WO2015125851A1 - Development of ligand screening system for neurotransmitter receptors - Google Patents

Abstract

Provided is a screening method for substances that bind to neurotransmitter receptors, have a group structure represented by formula (II) (in the formula, Rec-Nu represents a group obtained by separating a hydrogen atom from a neurotransmitter receptor (Rec-Nu-H). Nu represents a bivalent group obtained by separating a hydrogen atom from a nucleophile group (Nu-H) possessed by the neurotransmittor receptor. L² represents a bilavent linking group and Fl represents a marker group). A candidate substance, the fluorescence patterns of which vary between when an antagonist is bound and when an agonist is bound, and which is expected to interact with a marked neurotransmitter receptor is made to act on the receptor, the binding mode of the candidate substance and the receptor is detected by variation in the signals emitted by a marker, and screening is carried out based on those results.

Description

Development of a ligand screening system for neurotransmitter receptors

The present invention relates to a fluorescently labeled neurotransmitter receptor, a method for producing a labeled neurotransmitter receptor, a neurotransmitter receptor labeling agent, a compound useful as the labeling agent, and a neurotransmitter receptor ligand. The present invention relates to a screening method.

Receptors that receive neurotransmitters are proteins that are widely known as important drug targets. For example, glutamate is widely accepted as one of important neurotransmitters, and its receptor glutamate receptor is a protein essential for neurotransmission. Glutamate receptors are classified into AMPA, NMDA, and kainate types based on the characteristics of agonists. AMPA receptors are proteins essential for neuronal activity, particularly memory and learning. Causes various diseases such as illness, stroke, and Alzheimer's disease. Therefore, AMPA type receptors have been positioned as important drug targets. So far, a plurality of selective agonists have been developed, but there are problems such as subtype selectivity. The reason is that there is no known method that can evaluate the action of a drug on AMPA receptors at high throughput, and a highly efficient method for developing an active drug has been demanded.
For example, detection of a fluorescence response to glutamate using a ligand binding site that is a partial sequence of glutamate receptor has been reported (Non-patent Document 1), but there is no fluorescence response system using a full-length glutamate receptor. . Drug assays using full-length glutamate receptors typically involve evaluation of AMPA receptor activity (ion channel characteristics), but the evaluation method is complicated and has not been developed into a high-throughput evaluation method. Absent.

An object of the present invention is to provide a technique for clarifying a function as a ligand by assaying a neurotransmitter receptor ligand (agonist, partial agonist, antagonist) at high throughput.

The present invention relates to a fluorescently labeled neurotransmitter receptor, a method for producing a labeled neurotransmitter receptor, a neurotransmitter receptor labeling agent, a compound useful as the labeling agent, and a neurotransmitter receptor ligand. A screening method is provided.
Item 1. The following formula (II)

(In the formula, Rec-Nu represents a group in which a hydrogen atom has been eliminated from a neurotransmitter receptor (Rec-Nu-H). Nu represents a nucleophilic group (Nu-H) of the neurotransmitter receptor. A divalent group in which a hydrogen atom is eliminated from L ^2, L ² represents a divalent linking group, and Fl represents a labeling group.
It is expected that the labeled neurotransmitter receptor interacts with the receptor, which has a basic structure represented by the formula and changes the fluorescence pattern between when the antagonist is bound and when the agonist is bound. A substance that binds to a neurotransmitter receptor, characterized in that a candidate substance is allowed to act, and the binding mode of the candidate substance and the receptor is detected based on a change in a signal emitted by a label, and is performed based on the result. Screening method.
Item 2. Item 2. The screening method according to Item 1, wherein the candidate substance is allowed to act on cells having a labeled neurotransmitter receptor.
Item 3. Item 3. The screening method according to Item 1 or 2, wherein the receptor is an AMPA receptor.
Item 4. Formula (I) below

(In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
A compound represented by
Item 5. Formula (I)

(In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
A labeling agent for the neurotransmitter receptor represented by:
Item 6. Formula (I)

(In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
And the neurotransmitter receptor (Rec-Nu-H) is reacted with the compound represented by the following formula (II):

(In the formula, Rec-Nu represents a group in which a hydrogen atom is eliminated from the neurotransmitter receptor (Rec-Nu-H). Nu represents a hydrogen atom from a nucleophilic group possessed by the neurotransmitter receptor. (L ² represents a divalent linking group, and Fl represents a labeling group.)
A labeled neurotransmitter receptor having a basic structure represented by the above, wherein the signal pattern emitted by the label changes between when an antagonist is bound and when an agonist is bound. A method for producing a receptor.

Receptor proteins that receive ligands such as neurotransmitters are important drug targets. For example, glutamate is widely known as an important neurotransmitter, and its receptor glutamate receptor is an essential protein for neurotransmission. The present inventors have developed a technique for selectively labeling in the vicinity of a ligand binding site of a neurotransmitter receptor such as AMPA receptor which is a kind of glutamate receptor.

In the method of the present invention, it is possible to covalently fluorescently label a receptor expressed on the surface layer of a living cell. As a result of labeling a label such as a fluorophore in the vicinity of the ligand binding site of the receptor, it became possible to visualize the binding response of the ligand as a change in the signal emitted by the label such as fluorescence. As an unexpected result, a ligand of a neurotransmitter receptor whose structure is greatly changed by the binding of a ligand, an agonist that activates the receptor and an inactivatable antagonist, for example, a signal emitted by a label such as a fluorescence response is different, Agonists and antagonists can be distinguished, for example, as changes in fluorescence. This change can be used for a high-throughput detection system. From the above results, this research result means that a system capable of assaying agonists of any neurotransmitter receptor including, for example, AMPA receptor, at high throughput could be constructed.
Even with ligands of neurotransmitter receptors other than AMPA receptors, the agonists that activate the receptors and the inactivatable antagonists differ in the signal emitted by a label such as a fluorescent response, and any neurotransmitter receptor agonist. Antagonists can be distinguished, for example, as fluorescence changes, and selective agonists can be screened at high throughput.

LigandgDirected Acyl imidazole (LDAI) chemistry application and labeling agent molecular design In vitro (with purified protein) labeling. Labeling driven by ligand recognition is progressing in Invitro. Labeling with AMPA receptor overexpressing cells. Confirm selective and efficient labeling in HEK293T cells. Labeling time dependence of cell surface AMPA receptors. Confirm the progress of the labeling reaction over time. We evaluated the pigment dependence of the labeling behavior of neuronal endogenous AMPA receptors. Further selective labeling with hydrophilic Alexa488 type labeling agent. Imaging of neuronal endogenous AMPA receptors. Labeling of hippocampal cells isolated from rats. Successful visualization of neuronal endogenous AMPA receptors. Live imaging using chemical labeling with Alexa488 type labeling agent and HEK293T that transiently expresses GluR2 Ligand response evaluation of Alexa488 labeled AMPA receptor. After labeling with Alexa488 in HEK293T cells expressing GluR2, excess labeling agent was washed out, and fluorescence response was evaluated again with ligand (with NBQX added). • Evaluate ligand response in more detail. -Simultaneous evaluation of ligand binding / dissociation and channel activity.・ Evaluate ligand responses in nerve cells (synapses). Structure of labeling agent used Ligand function analysis with labeled AMPAR-1. It became clear that the fluorescence of AMPA receptor ligand was increased. Ligand function analysis by labeled AMPAR-2. Agonist and Antagonist showed different response patterns, and it became clear that it functions as a biosensor that distinguishes Agonist and Antagonist. Evaluate responses to various ligands Ligand-responsive dye dependence. Alexa488 and Oregon green show similar responses regardless of the function of the ligand, making them suitable for binding rate quantification. In addition, Alexa568 and ATTO655 are suitable for categorizing ligands because they respond by distinguishing the function / action mechanism of the ligand.

The ligand-directed acylimidazole compound (LDAI), which is a receptor labeling reagent used in the present invention, is shown below.

(Wherein L ¹ and L ² each independently represent a divalent linking group, Lg represents a ligand for a neurotransmitter receptor, Fl represents a labeling group, and R ¹ and R ² are the same or different. Represents a hydrogen atom or a substituent.)
The labeling reagent (labeling agent) of the present invention having an acylimidazole moiety binds to the neurotransmitter receptor (Rec-Nu-H) at the ligand (Lg) moiety, and in the receptor (Rec-Nu-H) Nucleophilic group (Nu-H) attacks the C atom of the carbonyl of acylimidazole, the N-CO bond is cleaved, the ligand having an imidazole group leaves the acceptor, and the labeling group (Fl) of the acceptor Nu and -CO-O- (L ² ) linked via _n2 group labeled receptor: Rec-Nu-CO-O- (L ² ) _n2 -Fl
(In the formula, Rec-Nu represents a group in which H is eliminated from the neurotransmitter receptor (Rec-Nu-H), and Nu is a group in which H is eliminated from the nucleophilic group (Nu-H) of the receptor. L ² , n 2 and Fl are as defined above.
Is obtained. A plurality of labeling groups (Fl) may be linked to a plurality of Nu of the acceptor via a —CO—O— (L ² ) _n2 group.

Examples of the nucleophilic group (Nu-H) include NH ₂ group at the terminal of Lys contained in the amino acid constituting the receptor, phenolic OH group of Tyr, SH group of Cys, imidazole group of His, etc. NH ₂ group at the terminal of Nu includes -NH-, -O-, -S-, etc., and -NH- is preferred.

As the labeling group (Fl), Alexa-350, Alexa-430, Alexa-488, Alexa-532, Alexa-546, Alexa-555, Alexa-568, Alexa-594, Alexa-633, Alexa-647, Alexa- 660, Alexa-680, Alexa-750, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, BODIPY 505/515, fluorescein inthiocyanate (FITC), Oregon green, eosin isothiocyanate, PE, ATTO655, CypHer5E, Rhodamine5B, BODIPY 580/605, Texas Red, APC, Indocyanine Green, etc. Preferred examples include Alexa-488, Oregon green, Alexa-546, Alexa-568, ATTO655, and CypHer5E.

When a photocrosslinking agent is used as a labeling group, the interacting protein can be bound to the receptor by irradiating light when the interacting protein binds to the labeled neurotransmitter receptor. It is useful for detecting protein-protein interactions in receptors.

When a peptide is used as a labeling group, the labeled neurotransmitter receptor on the membrane surface is useful for such functional modification because the function of the protein such as intracellular transport is modified. In addition, when a water-soluble polymer such as PEG is used as a labeling group, the labeled neurotransmitter receptor increases the stability of membrane proteins by increasing the blood half-life or increasing the degree of solubilization. Such an effect can be expected.

Examples of the photocrosslinking agent include aryl azides such as phenyl azide, highly reactive groups such as diazirine and benzophenone. Since these groups react rapidly with nearby compounds by light irradiation, they are useful for detecting protein-protein interactions between ligands and receptors.
Examples of the water-soluble polymer include polyethylene glycol (PEG).

Examples of the radioisotope include a radioisotope, preferably ³ H, ³⁵ S and the like.
Examples of peptides include membrane-permeable peptides such as tat peptides, various homing peptides, various peptide ligands, and the like.

As a neurotransmitter receptor (Rec-Nu-H) labeled with a labeling group, any neurotransmitter receptor is a target of labeling, and is not particularly limited. Examples include ligands:
Glutamate receptor: binds glutamate as a neurotransmitter. Depending on the drug to be bound, it is divided into NMDA receptor, AMPA receptor and kainate receptor.
・ Muscarinic acetylcholine receptor: acetylcholine, muscarinic / adenosine receptor: adenosine, caffeine / adrenergic receptor: adrenaline, noradrenaline / GABA receptor: GABA
Cannabinoid receptor: Cannabis component and anandamide Cholecystokinin receptor: Cholecystokinin Dopamine receptor: Dopamine histamine receptor: Histamine opioid receptor: Opium component such as cocaine, morphine, heroin and endogenous peptide Ligand (enkephalin, endorphins, etc.)
Serotonin receptor: Serotonin Somatostatin receptor: Somatostatin Nicotinic acetylcholine receptor: Acetylcholine, Nicotine glycine receptor: Glycine as a neurotransmitter, strykinin

Representative examples of receptor and ligand combinations are shown above, but the combination of the receptor to be labeled and the ligand that binds to the receptor is not limited to these, and the currently known neurotransmitter receptor and ligand combinations Either a combination or a combination of neurotransmitter receptors and ligands discovered in the future may be used.

In the compound of the general formula (I) of the present invention,
L1 and L2 may be the same or different, and examples thereof include a divalent linking group. Examples of the divalent linking group include —O—, —CO—, —COO—, —O—CO—, —NHCO—, —CONH—, — (CH ₂ ) _m1 — (m1 represents an integer of 1 to 6). ), Arylene groups (particularly phenylene such as ortho, meta, para), heteroarylene groups, —NH—, — (CH ₂ CH ₂ O) _m2 — (m2 represents an integer of 1 to 10), — (CH ₂ CH (CH ₃ ) O) _m2 — (m2 represents an integer of 1 to 10) and the like may be used, and these may be used alone or in combination of two or more of the same or different. The linking group may be constituted. In addition, (terminal) CO, COO, O—CO—, CONH, NHCO, NH, etc. of the divalent linking group are bonded to COOH, NH _2, etc. such as Lg, Fl, amide, urethane, urea, etc. Can be formed.
The introduction of Lg and Fl can be carried out by utilizing them when they have a functional group such as carboxylic acid (COOH) or an ester or active ester thereof, NH ₂ , SH, OH or the like. When Lg and Fl do not have an appropriate functional group, a linking acid functional group such as carboxylic acid (COOH) or its ester or active ester, NH2, SH, OH, etc. is introduced at the terminal and this functional group is used. Can be connected. Lg and Fl can be introduced, for example, according to the following schemes 1 and 2. Schemes 1 and 2 are methods for introducing Lg and Fl using amide bonds (CONH, NHCO), but Lg and Fl are introduced according to known methods using ether bonds, thioether bonds, amino bonds, and the like. can do.

Wherein R ¹ , R ² , L ¹ , L ² , Lg, Fl are as defined above. CONH-L ^1a and NHCO-L ^1a represent specific embodiments of L ¹ , L ^2a represents a divalent linking group, Y ¹ represents an activity such as OH, alkoxy, aryloxy, imidazolyl, 1-hydroxybenzotriazolyl (O-Bt), N-hydroxysuccinimidyl (OSu), etc. (Denotes a leaving group used as an ester. DSC stands for disuccinimidyl carbamate.)

In the schemes 1 and 2, the amide bond (CONH, NHCO) is formed by using a raw material amino compound (compound having —NH ₂ group) and carboxylic acid, ester or active ester compound (compound having —CO—Y1 group). The reaction proceeds advantageously by using about equimolar amounts and reacting at a temperature from room temperature to the boiling point of the solvent for 1 to 24 hours. Examples of the solvent include methanol, ethanol, methylene chloride, chloroform, carbon tetrachloride, 1,2-dichloroethane, benzene, toluene, acetone, methyl ethyl ketone, ether, tetrahydrofuran and the like.

In Scheme 2, DSC is ^Fl-CONH-L 2a -OH or ^Fl-NHCO-L 2a -OH1 mol using excess from 1 mole excess of a base (triethylamine from a catalytic amount of pyridine, dimethylaminopyridine, The target compound (2) can be obtained by reacting in the presence of diisopropylethylamine, DBU, etc.) from room temperature to a temperature at which the solvent boils for 1 to 24 hours.

The structures of AMPA receptor ligand (AMPAR ligand), NMDA receptor ligand (NMDAR ligand), GABA receptor ligand (GABAR ligand), and metabotropic glutamate receptor ligand (mGluR1 ligand) are shown below.

The AMPA receptor ligand can be bound to L ¹ by an amide bond, urethane bond or the like using a terminal amino group.
The NMDA receptor ligand can be bound to L ¹ by an amide bond, an ester bond or the like using a terminal α or β unsaturated carboxyl group.
GABA receptor ligands can alkylate a 7-membered secondary amino group (NH) and bind to L ¹ via an NC bond.

The mGluR1 ligand described above is a pyrimidine ring having a halogen atom introduced therein. A divalent linking group L ¹ such as NH—, O—, or S— can be introduced into this halogen atom position by nucleophilic substitution reaction.
In addition to these four receptor ligands, L ¹ can also be bound at a position where the function of the ligand is not lost.
Examples of combinations of two or more divalent linking groups include the following:

For example, the terminal amino (NH) group exemplified above may form an amide bond with a COOH group such as Fl or Lg.

Examples of the substituent for R ¹ to R ² include alkyl, cycloalkyl, alkoxy, alkenyl, halogen atom, OH, CN, NO ₂ , COOH, NH ₂ , phenyl, benzyl, acetylamino, acetyl, acetyloxy, methoxycarbonyl, And ethoxycarbonyl, butoxycarbonyl, trifluoromethyl and the like.

Alkyl includes linear or branched C _1-18 alkyl such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, hexyl, preferably _Examples thereof include C _1-6 alkyl, more preferably C _1-4 alkyl.

Cycloalkyl includes C _3-10 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, preferably C _3-8 cycloalkyl, more preferably C _5-6 cycloalkyl. A part of the ring may be substituted with a hetero element or may have a substituent.
Alkoxy includes linear or branched methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentyloxy, isopentyloxy, hexyloxy, polyethylene glycol derivatives, etc. C _1-18 alkoxy, preferably C _1-6 alkoxy, more preferably C _1-4 alkoxy.
R ¹ and R ² are preferably hydrogen atoms.

The compound of the present invention can be synthesized according to the following scheme 3.

(Wherein R ¹ , R ² , L ¹ , L ² , Lg, and Fl are as defined above.)
In the reaction, 1 mole of compound (2) and 1 mole to excess of base (Base) are used with respect to 1 mole of compound (1), and 1 to 2 boiling points of the solvent in the solvent from room temperature to the boiling temperature of the solvent. It proceeds advantageously by reacting for 24 hours. Solvents include chlorinated hydrocarbons such as methylene chloride, chloroform and dichloroethane, aromatic hydrocarbons such as benzene and toluene, esters such as ethyl acetate, ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone, ethers, diisopropyl Examples include ethers such as pyrether and tetrahydrofuran, aliphatic or alicyclic hydrocarbons such as hexane and cyclohexane, DMF, DMSO, dioxane, and N-methylpyrrolidone. Examples of the base include triethylamine, diisopropylethylamine, pyridine, dimethylaminopyridine, DBU and the like.

(A) Receptor labeling method with compound (I) The neurotransmitter receptor may be an isolated receptor, or compound (I) may be reacted directly with a membrane-bound receptor. Alternatively, cells expressing the receptor may be reacted with compound (I). In the present specification, “receptor” may include all of a receptor protein or a fragment thereof having a ligand binding site and a nucleophilic group, a fragment of a cell membrane containing the receptor, and a cell expressing the receptor. As the cell, a cell in which a receptor gene is introduced to highly express the receptor can be preferably used. When the receptor is composed of a plurality of proteins, not only a protein containing a ligand binding site but also a cell in which genes of all proteins constituting the receptor are introduced and highly expressed can be used. A cell in which the receptor is highly expressed is preferable because a signal (fluorescence intensity or a change thereof) increases during ligand screening.

The reaction between the receptor and compound (I) can be carried out in a solvent, buffer solution or cell culture medium. Compound (I) is preferably used in excess relative to the receptor. Compound (I) labels the receptor when the receptor and the ligand (Lg) are bound, but non-specific labeling is suppressed. After completion of the reaction, the compound (I) can be removed by washing, whereby excess compound (I) can be removed, and nonspecific fluorescent labeling can be suppressed. The reaction temperature is from room temperature to about 37 ° C., and the reaction time is about 30 minutes to 12 hours. The solvent is water or a solvent in which compound (I) is dissolved, for example, lower alcohols such as methanol, ethanol, propanol, acetone, tetrahydrofuran (THF), dioxane, N-methylpyrrolidone, dimethylformamide (DMF), acetonitrile, dimethylacetamide, dimethyl Water-miscible solvents such as sulfoxide (DMSO) can be mentioned. Examples of the buffer include HEPES buffer, phosphate buffer, Tris buffer, and the like.

The cell expressing the receptor may be a cultured cell, and a solution of compound (I) is applied to the receptor-expressing cell (preferably human cell) in the tissue section or the receptor-expressing cell in the living body of a non-human mammal. It may be supplied and fluorescently labeled.

(B) Screening method for candidate substance (ligand) that binds to receptor Labeled receptor and receptor ligand (agonist, antagonist, partial agonist) are transferred to a receptor in a solvent such as water or a medium for cell culture. By mixing candidate substances to be bound and observing the change in fluorescence, it is possible to evaluate how the candidate substance binds to the receptor.

For example, as shown in FIG. 13, when the labeling group is Alexa488 or OG (Oregon green), both the agonist and the competitive antagonist have increased fluorescence (↑, FIG. 10), and when the labeling group is Alexa568, In the case of competitive antagonists, the fluorescence decreases (↓), and in the case of agonists and antagonists, no change in fluorescence occurs. Furthermore, in the case of ATTO655, the fluorescence increases when the ligand is an agonist (↑), the fluorescence is transiently increased after a competitive antagonist (↑ ↓), and the fluorescence decreases when a non-competitive antagonist is present (↑ ↓). ↓).

As described above, the labeled receptor of the present invention can be combined with several labeled receptors as necessary, for example, by changing the signal emitted by the label such as fluorescence, the ligand becomes an agonist, competitive antagonist, non-competitive. When it is possible to detect whether a ligand is an antagonist (agonist + antagonist) at high throughput, for example, select Alexa488 having a large fluorescence change. A fluorescently labeled receptor can be selected.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
Materials and Methods All chemicals and biochemical reagents were purchased from commercial sources and used without further purification. Thin layer chromatography (TLC) was performed using aluminum sheets (Merck Inc.) pre-coated with silica gel 60 F _254, visualized by fluorescence quenching or ninhydrin stain. Purification by chromatography was performed using flash column chromatography on silica gel 60 N (neutral, 40-50 μm, Kanto Chemical Co.).

Physicochemical measurements: ¹ H-NMR spectra were recorded on a 400 MHz Varian Mercury spectrometer. Chemical shifts were normalized by residual solvent peak or tetramethylsilane (δ = 0 ppm). MALDI-TOF MS spectra were recorded with Autoflex III (Bruker Daltonics, Bremen, Germany) using α-cyano-4-hydroxycinnamic acid (CHCA) or sinapinic acid (SA) as matrix. High resolution mass spectra were measured with Exactive (Thermo Scientific, CA, USA) equipped with electrospray ionization (ESI). Reverse phase HPLC (RP-HPLC) was measured on a Hitachi Chromaster system equipped with a diode array and fluorescence detector and a YMC-Pack Triat C18 or ODS-A column. A linear gradient containing 0.1% TFA or 10 mM ammonium acetate (solvent A) and 0.1% aqueous TFA-containing acetonitrile or acetonitrile ((solvent B)) was used.

Compound 4
Under a nitrogen atmosphere, 2-nitro-4-trifluoromethylaniline (10.5 g, 50 mmol), triethylamine (9 ml, 64 mmol), and dimethylaminopyridine (20 mg) were dissolved in 100 ml of dry THF. In an ice bath, 8.88 g (64 mmol) of ethyl oxalyl chloride was added dropwise, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off, the residue was dissolved in ethyl acetate and washed with water. The obtained organic layer was dried over sodium sulfate, and the solvent was distilled off. Recrystallization from ethanol gave 12.6 g (41.1 mmol, 82%) of compound 4 as pale yellow crystals. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 12.0 (s, 1H), 9.03 (d, J = 8.8 Hz, 1H), 8.58 (s, 1H), 7.96 (d, J = 6.8 Hz, 1H), 4.49 (q, J = 7.2 Hz, 1H), 1.47 (t, J = 6.8 Hz, 3H).

Compound 5
Under a nitrogen atmosphere, compound 4 (5.2 g, 17 mmol) was dissolved in 75 ml of dry THF. 2.6 g (23 mmol) of tert-butoxypotassium was added little by little, and the mixture was stirred at room temperature for 30 minutes. Thereafter, 3.1 g (25 mmol) of ethyl bromoacetate was added dropwise, and the mixture was stirred at room temperature for 5 hours and further heated to reflux for 3 hours. After the solvent was distilled off, the residue was dissolved in ethyl acetate and washed with water. The obtained organic layer was dried over sodium sulfate, and the solvent was distilled off. Purification by column chromatography (silica, hexane / ethyl acetate = 6 / 1-5 / 1-4 / 1) gave 2.6 g (6.6 mmol 39%) of yellow oily compound 5. ¹ H-NMR (400 MHz, CDCl ₃ ) δ 8.31 (s, 1H), 7.96-7.91 (m, 2H), 4.48 (d,, J = 17.6 Hz 1H), 4.27 (d, J = 6.8 Hz, 1H ), 4.08 (d, J = 6.8 Hz, 1H), 1.32 (t, J = 6.8 Hz, 3H), 1.13 (t, J = 6.8 Hz, 3H).

Compound 6
Compound 5 (0.2 g, 0.51 mmol) was dissolved in 3 ml of acetic acid. Iron powder 0.17 g was added and heated under reflux for 1 hour. The reaction solution was air-cooled, the solvent was distilled off, water was added to the residue, and the mixture was washed with solid.The resulting solid was recovered and then recrystallized from ethanol to give 82.4 mg (0.26 mmol, 51%) of white Crystalline compound 6 was obtained. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.4 (s, 1H), 7.45 (s, 3H), 5.00 (s, 2H), 4.16 (q, J = 6.8 Hz, 2H), 1.20 (t , J = 7.2 Hz, 3H).

Compound 7
Compound 6 (82.4 mg, 0.26 mmol) was dissolved in 1 ml of concentrated sulfuric acid. Under an ice bath, 26.3 mg (0.26 mmol) of potassium nitrate was added and stirred for 1 hour. The reaction solution was added to ice water, and the precipitated solid was extracted with ethyl acetate. The solvent of the obtained organic layer was distilled off to obtain 88.0 mg (0.21 mmol) of crude compound 7 as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.7 (s, 1H), 8.22 (s, 1H), 7.65 (s, 1H), 5.02 (s, 2H), 4.17 (q, J = 6.8 Hz , 2H), 1.21 (t, J = 7.2 Hz, 3H).

Compound 8
Crude compound 7 (88 mg, 0.21 mmol) was dissolved in 0.6 ml of DMF. 9% 10% palladium carbon was added and stirred for 1 hour under hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off. The residue was washed with ethanol to obtain 87.2 mg (0.22 mmol, 100%) of crude compound 8 as a white solid. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.0 (s, 1H), 7.20 (s, 1H), 6.64 (s, 1H), 5.47 (s, 2H), 4.81 (s, 2H), 4.17 (q, J = 6.8 Hz, 2H), 1.22 (t, J = 7.2 Hz, 3H).

Compound 9
Crude compound 8 (87.2 mg, 0.22 mmol) and 2,5-dimethoxy-3-tetrahydrofurancarbaldehyde 35.2 mg (0.22 mol) were dissolved in 1.5 ml of acetic acid. After heating and refluxing for 20 minutes, the mixture was allowed to cool and the solvent was distilled off. After dissolving in 0.5 ml of methanol, 20 ml of chloroform was added for reprecipitation. The solid was collected by filtration, and after drying under reduced pressure, 80.9 mg (0.17 mmol, 77%) of white solid compound 9 was obtained. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 12.5 (s, 1H), 9.76 (s, 1H), 7.8-7.6 (m, 2H), 7.64 (s, 1H), 7.05 (s, 1H) , 6.63 (s, 1H), 4.98 (s, 2H), 4.14 (q, J = 6.8 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H).

Compound 10
Compound 9 (100 mg, 0.24 mmol) was dissolved in 3 ml of ethanol. Hydroxyamine hydrochloride 34 mg (0.49 mmol) and sodium acetate 40 mg (0.48 mmol) were added, followed by heating under reflux for 1.5 hours. The solid was washed with water, and the solid was collected by filtration. After drying under reduced pressure, 92 mg (0.22 mmol, 89%) of Compound 10 as a white solid was obtained. ^{From 1} H-NMR, the abundance ratio of the cis form to the trans form was 22:78.

Compound 11
Compound 10 (32 mg, 0.075 mmol) was dissolved in a mixed solution of ethanol 1 ml and concentrated hydrochloric acid 0.1 ml. 10% palladium carbon (15 mg) was added, and the mixture was stirred overnight under a hydrogen atmosphere. After removing palladium carbon by filtration, the solvent was distilled off to obtain 26 mg (0.64 mmol, 85%) of Compound 11. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.64 (s, 1H), 7.31 (s, 1H), 7.05 (s, 1H), 6.93 (s, 1H), 6.41 (s, 1H), 5.03 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 4.05 (s, 2H), 1.27 (t, J = 7.2 Hz, 3H).

Compound 12
Imidazole acetate hydrochloride 31 mg (0.19 mmol) was dissolved in 2 ml of dry DMF. WSC-HCl 45 mg (0.24 mmol), HOBt-H ₂ O 32 mg (0.24 mmol), compound 12 (70 mg, 0.16 mmol), DIEA 164 μl (0.94 mmol) were added, and the mixture was stirred at room temperature for 5 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH = 5/1 (NH ₃ 1%)) to obtain 31 mg (61 μmol) of Compound 12. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 7.60 (s, 2H), 7.23 (s, 1H), 6.98 (s, 1H), 6.80-6.77 (s, 2H), 6.21 (s, 1H) , 5.03 (s, 1H), 4.27-4.18 (m, 4H), 3.51 (s, 2H), 1.23 (t, J = 7.2 Hz, 3H).

Compound 13
Compound 12 (20 mg, 33 μmol) was dissolved in 0.5 ml of methanol, 284 μl (142 μmol) of 0.5 M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was neutralized by adding 1N hydrochloric acid and then dried under reduced pressure, and the obtained crude product was directly used in the next reaction. Used in the next reaction.

Compound 14
Oregon green (300 mg, 0.73 mmol) was dissolved in 5 ml of dry DMF, HATU 540 mg (1.31 mmol) and DIEA 0.36 ml (2.2 mmol) were added, and the mixture was stirred at room temperature for 4 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH / AcOH = 100/10/1 → 100/25/1) to obtain 173 mg (0.35 mmol) of Compound 14. ¹ H-NMR (400 MHz, DMSO-d ₆ ) δ 8.78 (m, 1H), 8.58 (m, 1H), 8.58 (m, 1H), 8.06 (m, 1H), 7.36 (m, 1H), 6.32 (m, 4H), 4.60 (m, 1H), 3.62-3.30 (m, 8H).

Compound 15
Compound 14 (55 mg, 0.11 mmol) was dissolved in 1.5 ml of dry DMF. Under a nitrogen atmosphere, 71 mg (0.28 mmol) of disuccinimidyl carbamate and 38.6 μl (0.28 mmol) of triethylamine were added and stirred at room temperature for 6 hours. After the solvent was distilled off, the residue was dissolved in 2 ml of dichloromethane and reprecipitated by adding 2 ml of diethyl ether. The obtained solid was dried under reduced pressure to obtain 40 mg of crude compound 15. The obtained crude product was directly used in the next reaction.

Compound 1 (Oregon Green)
Compound 13 (36 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, Compound 15 (25 mg, 39 μmol) and 11.4 μl (0.14 mmol) of pyridine were added, and the mixture was stirred at room temperature for 6 hours. Acetonitrile was added to the reaction solution for reprecipitation, and the obtained solid was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 1 (Oregon Green). MALDI-TOF-Mass ([M + H] = 1014.38 (obs.), 1015.21 (calc.))

Compound 16
Compound 11 (166 mg, 0.37 mmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, succinic anhydride (45 mg, 0.45 mmol) and DIEA 130 μl (0.74 mmol) were added, and the mixture was stirred at room temperature for 2.5 hours. After the solvent was distilled off, the residue was washed with water to obtain 92 mg (0.18 mmol) of Compound 16. ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.60 (s, 1H), 7.28 (s, 1H), 6.81 (d, J = 7.2 Hz, 2H), 6.23 (s, 1H), 5.01 (s, 1H), 4.25-4.20 (m, 4H), 2.59-2.42 (m, 4H), 1.24 (t, J = 5.2 Hz, 3H).

Compound 17
Compound 16 (92 mg, 0.18 mmol) was dissolved in 5 ml of dry DMF. WSC-HCl 45 mg (0.24 mmol), HOBt-H ₂ O 32 mg (0.24 mmol), histamine hydrochloride 43 mg (0.24 mmol) and DIEA 158 μl (0.91 mmol) were added, and the mixture was stirred at room temperature for 3 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH = 5/1 (NH ₃ 1%)) to obtain 72 mg (0.12 mmol) of Compound 17. ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.59 (s, 1H), 7.55 (s, 1H), 7.24 (s, 1H), 6.85-6.77 (m, 3H), 6.21 (t, J = 4.2 Hz, 1H), 5.02 (s, 1H), 4.29-4.20 (m, 4H), 2.77-2.42 (m, 8H), 1.25 (t, J = 5.4 Hz, 3H).

Compound 18
Compound 17 (20 mg, 33 μmol) was dissolved in 0.5 ml of methanol, 284 μl (142 μmol) of 0.5 M aqueous lithium hydroxide solution was added, and the mixture was stirred at room temperature for 3 hours. The reaction mixture was neutralized by adding 1N hydrochloric acid and then dried under reduced pressure, and the obtained crude product was directly used in the next reaction. Used in the next reaction.
Compound 2 (Oregon Green)
Compound 18 (33 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, Compound 15 (21 mg, 33 μmol) and 10.6 μl (0.13 mmol) of pyridine were added, and the mixture was stirred at room temperature for 6 hours. Acetonitrile was added to the reaction solution for reprecipitation, and the obtained solid was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (Oregon Green). ESI-Mass ([M + Na] = 1123.2507 (obs.), 1123.2504 (calc.))

Compound 19
Compound 11 (130 mg, 0.29 mmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, adipic anhydride (45 mg, 0.45 mmol) (ref. 1) and 102 μl (0.58 mmol) of DIEA were added, and the mixture was stirred at room temperature for 3 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH = 5/1 (NH ₃ 1%)) to obtain 137 mg (0.25 mmol) of Compound 19. ¹ H-NMR (400 MHz, CD ₃ OD) δ 7.90 (s, 1H), 7.28 (s, 1H), 6.82-6.73 (m, 2H), 6.20 (d, J = 7.2 Hz, 1H), 5.03 ( s, 2H), 4.24-4.19 (m, 4H), 2.32-2.28 (m, 4H), 1.71-1.56 (m, 4H), 1.24 (t, J = 7.2 Hz, 3H).

Compound 20
Compound 19 (137 mg, 0.25 mmol) was dissolved in 5 ml of dry DMF. WSC-HCl 63 mg (0.33 mmol), HOBt-H ₂ O 45 mg (0.33 mmol), histamine hydrochloride 61 mg (0.33 mmol) and DIEA 221 μl (1.27 mmol) were added, and the mixture was stirred at room temperature for 2 hours. After the solvent was distilled off, purification was performed by column chromatography (silica, CHCl ₃ / MeOH = 5/1 (NH ₃ 1%)) to obtain 72 mg (0.12 mmol) of crude compound 20. The obtained crude product was directly used in the next reaction.

Compound 21
Crude compound 20 (33 mg) was dissolved in 0.5 ml of methanol, and 210 μl (105 μmol) of 0.5 M aqueous lithium hydroxide solution was added and stirred at room temperature for 5 hours. After neutralizing with 1N hydrochloric acid, the mixture was dried under reduced pressure. The obtained solid was purified by HPLC (acetonitrile (0.1% TFA) / water (0.1% TFA)) and lyophilized to obtain Compound 21. ¹ H-NMR (400 MHz, CD ₃ OD) δ 8.80 (s, 1H), 7.60 (s, 1H), 7.38 (s, 1H), 7.23 (s, 1H), 6.82-6.78 (m, 2H), 6.21 (s, 1H), 5.01 (s, 2H), 4.24 (s, 2H), 3.47 (t, J = 5.4 Hz, 2H), 2.90 (t, J = 5.4 Hz, 2H), 2.22-2.14 (m , 4H), 1.62-1.55 (m, 4H).

Compound 3 (Oregon Green)
Compound 21 (18 mg, 26 μmol) was dissolved in 0.5 ml of dry DMF. Under a nitrogen atmosphere, Compound 15 (17 mg, 26 μmol) and 10.6 μl (0.13 mmol) of pyridine were added, and the mixture was stirred at room temperature for 6 hours. Acetonitrile was added to the reaction solution for reprecipitation, and the obtained solid was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 3 (Oregon Green). ESI-Mass ([M + Na] = 1129.3009 (obs.), 1129.2997 (calc.))

Compound 23
Compound 17 (18 mg, 26 μmol) was dissolved in 0.5 ml of dry DMF. Under a nitrogen atmosphere, Compound 22 (15.6 mg, 45 μmol) (ref. 2) and 10 μl (0.12 mmol) of pyridine were added, and the mixture was stirred at room temperature for 6 hours. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 23 (5.73 mg, 7 μmol). MALDI-TOF-Mass ([M + H] = 807.326 (obs.), 807.292 (calc.))

Compound 24
Compound 23 (5.738 mg, 7 μmol) was suspended in 1.0 ml of dry dichloromethane. Under a nitrogen atmosphere, 0.5 ml of TFA was added and stirred at room temperature for 1 hour. After distilling off the solvent, TFA was carefully removed by azeotropy with toluene to obtain crude compound 24. The obtained product was directly used for the next reaction.

Compound 2 (Alexa488)
Crude compound 24 (1.8 mg, 2 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, Alexa488-NHS-ester (1 mg) and DIEA 1 μl (6 μmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (Alexa488) (1.0 mg). MALDI-TOF-Mass ([MH] = 1221.619 (obs.), 1221.217 (calc.))

Compound 2 (ATTO655)
Crude compound 24 (1.8 mg, 2 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, ATTO655-NHS-ester (1 mg, 1 μmol) and DIEA 1 μl (6 μmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (ATTO655) (1.0 mg). ESI-Mass ([M + H] = 1216.4352 (obs.), 1216.4380 (calc.))

Compound 2 (Alexa546)
Crude compound 24 (1.8 mg, 2 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, Alexa546-NHS-ester (1 mg, 1 μmol) and DIEA 1 μl (6 μmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (Alexa546) (0.92 mg).

Compound 2 (Alexa568)
Crude compound 24 (0.88 mg, 1.24 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, Alexa568-NHS-ester (0.98 mg, 1.24 μmol) and DIEA 0.65 μl (3.72 μmol) were added, and the mixture was stirred at room temperature for 5 hours. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (Alexa568). MALDI-TOF-Mass ([M + H] = 1383.7 (obs.), 1383.4 (calc.))

Compound 2 (CypHer5E)
Crude compound 24 (0.88 mg, 1.24 μmol) was dissolved in 1.0 ml of dry DMF. Under a nitrogen atmosphere, CypHer5E-NHS-ester (2.0 mg, 2.0 μmol) and DIEA 2 μl (12 μmol) were added, and the mixture was stirred at room temperature overnight. The reaction solution was purified by HPLC (acetonitrile / 10 mM ammonium acetate aqueous solution) and lyophilized to obtain the target compound 2 (CypHer5E). ESI-Mass ([M + H] = 1439.4249 (obs.), 1439.4240 (calc.))

Biochemical experiments: SDS-PAGE and Western blotting were performed using a Bio-Rad Mini-Protean III electrophoresis apparatus. Fluorescence and chemiluminescence signals were detected with a ChemiDoc XRS system equipped with a 520DF30 filter (ChemiDoc, Bio-Rad laboratory) and Imagequant LAS 4000 (GE Healthcare). As the confocal laser microscope, FV1000 (Olympus) or LSM710 (Carl Zeiss) was used.

In vitro labeling of AMPAR ligand binding domain S1S2 AMPAR ligand binding domain S1S2 (3 μM) (ref. 3) in the presence or absence of NBQX (150 μM) in HEPES buffer (50 mM, pH 7.4, 100 mM NaCl) Labeling agent (6 μM) was added to and incubated at 37 ° C. The sample was collected at an arbitrary time, and the labeling rate was determined by SDS-PAGE.

Cell culture and gene transfer
HEK293T cells 10% FBS, penicillin (100 units / mL) and Dulbecco was added streptomycin (100 μg / mL)'s Modified Eagle Medium (DMEM, glucose 4.5 g / L) in a medium, in a humidified atmosphere of 5% CO ₂ For all experiments cultured in, cells were harvested from subconfluent (<80%) with trypsin-EDTA solution or cell scraper method and resuspended in fresh medium. Subculture was performed every 2-3 days. The gene transfer of glutamate receptor (GluR2) was performed using lipofectamine 2000 (Invitrogen) according to the attached manual. For labeling, cells 36 to 48 hours after gene introduction were used.

Labeling of GluR2 in HEK293T cells Labeling agent (2 μM) was added to HEK293T cells transfected with the above gene in the presence or absence of NBQX (50 μM) in DMEM medium (glutamax, 25 mM HEPES, FBS (-)). ) Was added and incubated at 17 ° C. for 4 hours. Cells were washed twice with HBS and lysed on ice using RIPA (radioimmunoprecipitation assay) buffer containing 1% protease inhibitor cocktail set III (Calbiochem®). The resulting solution was mixed with 2 × SDS-PAGE buffer (pH 6.8, 125 mM Tris · HCl, 20% glycerol, 4% SDS and 0.01% bromophenol blue, 100 mM DTT) and vortexed at room temperature for 30 minutes. The obtained sample was developed by SDS-PAGE and transferred to an Immun-Blot PVDF membrane (Bio-Rad). The labeled product was detected with an anti-Fluorescein antibody (Invitrogen, × 2000) and an anti-rabbit IgG antibody-HRP complex (GE Healthcare, × 3000). Immunodetection of GluR2 was performed using anti-HA antibody (Abcam) and anti-rabbit IgG antibody-HRP complex (GE Healthcare, × 5000). The HRP signal was detected with LAS 4000 imaging system (FujiFilm) using ECL prime western blotting detection reagent (GE Healthcare).

Labeling of Endogenous AMPA Receptors in Cultured Cerebellar Granule Cells Neurobasal medium (glutamax, 10 mM) was obtained from cultured neurons derived from cerebellar granule cells (ref. 4) isolated from 7-day-old rats and cultured for 11 days. A labeling agent (0.3 μM) was added in the presence or absence of NBQX (50 μM) in HEPES, FBS (−), 25 mM KCl) and incubated at 17 ° C. for 4 hours. Cells were washed twice with HBS and lysed on ice using RIPA (radioimmunoprecipitation assay) buffer containing 1% protease inhibitor cocktail set III (Calbiochem®). The resulting solution was mixed with 2 × SDS-PAGE buffer (pH 6.8, 125 mM Tris · HCl, 20% glycerol, 4% SDS and 0.01% bromophenol blue, 100 mM DTT) and vortexed at room temperature for 30 minutes. The obtained sample was developed by SDS-PAGE and transferred to an Immun-Blot PVDF membrane (Bio-Rad). Labeled products were detected with anti-Fluorescein antibody (Invitrogen, × 2000) or anti-Alexa488 antibody (Invitrogen, × 1000) and anti-rabbit IgG antibody-HRP complex (GE Healthcare, × 3000). Immunodetection of GluR1 was performed using anti-GluR1 antibody (millipore) and anti-mouse IgG antibody-HRP complex (GE Healthcare, × 3000). The HRP signal was detected with LAS 4000 imaging system (FujiFilm) using ECL prime western blotting detection reagent (GE Healthcare).

Labeling of Endogenous AMPA Receptors in Cultured Hippocampal Pyramidal Cells Cultured neurons from hippocampal pyramidal cells (ref. 4) isolated from embryonic day 18 rats and cultured for 18 days were treated with Neurobasal medium (glutamax, Labeling agent (1 μM) was added in the presence or absence of NBQX (50 μM) in 10 mM HEPES, FBS (−), 25 mM KCl) and incubated at 17 ° C. for 4 hours. Cells were washed twice with HES and fixed with 4% aqueous paraformaldehyde. After blocking with normal goat serum, immunostaining was performed with anti-GluR2 antibody (millipore, × 1000) and anti-mouse IgG antibody-Alexa546 modification (GE Healthcare, × 3000). After encapsulating with an encapsulant, labeled fluorescence and immunostaining were observed with a confocal laser microscope.

Labeling and live cell imaging of GluR2 in HEK293T cells In the presence or absence of NBQX (50 μM) in DMEM medium (glutamax, 25 mM HEPES, FBS (-)) on HEK293T cells already transfected with GluR2 as described above Labeling agent (2 μM) was added and incubated at 17 ° C. for 4 hours. The cells were washed twice with HBS and observed with a fluorescent microscope or a confocal microscope. In fluorescence detection, liquid exchange was performed by refluxing the HBS solution, agonist, and antagonist solution using a fluorescence microscope (IX-71) connected to a CCD camera (ORCA-Flash).

Reference (ref)
1. Napoli, A. et al, Anal. Chem., 82, 5552-5560 (2010)
2. Armstrong, N. et al, Neuron, 28, 165-181 (2000)
3. Kiyonaka, S. et al, Nat. Neurosci., 10, 691-701 (2007)
4). Beaudoin III, GMJ et al, Nat.Protoc., 7, 1741-1754 (2012)

Experimental section on synthesis of NMDA receptor labeling agent
Synthesis of LDAI reagent 4a

Compound 11a
An ice-cold solution of NaNO ₂ (3.2 g, 43 mmol) was added dropwise to a concentrated hydrochloric acid (80 mL) solution of 3,5-dichloroaniline (7.0 g, 43 mmol) at 0 ° C. over 10 minutes with stirring. The reaction mixture was stirred at 0 ° C. for 30 minutes. A solution of SnCl ₂ (43 g, 225 mmol) in concentrated hydrochloric acid (40 mL) was added dropwise to the reaction mixture at 0 ° C. A white precipitate was collected and suspended in water (500 mL). After neutralizing to pH 7.0 with 1N NaOH, AcOEt (500 mL) was added to extract the product into the organic layer. The organic layer was washed with saturated brine (100 mL × 2) and dried over Na ₂ SO ₄ . The solution was then filtered and the solvent was evaporated to give compound 11a (2.65 g, 15 mmol, 35%) as an orange solid.
¹ H NMR (400 MHz; CDCl ₃ ): δ 6.75 (t, J = 2.0 Hz, 1H), 6.71 (d, J = 2.0 Hz, 2H), 5.21 (br, 1H)

Compound 12a
Compound 11a (2.5 g, 14 mmol) was added to a solution of ethyl pyruvate (1.56 mL, 14 mmol) in AcOH (80 mL) and stirred at 100 ° C. for 30 minutes under N ₂ atmosphere. The reaction was monitored by TLC (AcOEt: hexane = 1: 3) and the reaction mixture was cooled to room temperature. The solvent was removed and AcOEt (80 mL) was added to the crude compound. The organic layer was washed with saturated aqueous NaHCO ₃ solution (80 mL x2) and saturated brine (80 mL x2), dried over Na ₂ SO ₄ and compound 12a (3.66 g, 13.3 mmol, 94% (E: Z = 5 : 2)) was obtained as a colorless oil.
¹ H NMR (400 MHz; CDCl ₃ ): δ 11.80 (s, 1H, Z), 10.10 (s, 1H, E), 7.30 (s, 1H, Z), 7.20 (s, 1H, E), 7.00- 7.02 (m, 1H, Z, 1H, E), 4.16-4.27 (m, 1H, Z, 1H, E), 2.09 (s, 1H, Z), 2.04 (s, 1H, E), 1.23-1.29 ( m, 3H, Z, 3H, E)

Compound 13a
Polyphosphonic acid (10 mL) was slowly added to compound 12a (E: Z = 5: 2 mixture, 3.6 g, 13 mmol). The reaction mixture was stirred at 125 ° C. for 30 minutes. To the reaction mixture was added 100 mL of cold water, and a saturated aqueous NaHCO ₃ solution was added to adjust the pH to 7. The product was extracted with AcOEt (60 mL x2). The organic layer was washed with saturated brine (60 mL) and dried over Na ₂ SO ₄ to obtain compound 13a (2.01 g, 7.8 mmol, 60%).
¹ H NMR (400 MHz; CDCl ₃ ): δ 12.4 (s, 1H), 7.44 (s, 1H), 7.28 (s, 1H), 7.11 (s, 1H), 4.36 (q, J = 7.20 Hz, 2H ), 1.33 (t, J = 7.20 Hz, 3H)

Compound 14a
To a stirred solution of compound 13a (2.01 g, 7.79 mmol) in 1,2-dichloroethane (30 mL) was added N-methylformanilide (1.6 mL, 12.9 mmol) and phosphorus oxychloride (1.1 mL, 12.1 mmol), The mixture was stirred at 80 ° C. for 9 hours under N ₂ atmosphere. The reaction was monitored by TLC (AcOEt: hexane = 1: 1), and 80 mL of 50% NaOAc aqueous solution and 5 g of ice were added to the reaction solution. This solution was recrystallized by keeping it at 4 ° C overnight. The orange precipitate was collected and washed with water and 1,2-dichloroethane to give compound 14a (1.62 g, 5.66 mmol, 73%) as an orange powder.
¹ H NMR (400 MHz; CDCl ₃ ): δ 10.80 (s, 1H), 9,37 (br, 1H), 7.39 (s, 1H), 7.35 (s, 1H), 4.52 (q, J = 7.2 Hz , 2H), 1.50 (t, J = 7.2 Hz, 3H)

Compound 15a
Compound 14a (57 mg, 0.199 mmol) was mixed with acetic acid and 2- (trimethylphosphoranylidene)-, 1,1-dimethylethyl ester (97 mg, 0.259 mmol) in a 1: 1 mixture of CH ₃ CN and dioxane (5 mL). mmol). The reaction mixture was stirred at 70 ° C. for 5 hours under N ₂ atmosphere. The reaction was monitored by TLC (AcOEt: hexane = 1: 3) and the reaction mixture was cooled to room temperature. The solvent was distilled off, and the crude residue was purified by silica gel column chromatography (AcOEt: hexane = 1: 3) to obtain compound 15a (44 mg, 0.115 mmol, 58%) as a white solid.
¹ H NMR (600 MHz; (CD ₃ ) ₂ SO): δ 12.70 (br, 1H), 8.28 (d, J = 16.0 Hz, 1H), 7.49 (s, 1H), 7.31 (s, 1H), 6.46 (d, J = 16.0, 1H), 4.35 (q, J = 7.2 Hz, 2H), 1.47 (s, 9H), 1.34 (t, J = 7.2 Hz, 3H)

Compound 16a
To a stirred solution of compound 15a (1.0 g, 2.60 mmol) in CH ₂ Cl ₂ (10 ml) was added TFA (10 ml). The mixture was stirred at room temperature for 5 minutes, and the reaction was monitored by TLC (AcOEt: hexane = 1: 3) to confirm the completion of the reaction. After azeotropic removal of TFA with toluene, compound 16a (828 mg, 2.52 mmol, 97%) was obtained as a white powder.
¹ H NMR (600 MHz; (CD ₃ ) ₂ SO): δ 8.25 (d, J = 16.0 Hz, 1H), 7.49 (s, 1H), 7.30 (s, 1H), 6.42 (d, J = 16.0 Hz ), 4.35 (q, J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H)

Compound 17a
To a stirred solution of compound 16a (36 mg, 110 μmol, 1.0 eq) in dry DMF (4 mL), TEA (62 μL, 440 μmol, 4.0 eq), histamine-2HCl (24 mg, 110 μmol, 1.0 eq), HBTU (50 mg, 132 μmol, 1.2 eq) was added and stirred for 2 hours under N ₂ atmosphere. The reaction was monitored by TLC (CHCl ₃ : MeOH = 10: 1 + 1% NH ₃ aq) and the solvent was distilled off. The crude residue was purified by silica gel column chromatography (CHCl ₃ : MeOH = 10: 1 + 1% NH ₃ aq → 2% NH ₃ aq) to obtain compound 17a (47 mg, 110 μmol, 100%) as a white solid. It was. ¹ H NMR (600 MHz; (CD ₃ ) ₂ SO): δ 12.50 (br, 1H), 11.82 (br, 1H), 8.19 (s, 1H), 8.33 (d, J = 16.0, 1H), 7.51 ( s, 1H), 7.47 (s, 1H), 7.28 (s, 1H), 6.86 (br, 1H), 6.50 (d, J = 16.0 Hz, 1H), 4.33 (q, J = 7.2 Hz, 2H), 3.38 (t, J = 6.8 Hz, 2H), 2.67 (t, J = 6.8 Hz, 2H), 1.32 (t, J = 7.2 Hz, 3H)

Compound 4a
A solution of compound 17a (47 mg, 110 μmol) in MeOH (2 mL) and 1N aqueous NaOH (1.1 mL, 1.1 mmol, 10 eq) was stirred at 60 ° C. overnight. The reaction was monitored by TLC (CHCl ₃ : MeOH = 10: 1 + 1% NH ₃ aq) and MeOH was distilled off. After neutralizing the solution with 1N HCl to pH 5-6, water was distilled off. DMF (3 mL) was added and the solution was filtered to remove NaCl to give compound 18a as an orange oil (47 mg, 119 μmol, 97%).

To a stirred solution of compound 18a (6 mg, 16 μmol, 1.0 eq) in dry DMF (0.5 mL), add compound 8 (10 mg, 16 μmol, 1.0 eq), dry pyridine (5 μL, 63 μmol, 4.0 eq). In addition, the mixture was stirred for 12 hours under N ₂ atmosphere. The reaction was monitored by TLC (CHCl3: MeOH = 10: 1) and the solvent was distilled off. The crude residue was RP-HPLC (column; YMC-packODS-A, 250 x 25 mm, mobile phase; CH ₃ CN; 10 mM NH ₄ OAc = 0: 100 → 70:30 (linear gradient over 70 minutes, flow rate; Purification by 10 mL / min, detection; UV (220 nm)) gave Compound 4a (6 mg, 6.5 μmol, 42%) as a yellow powder.
¹ H NMR (600 MHz; (CD ₃ ) ₂ SO): δ 8.84 (t, J = 6.0 Hz, 1H), 8.44 (s, 1H), 8.28 (d, J = 14.6 Hz, 1H), 8.11 (s , 1H), 8.09 (d, J = 7.8 Hz, 1H), 7.76 (s, 1H), 7.39 (s, 1H), 7.29 (s, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.09 (s, 1H), 6.85 (d, J = 14.6 Hz, 1H), 7.09 (s, 1H), 6.85 (d, J = 14.6 Hz, 1H), 6.69 (br, 2H), 6.45 (d, J = 11.4 Hz, 2H), 4.48 (t, J = 4.8 Hz, 2H), 3.78 (t, J = 4.8 Hz. 2H), 3.66 (t. J = 6.0 Hz, 2H), 3.47-3.51 (m, 2H) , 3.32-3.34 (m, 2H), 2.58 (t, J = 7.2 Hz, 2H)
HR-ESI MS m / z calcd for [M + H] ⁺ 918.1387, found 918.1373

Synthesis of GABA receptor labeling agent synthesis term compound 2b

tert-Butyl (2- (2-(((perfluorophenoxy) carbonyl) oxy) ethoxy) ethyl) carbamate (2b)
Bis (perfluorophenyl) carbonate (1.15 g, 2.93 mmol), TBAF (190 mg) in a stirred solution of tert-butyl (2- (2-hydroxyethoxy) ethyl) carbamate (500 mg, 2.44 mmol) in THF (24 ml) , 0.73 mmol). The reaction mixture was stirred for 24 hours at room temperature. The solution was diluted with CHCl ₃ and washed with 1N NaOH. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel column chromatography (hexane: AcOEt = 10: 1 → hexane: AcOEt = 2: 1) to obtain compound 2b (653 mg, 1.579 mmol, 64%) as a clear oil. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 4.90 (s, 1H), 4.47 (m, 2H), 3.79 (m, 2H), 3.59 (t, 2H, J = 5.6 Hz), 3.36 (m, 2H), 1.46 (s, 9H).
Synthesis of compound 1b

tert-butyl 4- (7-chloro-5- (2-fluorophenyl) -2-oxo-2,3-dihydro-1H-benzo [e] [1,4] diazepin-1-yl) butanoate (3b)
7-Chloro-5- (2-fluorophenyl) -1,3-dihydro-2H-benzo [e] [1,4] diazepin-2-one (200 mg, 0.69 mmol) in dry DMF (700 μl) To the stirred solution of NaH (38 mg, 1.0 mmol) was added at 0 ° C. The reaction mixture was stirred at 0 ° C. for 5 minutes. A solution of tert-butyl 4-bromobutanoate (185 mg, 0.83 mmol) in dry DMF (700 μl) was then added. The reaction mixture was stirred at room temperature for 1 hour. The solution was diluted with CHCl ₃ and washed with H ₂ O. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel column chromatography (hexane: AcOEt = 4: 1 → hexane: AcOEt = 2: 1) to obtain compound 3b (125 mg, 0.29 mmol, 42%) as a transparent solid.
¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.67 (t, 1H, J = 8.4 Hz), 7.49 (m, 2H), 7.40 (d, 1H, J = 8.8 Hz), 7.29 (m, 1H) , 7.17 (s, 1H), 7.07 (t, 1H, J = 9.6 Hz), 4.87 (d, 1H, J = 10.4 Hz), 4.40 (m, 1H), 3.78 (d, 1H, J = 10.4 Hz) , 3.67 (m, 1H), 2.15 (m, 2H), 1.86 (m, 1H), 1.72 (m, 1H), 1.39 (s, 9H).

tert-butyl (16- (7-chloro-5- (2-fluorophenyl) -2-oxo-2,3-dihydro-1H-benzo [e] [1,4] diazepin-1-yl) -13- Oxo-3,6,9-trioxa-12-azahexadecyl) carbamate (4b)
To a stirred solution of compound 3b (18 mg, 0.042 mmol) in dry CH ₂ Cl ₂ (1 ml) was added TFA (0.5 ml). The reaction mixture was stirred at room temperature for 1 hour. After azeotropic removal of TFA with toluene, the residue was dissolved in dry DMF (420 μl). Then tert-butyl ((2- (2- (2-aminoethoxy) ethoxy) ethoxy) methyl) carbamate (13 mg, 0.063 mmol), COMU (27 mg, 0.063 mmol), DIPEA (73 μl, 0.42 mmol) Was added to this solution. The reaction mixture was stirred at room temperature for 1 hour. The solution was diluted with CHCl ₃ and washed with 1N NaOH. The organic layer was dried over Na ₂ SO ₄ , filtered and evaporated. The residue was purified by silica gel column chromatography (hexane: AcOEt = 2: 1 → CHCl 3: MeOH = 10: 1) Compound 4b was purified by as a (24 mg, 0.037 mmol, 88 %) a transparent solid. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.56-7.67 (m, 4H), 7.34 (m, 1H), 7.18 (m, 1H), 7.14 (s, 1H), 4.69 (d, 1H, J = 10.8 Hz), 4.34 (m, 1H), 3.88 (d, 1H, J = 10.8 Hz), 3.79 (m, 1H), 3.18-3.67 (m, 16H), 2.14 (m, 2H), 1.85 (m , 1H), 1.70 (m, 1H), 1.42 (s, 9H).

N- (1- (1H-imidazol-4-yl) -2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl) -4- (7-chloro-5- (2-fluoro Phenyl) -2-oxo-2,3-dihydro-1H-benzo [e] [1,4] diazepin-1-yl) butanamide (5b)
To a stirred solution of compound 4b (24 mg, 0.037 mmol) in dry CH ₂ Cl ₂ (1 ml) was added TFA (0.5 ml). The reaction mixture was stirred at room temperature for 1 hour. After azeotropic removal of TFA with toluene, the residue was dissolved in dry DMF (370 μl). Next, 2- (1H-imidazol-4-yl) acetic acid (6 mg, 0.055 mmol), EDC (10 mg, 0.055 mmol), HOBT (8 mg, 0.055 mmol), DIPEA (64 μl, 0.37 mmol) Added to the solution. The reaction mixture was stirred at room temperature for 1 hour. The solution was purified by silica gel column chromatography (CHCl ₃ : MeOH: NH ₄ OH aq. = 10: 1: 1) to obtain compound 5b (18 mg, 0.027 mmol, 74%) as a transparent solid. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 7.62 (t, 1H, J = 6.4 Hz), 7.53 (s, 1H), 7.41-7.49 (m, 4H), 7.12 (s, 1H), 7.06 ( t, 1H, J = 9.2 Hz), 6.84 (s, 1H,), 4.81 (d, 1H, J = 10.4 Hz), 4.29 (m, 1H), 3.77 (d, 1H, J = 10.4 Hz), 3.67 (m, 1H), 3.51 (s, 1H), 2.05 (m, 2H), 1.91 (m, 1H), 1.65 (m, 1H).

2- (2-((tert-butoxycarbonyl) amino) ethoxy) ethyl 4- (19- (7-chloro-5- (2-fluorophenyl) -2-oxo-2,3-dihydro-1H-benzo [ e] [1,4] diazepin-1-yl) -2,16-dioxo-6,9,12-trioxa-3,15-diazanonadecyl) -1H-imidazole e-1-carboxylate (6b)
Compound 2b (17 mg, 0.04 mmol) was added to a stirred solution of compound 5b (18 mg, 0.027 mmol) in dry DMF (270 μl). The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by silica gel column chromatography (CHCl ₃ only → CHCl ₃ : MeOH = 10: 1) to obtain compound 6b (15 mg, 0.017 mmol, 63%) as a transparent solid. ¹ H-NMR (400 MHz, CDCl ₃ ): δ 8.10 (s, 1H), 7.62 (t, 1H, J = 7.6 Hz), 7.41-7.49 (m, 3H), 7.35 (s, 1H), 7.26 ( m, 1H), 7.13 (s, 1H), 7.06 (t, 1H, J = 9.2 Hz), 4.82 (d, 1H, J = 10.8 Hz), 4.52 (m, 2H), 4.32 (m, 1H), 3.30-3.77 (m, 28H), 2.09 (m, 2H), 1.94 (m, 1H), 1.70 (m, 1H), 1.42 (s, 9H).

2- (2- (2 ', 7'-difluoro-3', 6'-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9'-xanthene] -5-carboxamido) ethoxy) ethyl 4- (19- (7-Chloro-5- (2-fluorophenyl) -2-oxo-2,3-dihydro-1H-benzo [e] [1,4] diazepin-1-yl) -2,16-di Oxo-6,9,12-trioxa-3,15-diazanonadecyl) -1H-imidazole-1-carboxylate (1b)
To a stirred solution of compound 6b (1 mg, 1.1 μmol) in dry CH ₂ Cl ₂ (0.5 ml) was added TFA (0.5 ml). The reaction mixture was stirred at room temperature for 1 hour. After azeotropic removal of TFA with toluene, the residue was dissolved in dry DMF (100 μl). Then, 2,5-dioxopyrrolidin-1-yl 2 ′, 7′-difluoro-3 ′, 6′-dihydroxy-3-oxo-3H-spiro [isobenzofuran-1,9′-xanthene] -5- Carboxylate (1 mg, 2.0 μmol), DIPEA (20 μl, 0.11 mmol) was added to this solution. The reaction mixture was stirred at room temperature for 1 hour. This solution was purified by HPLC to give compound 1b (0.8 mg, 0.7 μmol, 64%) as a yellow solid. HR-ESI MS: calcd for C ₅₈ H ₅₅ ClF ₃ N ₇ O ₁₅ [M + H] ⁺ = 1182.3475: obsd 1182.3497.

Synthesis of experimental labeling agent (Fl-AI-C4-PTMB) for synthesis of metabotropic glutamate receptor (mGluR1) labeling agent

Synthesis of compound 11c

Compound 10c (171 mg, 0.452 mmol, 1.0 eq), HBTU (206 mg, 0.542 mmol, 1.2 eq), TEA (315 μL, 2.26 mmol, 5.0 eq) and 2- (2- in dry DMF (13 mL) A mixture of aminoethoxy) ethanol (90.0 μL, 0.904 mmol, 2.0 eq) was stirred at room temperature for 5 hours under nitrogen atmosphere. TEA (189 μL, 1.36 mmol, 3.0 eq) was then added to the solution and stirred at room temperature for 2 hours 30 minutes under a nitrogen atmosphere. Thereafter, the solution was distilled off. The residue was purified by silica gel column chromatography (CHCl ₃ : Methanol: = 10: 1 (AcOH 0.5%)) to obtain a yellow solid compound 11c (122 mg, 58.2%).

Synthesis of compound 12c

A mixture of Compound 11c (10.0 mg, 21.6 μmol, 1.0 eq), TEA (7.5 μL, 53.9 μmol, 2.5 eq) and DSC (13.8 mg, 53.9 μmol, 2.5 eq) in dry DMF (0.5 mL) under a nitrogen atmosphere For 5 hours at room temperature. Thereafter, the solution was distilled off to obtain Compound 12c, which was stored at -80 ° C.
Synthesis of spacer 2 (compound 14c)

Boc-Ape (5) -OH (215 mg, 0.99 mmol, 1.1 eq), HBTU (410 mg, 1.08 mmol, 1.0 eq), DIEA (744 μL, 4.50 mmol, 5.0 eq) in dry DMF (10 mL) And a mixture of histamine dihydrochloride (100 mg, 0.543 mmol, 0.60 eq) was stirred at room temperature for 3 hours and 25 minutes under a nitrogen atmosphere. The solvent was distilled off under reduced pressure. The residue was then extracted with CHCl ₃ (30 mL), and the organic layer was washed with 0.1N NaOH (30 mL × 3) and 30 mL saturated brine, dried over Na ₂ SO ₄ and evaporated under reduced pressure. did. The residue was purified by silica gel column chromatography (CHCl ₃ : methanol: NH ₃ water = 10: 1: 1%) to obtain Compound 14c.

Synthesis of compound 15c

To a solution of compound 14c (424 mg, 1.36 mmol, 1.0 eq) in 8 mL of CH ₂ Cl ₂ was added 2 mL of TFA on ice and stirred at room temperature for 2 hours and 15 minutes. Thereafter, the solvent was removed, and residual TFA was further removed by azeotropy with toluene (three times) to obtain Compound 15c (623 mg, 105%). The product was used in the next reaction without further purification.

Synthesis of compound 4c

4,6-dichloropyrimidine (300 mg, 2.01 mmol 1.0 eq), tributyl (1-ethoxyvinyl) tin (0.679 mL, 2.01 mmol, 1.0 eq), Pd (PPh ₃ ) ₄ (in dry DMF (6.0 mL) 69.7 mg, 0.0603 mmol, 0.03 eq), a mixture of cesium fluoride (611 mg, 4.02 mmol, 2.0 eq) and copper (I) iodide (38.3 mg, 2.01 mmol, 0.1 eq) under N ₂ atmosphere at 80 ° C For 8 hours. The reaction was quenched with CH ₂ Cl ₂ / H ₂ O (1/1, v / v, 10 mL) and the suspension was filtered through Celite with CH ₂ Cl ₂ . The organic layer was washed with water (50 mL × 3) and 50 mL saturated brine, and dried over Na ₂ SO ₄ overnight. Thereafter, the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography (hexane: ethyl acetate = 25: 1) to give compound 4c (38.1 mg, 10.3%) as a colorless solid.

Synthesis of compound 5c

To a solution of compound 4c (104 mg, 0.562 mmol 1.0 eq) in THF / H ₂ O (1.8 mL; 1/1, v / v) was added NBS (110 mg, 0.618 mmol 1.1 eq) at room temperature and 2 Stir for hours. N-methylthiourea (50.7 mg, 0.562 mmol 1.0 eq) was added to the reaction mixture and the mixture was stirred at room temperature for 2 hours. The reaction mixture was diluted with water (2 mL) and the resulting solid was filtered and recrystallized with hot THF / water. The supernatant liquid was purified by silica gel column chromatography (hexane: ethyl acetate: NH ₃ water = 5: 1: 0.01) to obtain compound 5c (total amount: 44.0 mg, 34.5%) as a colorless solid.

Synthesis of compound 6c

4-methoxybenzoyl chloride (20.9 μL, 0.174 mmol 1.5 eq) in a solution of compound 5c (26.3 mg, 0.116 mmol 1.0 eq) and Et3N (97.0 μL, 0.696 mmol 6.0 eq) in toluene (0.6 mL) at room temperature in N ₂ atmosphere Added below. The reaction mixture was refluxed at 100 ° C. for 3 hours and 50 minutes. The mixture reaction was cooled with water and extracted with CH ₂ Cl ₂ . The organic layer was dried over Na ₂ SO ₄ and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH ₂ Cl ₂ , then CH ₂ Cl ₂ : ethyl acetate = 4: 1) to obtain compound 6c (32.5 mg, 80.3%) as a colorless solid.

Synthesis of compound 8c

To a solution of compound 6c (50.0 mg, 0.143 mmol 1.0 eq) and K ₂ CO ₃ (98.8 mg, 0.715 mmol 5.0 eq) in DMF (3 mL) was added compound 15c (94.0 mg, 0.215 mmol 1.5 eq) at room temperature. It was. The reaction mixture was heated to 80 ° C. for 4 hours 30 minutes. The mixture was purified by silica gel column chromatography (CHCl ₃ : Methanol: NH ₃ water 10: 1: 1%) to obtain Compound 8c (32.6 mg, 43.6%) as a colorless solid.

Synthesis of compound 2c

Compound 8c (10.0 mg, 11.0 μmol, 1.0 eq) and 0.5 mL pyridine were added to crude compound 12c in 0.5 mL dry DMF at room temperature under a nitrogen atmosphere. The mixture was stirred at room temperature for 7 hours under a nitrogen atmosphere. After that, the crude solution was HPLC (A: B = 5:95 → 70:30 for 55 min (linear gradient) → 100: 0 for 5 min (linear gradient), A = CH ₃ CN, B = 10 mM AcONH ₄ aq ). The desired product eluted at 45 min to give compound 2c (Fl-AI-C4-PTMB) as an orange solid (4.6 mg, 41.4%). The product was identified by ¹ H-NMR and MALDI ([M + H] = 1012.7 (obs.), 1012.3 (cal.)).

Claims (6)

1. The following formula (II)
   

   

   (In the formula, Rec-Nu represents a group in which a hydrogen atom has been eliminated from a neurotransmitter receptor (Rec-Nu-H). Nu represents a nucleophilic group (Nu-H) of the neurotransmitter receptor. A divalent group in which a hydrogen atom is eliminated from L ^2, L ² represents a divalent linking group, and Fl represents a labeling group.
   It is expected that the labeled neurotransmitter receptor interacts with the receptor, which has a basic structure represented by the formula and changes the fluorescence pattern between when the antagonist is bound and when the agonist is bound. A substance that binds to a neurotransmitter receptor, characterized in that a candidate substance is allowed to act, and the binding mode of the candidate substance and the receptor is detected based on a change in a signal emitted by a label, and is performed based on the result. Screening method.
2. The screening method according to claim 1, wherein the candidate substance is allowed to act on cells having a labeled neurotransmitter receptor.
3. The screening method according to claim 1 or 2, wherein the receptor is an AMPA receptor.
4. Formula (I) below
   

   

   (In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
   A compound represented by
5. Formula (I) below
   

   

   (In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
   A labeling agent for the neurotransmitter receptor represented by:
6. Formula (I) below
   

   

   (In the formula, L ¹ and L ² each independently represent a divalent linking group, and Lg represents a ligand for a neurotransmitter receptor (Rec-Nu-H) having a nucleophilic group (Nu-H). Fl represents a labeling group, and R ¹ and R ² are the same or different and represent a hydrogen atom or a substituent.)
   And the neurotransmitter receptor (Rec-Nu-H) is reacted with the compound represented by the following formula (II):
   

   

   (In the formula, Rec-Nu represents a group in which a hydrogen atom is eliminated from the neurotransmitter receptor (Rec-Nu-H). Nu represents a hydrogen atom from a nucleophilic group possessed by the neurotransmitter receptor. (L ² represents a divalent linking group, and Fl represents a labeling group.)
   A labeled neurotransmitter receptor having a basic structure represented by the above, wherein the signal pattern emitted by the label changes between when an antagonist is bound and when an agonist is bound. A method for producing a receptor.

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