu Photonics Quantaurus-QY PBS - measured in ().

The maximum absorption wavelength and fluorescence wavelength were about the same as NBD-pAP and NBD-pAMP, and the fluorescence quantum yield was about 5% in PBS, which was about the same brightness as NBD-pAMP.

(2) Cell imaging Next, NBD-lys and NBD-amino-ala were ^{applied to A549 cells, which are LAT1 highly expressing cells, in a Na +} free buffer solution having a pH of 5.3, and evaluated as to whether they could be used as a LAT1 substrate (Fig.). 15).

FIG. 15 shows a fluorescence image of A549 cells treated with 5 μM NBD-lys and NBD-amino-ala. Cells were pre-incubated in pH 5.3 Na ^{+ free buffer for 30 minutes and then incubated in pH 5.3 Na +} free buffer containing 5 μM NBD-lys and NBD-amino-ala for 30 minutes (5 mM). In the presence or absence of BCH). Images were obtained with the Leica SP8. The imaging conditions are as follows: Ar 20%, line average 5, HC PL APO CS2 40 × / 1.30 OIL, Ex. 488 nm 1% Em. HyD3 534-634 nm, 100% gain offset-0.01.

As the results obtained in FIG. 15 show, all the compounds were taken up by A549 cells at the same concentration (5 μM) as NBD-pAMP, and the uptake was inhibited by the addition of the LAT inhibitor BCH. Therefore, it was suggested that NBD-lys and NBD-amino-ala into which lysine and 3-amino-alanine were introduced as amino acid structures were also incorporated into cells as LAT1 substrates. That is, in the substrate recognition of LAT1 for a compound having NBD as a fluorescent mother nucleus, it was shown that the structure of the amino acid side chain site is acceptable even if it does not have an aryl group.

INDUSTRIAL APPLICABILITY The present invention provides fluorescent substrates NBD-pAP, NBD-pAMP, NBD-lys, NBD-amino-ala, etc., which are taken up into cells by LAT1, which is an amino acid transporter whose expression is upregulated in many cancer types. can do.
Since LAT1 is a transporter that has been reported to be involved in a wide range of life phenomena including diseases such as cancer, many researchers are paying attention to the elucidation of the function of LAT1 and the development of medical tools using LAT1. Is collecting. Therefore, the results of the present invention that succeeded in developing a fluorescent substrate that is taken up via LAT1 are not only applied to clinical applications such as intraoperative imaging of cancers such as brain tumors, but also related to the search for inhibitors for LAT1 and LAT1. It is thought that it will also contribute to the elucidation of life phenomena.

Claims (11)

1. A compound represented by the following general formula (I) or a salt thereof.
   

   

   (During the ceremony,
   R ¹ is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms:
   X represents ^{NR 2}
   Here, R ² is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms:
   Y is a nitro group (-NO ₂ ), a sulfonic acid group (-SO ₃ H) or a sulfonamide group (-SO ₂ NR'R''), where R'and R'' are independent of each other. In addition, it is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms;
   L is selected from the group consisting of alkylene, arylene, and a group composed of an arbitrary bond of alkylene and arylene. )
2. The compound according to claim 1, or a salt thereof, wherein L is selected from the following.
   -(CR ^a R ^b ) _n- , ^{-Ar-, *} -Ar- (CR ^a R ^b ) _m- , ^* -(CR ^a R ^b ) _s -Ar-, ^* -(CR ^a R ^b ) _s- Ar- (CR ^a R ^b ) _m-
   (Ar represents an arylene, ^Ra and R ^b are each independently and independently at each appearance, a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, n is an integer of 1 to 8, and m is an integer of 1 to 8. It is an integer of 1 to 5, s is an integer of 1 to 8, and * indicates the side to be combined with X.)
3. The compound according to claim 1 or 2, or a salt thereof, wherein L is selected from the following.
   ^{_{-CH 2 -, * -Bz-CH}} 2 -, - (CH 2) 4 -, * -CH 2 -Bz-CH 2 -
   (Bz represents a benzene ring, and * indicates the side bonded to X.)
4. The compound according to claim 3, or a salt thereof, wherein L is ^* -Bz-CH _2-.
5. The compound according to claim 3, or a salt thereof, wherein L is -CH _2-.
6. L is, - _{(CH 2) 4} - The compound or a salt thereof according to claim 3.
7. The compound according to claim 3, or a salt thereof, wherein L is ^* -CH _2- Bz-CH _2-.
8. The compound according to any one of claims 1 to 7, or a salt thereof, wherein Y is a nitro group (-NO _2).
9. A fluorescent probe containing the compound according to any one of claims 1 to 8 or a salt thereof.
10. It is a method for detecting the intracellular uptake by LAT1.
    (A) A step of introducing a fluorescent probe containing the compound according to any one of claims 1 to 8 or a salt thereof into the cell, and (b) measuring the fluorescence emitted by the compound or the salt thereof in the cell. Method involving steps.
11. A method of visualizing cancer cells or tissues
    (A) A step of applying a fluorescent probe containing the compound according to any one of claims 1 to 8 or a salt thereof to a cell or tissue, (b) Fluorescence measures the cell or tissue to which the fluorescent probe is applied. A method comprising the step of irradiating with an appropriate wavelength, thereby observing or measuring the fluorescence emitted within the cell or tissue.

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