Novel fluorescent substrates and uses thereof

Abstract

The present disclosure relates to a novel fluorescent substrate, which is a compound or isomer of formula (I), and its use. A high-sensitivity detection reagent combination and detection method are also provided, which comprises the fluorescent substrate compound or isomer of formula (I) and an enzyme-labeled antibody. The fluorescent substrate and detection method can also be widely used for detecting cells with low expression of specific antigens. Compared with the traditional fluorescent secondary antibody detection method and the TSA-FITC combination method, the detection efficiency of the method of the present disclosure is higher, and the detection sensitivity and stability are higher.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological detection, in particular to a novel fluorescent substrate and its use. BACKGROUND

[0002] In the field of disease diagnosis, it is of great significance to detect the markers of target cells with high specificity and high sensitivity. Fluorescence analysis method has extremely high spatial and temporal resolution, and can realize effective detection of biomolecules such as nucleic acids and proteins of target cells, and has a wide range of biomedical applications. However, the content of some disease-related biomolecules (such as circulating epithelial cells, circulating endothelial cells, immune cells, and other tumor biomarkers) in the body is very low, and the conventional fluorescence detection technology cannot accurately analyze them, so it is still necessary to design and construct a fluorescence biosensor with better selectivity and higher sensitivity.

[0003] The fluorescence antibody used for immunofluorescence detection currently has generally low detection sensitivity, especially when used for fluorescence microscope scanning analysis, it is difficult to detect some samples with low expression using conventional fluorescence antibody.

[0004] Tyramide signal amplification (TSA) is an enzyme-mediated amplification technology. In the presence of hydrogen peroxide, horseradish peroxidase (HRP) can catalyze the conversion of tyramide into an oxygen active free radical, which can quickly covalently bind to proteins or HRP-linked antibodies. The fluorescence dye is combined on the tyramide molecule to form a covalent bond binding site, thereby stably and efficiently enriching the fluorescent dye around the target cells and depositing it on the target cells, so that the detection signal is amplified by several orders of magnitude. However, the fluorescent substrate TSA-FITC (FITC) used in this method currently has low detection sensitivity, and the detection sensitivity needs to be further improved, which is mainly because the fluorescence signal intensity of FITC fluorescent dye itself is not enough. Currently, there are also some other fluorescent dyes such as Alexa Fluor488, Dylight488, iFluor488, etc., but these fluorescent dyes are imported reagents and have high cost. SUMMARY

[0005] The purpose of the present disclosure is to provide a fluorescent substrate with larger stokes shift and higher signal-to-noise ratio, and the fluorescent substrate has good light stability and can be used in TSA detection to obtain better imaging effect.

[0006] In one aspect, the present disclosure provides a compound represented by formula (I) or a stereoisomer thereof,

[0007]

[0008] wherein,

[0009] R1, R2, R4, R5, R6are independently selected from hydrogen, halogen, C1-C9alkyl, C1-C9alkoxy. In some embodiments, the alkyl is C1-C3alkyl, and the alkoxy is C1-C3alkoxy.

[0010] R3is selected from hydrogen, halogen, C1-C9alkyl, C1-C9alkoxy, -C1-C9alkyl- unsubstituted aryl or substituted aryl, -C1-C9alkyl-unsubstituted heteroaryl or substituted heteroaryl, -alkenyl-unsubstituted aryl or substituted aryl, -alkenyl- unsubstituted heteroaryl or substituted heteroaryl. In some embodiments, the alkyl is C1-C3alkyl. In some embodiments, the alkoxy is C1-C3alkoxy. In some embodiments, the alkenyl is selected from C2-C9alkenyl. In some embodiments, the alkenyl is preferably C2-C5alkenyl. In some embodiments, R3is selected from -CH=CH-CH=CH-CH2-, -CH2-CH=CH-.

[0011] In some embodiments, the substituted aryl or heteroaryl is substituted with 1-3 substituents independently selected from halogen, C1-C3alkyl, C1-C3alkoxy, haloC1-C3alkyl, haloC1-C3alkoxy, aryl, heteroaryl.

[0012] The compound provided by the present disclosure can be used as a fluorescent substrate, can achieve efficient fluorescent detection, can replace FITC in TSA detection, and has relatively higher detection sensitivity, can efficiently detect low-expression markers, and has obvious advantages.

[0013] The fluorescent substrate provided by the present disclosure improves the imaging quality of low-abundance targets through tyramide signal amplification technology, and the absorption wavelength range covered by the provided fluorescent substrate is 400-600 nm, and the preferred range is 488-594 nm. In actual application, fluorescent substrates with different absorption wavelengths can be selected according to different detection conditions and requirements.

[0014] In one aspect, the present disclosure also provides fluorescent substrates BDP-A, BDP-B, and BDP-C for absorption wavelengths near 488 nm, and fluorescent substrates BDP-D, BDP-E, and BDP-F for absorption wavelengths near 594 nm. Compared with the traditional fluorescent secondary antibody detection method, the detection sensitivity of BDP-A, BDP-B, BDP-C, BDP-D, BDP-E, and BDP-F is higher, and low-expression markers can be detected.

[0015] In another aspect, the present disclosure also provides a reagent combination comprising the compound or a stereoisomer thereof.

[0016] In another aspect, the present disclosure also provides a kit comprising the compound or stereoisomer thereof or the reagent combination.

[0017] In another aspect, the present disclosure also provides a method for labeling a molecular target, comprising the step of contacting the molecular target with the compound or stereoisomer thereof or the reagent combination.

[0018] In another aspect, the present disclosure also provides a method for imaging a sample, comprising contacting the sample with the compound or stereoisomer thereof or the reagent combination.

[0019] In another aspect, the present disclosure also provides the use of the compound or stereoisomer thereof, the reagent combination or the kit in the preparation of a fluorescent detection reagent or a cell imaging reagent.

[0020] The beneficial effects achieved by the present disclosure are at least as follows:

[0021] 1. The present disclosure provides a new compound or stereoisomer thereof, which can be used as a fluorescent substrate to achieve high-sensitivity fluorescent detection and can be widely used for detecting cells with low expression of specific antigens. The use of the fluorescent substrate detection system of the present disclosure has higher detection sensitivity while not affecting specificity.

[0022] 2. The reagent combination and detection method provided by the present disclosure have higher detection sensitivity and detection stability compared to traditional fluorescent secondary antibody methods and TSA-FITCT methods. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The fluorescence intensity changes of compounds BDP-A, BDP-B and BDP-C under continuous irradiation of green light are shown (PBS, 4 μM).

[0024] Figure 2 The fluorescence responses of compounds BDP-A, BDP-B and BDP-C to different biological factors are shown.

[0025] Figure 3 The fluorescence intensity changes of compounds BDP-D, BDP-E and BDP-F under continuous irradiation of green light are shown (PBS, 4 μM).

[0026] Figure 4 The fluorescence responses of compounds BDP-D, BDP-E and BDP-F to different biological factors are shown.

[0027] Figure 5 The comparison of fluorescent substrate BDP-A, fluorescent substrate FITCT and fluorescent secondary antibody detection results is shown.

[0028] Figure 6 Fluorescent substrate BDP-D, BDP-E, BDP-F and fluorescent secondary antibody detection results are shown.

[0029] Figure 7 Fluorescent substrate FITC, BDP-A, BDP-F fluorescence detection stability comparison is shown. DETAILED DESCRIPTION

[0030] As used herein, unless otherwise indicated, "alkyl" means a straight or branched chain saturated aliphatic hydrocarbon group including all isomers and having the specified number of carbon atoms.

[0031] Common abbreviations for alkyl groups are used throughout the specification, such as methyl can be represented by "Me" or CH3, ethyl can be represented by "Et" or CH2CH3, propyl can be represented by "Pr" or CH2CH2CH3, butyl can be represented by "Bu" or CH2CH2CH2CH3, and the like. For example, "C 1-6 "alkyl" (or "C 1- "C6alkyl" means a straight chain or branched chain alkyl group including all isomers and having the specified number of carbon atoms. 1-6 "alkyl" includes all hexyl and pentyl alkyl isomers as well as n-, i-, s- and t-butyl, n- and i-propyl, ethyl and methyl. The term "alkyl" means a straight chain or branched chain aliphatic hydrocarbon group which can be straight chain or branched and which has the specified number of carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and t-butyl, pentyl, hexyl and the like. 1-4 "alkyl" includes all hexyl and pentyl alkyl isomers as well as n-, i-, s- and t-butyl, n- and i-propyl, ethyl and methyl. The term "alkyl" means a straight chain or branched chain aliphatic hydrocarbon group which can be straight chain or branched and which has the specified number of carbon atoms. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec- and t-butyl, pentyl, hexyl and the like.

[0032] The term "alkoxy" represents a straight chain or branched chain alkyl group of the specified number of carbon atoms attached through an oxygen bridge.

[0033] The term "alkenyl" means an aliphatic hydrocarbon group containing at least one carbon-carbon double bond, which can be straight-chain or branched and which has the specified number of carbon atoms. Preferred alkenyl groups contain one carbon-carbon double bond, and up to four non-aromatic carbon-carbon double bonds can be present. Alternative examples of alkenyl groups include ethenyl, propenyl, n-butenyl, 2-methyl-l-butenyl, 3-methylbut-2-enyl, n-pentenyl, octenyl, and decenyl.

[0034] The term "aryl" means aromatic monocyclic and polycyclic ring systems in which the individual carbon rings in polycyclic ring systems are fused or linked by single bonds. Suitable aryl groups include phenyl, naphthyl, 2,3-dihydro-lH-indenyl, and biphenyl.

[0035] "Heterocycle," "heterocyclic" or "heterocyclyl" means a monocyclic or bicyclic 3-12 membered ring system in which at least one ring is non-aromatic (saturated or partially unsaturated) and which contains at least one heteroatom selected from O, S and N. In bicyclic ring systems, the second ring can be heteroaryl, heterocyclyl or a saturated, partially unsaturated or aromatic carbocyclic ring, and the point of attachment to the rest of the molecule can be on either ring. For bicyclic systems, the rings are fused (e.g., quinoline), at a ring carbon atom (e.g., 1,4-dioxaspiro[4.5]decane), or a bridged group (e.g., 8-azabicyclo[3.2.1]octyl). Thus "heterocyclyl" includes heteroaryl, as well as dihydro and tetrahydro analogs thereof. Attachment of a heterocyclyl substituent can be through a carbon atom or through a heteroatom.

[0036] "Heterocycle," "heterocyclic" or "heterocyclyl" means a monocyclic or bicyclic 3-12 membered ring system in which at least one ring is non-aromatic (saturated or partially unsaturated) and which contains at least one heteroatom selected from O, S and N. In bicyclic ring systems, the second ring can be heteroaryl, heterocyclyl or a saturated, partially unsaturated or aromatic carbocyclic ring, and the point of attachment to the rest of the molecule can be on either ring. For bicyclic systems, the rings are fused (e.g., quinoline), at a ring carbon atom (e.g., 1,4-dioxaspiro[4.5]decane), or a bridged group (e.g., 8-azabicyclo[3.2.1]octyl). Thus "heterocyclyl" includes heteroaryl, as well as dihydro and tetrahydro analogs thereof. Attachment of a heterocyclyl substituent can be through a carbon atom or through a heteroatom.

[0037] The term "heteroaromatic" (or "heteroaryl") generally refers to a heterocycle as defined above in which the entire ring system (whether mono- or polycyclic) is an aromatic ring system. The term "heteroaromatic ring" refers to a 5- or 6-membered monocyclic aromatic ring or a 7- to 12-membered bicyclic ring which is composed of carbon atoms and one or more heteroatoms selected from N, O, and S. For heteroaryl groups in which only one of the rings of the bicyclic ring needs to be heteroaromatic, the second ring can be heteroaromatic or aromatic, saturated, or partially unsaturated, and the point of attachment to the rest of the molecule can be on either ring. In the case of substituted heteroaryl rings containing at least one nitrogen atom (e.g., pyridine), such substitution can be those which lead to the formation of N-oxides. Examples of heteroaryl groups include, but are not limited to, furanyl, thienyl (or thiazolyl), pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolyl, isoquinolyl, naphthylidinyl, benzothienyl, benzofuranyl, benzimidazole, benzopyrazolyl, indolyl, isoindolyl, indolizinyl, indazolyl, purinyl, quinolizinyl, phtalazinyl, quinoxalyl, quinazolinyl, benzoxazolyl, benzisoxazolyl, 5,6,7,8-tetrahydroquinolinyl, imidazo[l,2-a]pyridinyl, imidazo[l,2-a]pyrimidinyl, 5,6-dihydropyrrolo[l,2-b]pyrazolyl, pyrrolo[3,2-c]pyridinyl, pyrrolo[2,3-b]pyridinyl, thieno[2,3-b]pyrrolyl, furopyridine and thienopyridine. The term "substituted" (e.g., as in "aryl optionally substituted with one or more substituents") includes mono- and disubstitution with the indicated substituents, as is chemically permissible.

[0038] The term "TSA" refers to the high sensitivity detection of target analytes by the catalysis of tyrosinamide by tyrosinase, which deposits fluorescein conjugated tyrosinamide substrate around the target analyte, which is amplified by the signal, thereby achieving high sensitivity detection of the target analyte. In the present disclosure, specific antibodies are first screened for the target analyte and labeled with horseradish peroxidase, and fluorescein is attached to p-hydroxyphenethylamine, which is attached to the tyrosine residues of the protein by the catalysis of horseradish peroxidase, thereby enriching the fluorescein around the target analyte, which achieves high sensitivity detection of the target analyte.

[0039] The term "quantum yield" refers to the ratio of the number of fluorescent photons emitted by a fluorescent substance to the number of photons of excitation light absorbed by the substance, and is used to measure the efficiency of light emission from a sample. The value of the quantum yield of fluorescence is determined primarily by the structure and properties of the compound, and also by the environmental factors in which the compound is placed. The quantum yield of fluorescence only reflects the light emission efficiency of the fluorescent substrate in the solution. The fluorescence signal intensity of the detection sample is related to both the characteristics of the fluorescein and the amount of fluorescein labeled in the sample.

[0040] In one aspect, the present disclosure is a compound of formula (I) or a stereoisomer thereof,

[0041]

[0042] wherein,

[0043] R1, R2, R4, R5, R6are independently selected from hydrogen, halogen, C1-C9 alkyl, C1-C9 alkoxy. In some embodiments, the alkyl is C1-C3 alkyl, and the alkoxy is C1-C3 alkoxy.

[0044] R3is selected from hydrogen, halogen, C1-C9 alkyl, C1-C9 alkoxy, -C1-C9 alkyl- unsubstituted aryl or substituted aryl, -C1-C9 alkyl-unsubstituted heteroaryl or substituted heteroaryl, -alkenyl-unsubstituted aryl or substituted aryl, -alkenyl- unsubstituted heteroaryl or substituted heteroaryl. In some embodiments, the alkyl is C1-C3 alkyl. In some embodiments, the alkoxy is C1-C3 alkoxy. In some embodiments, the alkenyl is selected from C2-C9 alkenyl. In some embodiments, the alkenyl is C2-C5 alkenyl. In some embodiments, R3is selected from -CH=CH-CH=CH-CH2-, -CH2-CH=CH-.

[0045] In some embodiments, the substituted aryl or heteroaryl is substituted with 1-3 substituents independently selected from halogen, C1-C3 alkyl, C1-C3 alkoxy, haloC1-C3 alkyl, haloC1-C3 alkoxy, aryl, heteroaryl.

[0046] In some embodiments, the compound is a compound of formula (II),

[0047]

[0048] wherein,

[0049] R1, R2, R4, R5, R6are independently selected from hydrogen, halogen, C1-C9 alkyl, C1-C9 alkoxy. In some embodiments, the alkyl is C1-C3 alkyl, the alkoxy is C1-C3 alkoxy.

[0050] A is selected from unsubstituted heteroaryl or substituted heteroaryl and unsubstituted aryl or substituted aryl.

[0051] In some embodiments, the substituted aryl or heteroaryl is substituted with 1-3 substituents independently selected from halogen, C1-C3 alkyl, C1-C3 alkoxy, halo C1-C3 alkyl, halo C1-C3 alkoxy, aryl, heteroaryl.

[0052] In some embodiments, A is 2,6-dimethoxy-phenyl, 4-N,N-dimethyl-anilino, indolyl.

[0053] In some embodiments, the compound is a compound shown as A, B, C, D, E, or F.

[0054]

[0055] In another aspect, the present disclosure also provides a reagent combination comprising the compound or a stereoisomer thereof.

[0056] In some embodiments, the reagent combination further comprises an enzyme-labeled antibody.

[0057] In some embodiments, the enzyme is selected from any one of dehydrogenase, oxidase, peroxidase, oxygenase. In some embodiments, the enzyme is any one of horseradish peroxidase, catalase, superoxide dismutase.

[0058] In some embodiments, the enzyme is horseradish peroxidase.

[0059] In some embodiments, the antibody is one or more of anti-HER2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-HER3 antibody, anti-MUC1 antibody, anti-PD-L1 antibody, anti-EpCAM antibody, anti-EGFR antibody, anti-c-met antibody, anti-FR (folate receptor) antibody, anti-CEA antibody, anti-PMSA antibody, anti-AR-V7 antibody, anti-CK antibody, anti-PLAP antibody, anti-GPC3 antibody, anti-CD31 antibody, anti-CD44 antibody, anti-Vimentin antibody, anti-cadherin antibody, etc.

[0060] In some embodiments, the reagent combination further comprises a blocking agent. In some embodiments, the blocking agent is peroxide. In some embodiments, the peroxide is H2O2.

[0061] In some embodiments, the reagent combination further comprises a buffer. In some embodiments, the buffer is a PBS solution or a Tris-HCl solution or a Hepes solution. In some embodiments, the buffer is a PBS solution or a Tris-HCl solution or a Hepes solution containing FBS or BSA. In some embodiments, the buffer is a PBS solution containing FBS or BSA. In some embodiments, the buffer is a Tris-HCl solution containing FBS or BSA. In some embodiments, the buffer is a Hepes solution containing FBS or BSA.

[0062] In another aspect, the present disclosure also provides a kit comprising the compound or a stereoisomer thereof or the reagent combination.

[0063] In another aspect, the present disclosure also provides a method of labeling a molecular target, the method comprising the step of contacting the molecular target with the compound or a stereoisomer thereof or the reagent combination.

[0064] In another aspect, the present disclosure also provides a method of imaging a sample, the method comprising contacting the sample with the compound or a stereoisomer thereof or the reagent combination.

[0065] In some embodiments, the sample is a cell-containing or cell-fragment-containing sample; more preferably, the sample is blood, plasma, urine, saliva, pleural effusion, peritoneal effusion, cerebrospinal fluid, tissue digestion sample.

[0066] In some embodiments, the method comprises the following steps:

[0067] (1) sample blocking: taking a sample, adding a blocking agent, incubating, obtaining a blocked sample;

[0068] (2) antibody incubation: mixing an enzyme-labeled antibody with the blocked sample obtained in step (1), incubating, obtaining an antibody-sample complex solution;

[0069] (3) substrate incubation: contacting the compound or a stereoisomer thereof or the reagent combination with the antibody-sample complex solution obtained in step (2), incubating, obtaining a fluorescent substrate-antibody-sample complex solution;

[0070] (4) sample imaging: taking a sample and dropping the fluorescent substrate-antibody-sample complex onto a glass slide, baking, adding DAPI staining, and scanning and analyzing using a fluorescence microscope.

[0071] In some embodiments, the concentration of the compound is at least 0.05 pg / mL, 0.1 pg / mL, or 0.15 pg / mL. In some embodiments, the concentration of the compound is 0.15 pg / mL-2.5 pg / mL. In some embodiments, the concentration of the compound is 0.3 pg / mL-1.25 pg / mL. In some embodiments, the concentration of the compound is 0.6 pg / mL-1.25 pg / mL.

[0072] In another aspect, the present disclosure also provides use of the compound or stereoisomer thereof, the reagent combination or the kit in preparation of a fluorescent detection reagent, a cell imaging reagent.

[0073] Experimental materials

[0074] 1. Sources of reagents, drugs, cell lines

[0075] Fluorescein isothiocyanate, p-hydroxyphenethylamine, methanol, dichloromethane, FITC T (fluorescein isothiocyanate ester), 2-pyrrole carboxaldehyde, methoxalyl triphenylphosphonium, anhydrous magnesium sulfate, 10% palladium on carbon, 3,5-dimethyl-2-pyrrole carboxaldehyde, phosphorus oxychloride, boron trifluoride ether, n-hexane, tetrahydrofuran, concentrated hydrochloric acid, N,N-dimethylformamide, DMSO, triethylamine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, saturated sodium bicarbonate solution, p-dimethylaminobenzaldehyde, acetic acid, pyrrolidine, petroleum ether, p-2,4-dimethoxybenzaldehyde, 3-indole carboxaldehyde, 3% hydrogen peroxide solution, fluorescent secondary antibody (anti-HRP IgG Alexa Fluor 594, Jackson ImmunoResearch, item number 123-585-021), fluorescent secondary antibody (anti-HRP IgG Alexa Fluor 488, Jackson ImmunoResearch, item number 123-545-021).

[0076] MCF7 lung cancer cells (HER2 low expression cells): Shanghai Cell Bank of Chinese Academy of Sciences, item number: SCSP-531.

[0077] SKBR3 breast cancer cells (HER2 high expression cells): Shanghai Cell Bank of Chinese Academy of Sciences, item number: TCHu225.

[0078] ZR-75-1 (human breast cancer cells): Shanghai Cell Bank of Chinese Academy of Sciences, item number: TCHu126.

[0079] A549 (human lung adenocarcinoma cells): Shanghai Cell Bank of Chinese Academy of Sciences, item number: TCHu150.

[0080] The above cell line cells can be proliferated by DMEM culture containing 10% serum (FBS) and 1% double antibiotic.

[0081] The above experimental materials can be commercially available.

[0082] 2. Model of detection equipment used in the examples

[0083] UV spectrophotometer (Shimadzu, UV-1280); fluorescence spectrophotometer (Hitachi, F-6000); multifunctional microplate analysis system (Biotek, 800TS).

[0084] Example 1: 3-(5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinine-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-A)

[0085] The synthetic route of the fluorescent substrate BDP-A is shown below.

[0086]

[0087] Reagents and reaction conditions

[0088] (a) 1) Ph3P=CH-COOMe, DCM, rt; 2) Pd / C, H2, MeOH, rt;

[0089] (b) 3,5-dimethyl-2-pyrrolecarboxaldehyde, POCl3, DCM, BF3·OEt2, DIPEA;

[0090] (c) 4.5 M HCl, THF, DCM, rt;

[0091] (d) tyramine, Et3N, HOBt, EDC·HCl, DMF, rt.

[0092] (1) Step a: synthesis of intermediate 1

[0093] Take 50 ml single neck flask, add 124 mg 2-pyrrolecarboxaldehyde, 870 mg methoxyformyltriphenylphosphonium and 6 mL dichloromethane to it, stir the reaction at room temperature, after the reaction is completed, add mixed solution (water: ether = 1:1) to extract and collect the organic phase, wash the organic phase with water twice, dry with anhydrous magnesium sulfate, concentrate under reduced pressure, and purify by flash column chromatography (DCM: PE = 5:1) to obtain about 169 mg of light yellow solid with a yield of about 86%. 1H NMR (400 MHz, Chloroform-d) δ 7.60 (d, J = 15.9 Hz, 1H), 6.96 (td, J = 2.8, 1.3 Hz, 1H), 6.61 - 6.57 (m, 1H), 6.31 (dt, J = 3.7, 2.5 Hz, 1H), 6.05 (d, J = 15.9 Hz, 1H), 3.80 (s, 3H).

[0094] In the reaction bottle of the hydrogen reaction generator, 169 mg of the above yellowish solid and 8 mL of methanol were added, 10% palladium-carbon was added, hydrogen was filled (hydrogen generator, 1 bar), and stirring was carried out at room temperature. After the reaction was completed, the palladium-carbon was removed by filtration with diatomite, and concentrated under reduced pressure. Separation and purification were carried out using flash column chromatography (PE:EA = 5:1) to obtain intermediate 1 as a yellowish liquid at about 146 mg with a yield of about 85%. 1 H NMR (400 MHz, Chloroform-d) δ 6.71 (td, J = 2.7, 1.5 Hz, 1H), 6.15 (q, J = 2.9 Hz, 1H), 5.97 (td, J = 3.4, 3.0, 1.7 Hz, 1H), 3.74 (s, 3H), 2.96 (t, J = 6.8 Hz, 2H), 2.69 (t, J = 6.8 Hz, 2H). 13 C NMR (101 MHz, Chloroform-d) δ 174.58, 130.95, 116.86, 108.04, 105.50, 51.86, 34.36, 22.58.

[0095] (2) Step b: synthesis of intermediate 2

[0096] A 50 mL two-necked flask was taken, 146 mg of intermediate 1, 129 mg of 3,5-dimethyl-2-pyrrolecarboxaldehyde were dissolved in 8 mL of dichloromethane, and protected by nitrogen. After being stirred at 0°C until completely dissolved, 160 mg of phosphorus oxychloride was added by syringe, and stirred at 0°C for 30 minutes. The reaction was transferred to room temperature and stirred for 6 hours. The reaction solution was cooled to 0°C, 539 mg of boron trifluoride etherate and 516 mg of N,N-diisopropylethylamine were added by syringe in turn, and stirred at room temperature for 12 hours until the reaction was completed. Tap water was added, mixed uniformly, filtered with diatomite, and the organic phase was collected by liquid separation. The aqueous phase was washed with dichloromethane, and the organic phase was collected. After being concentrated under reduced pressure, separation and purification were carried out using flash column chromatography (PE:EA = 5:1), and recrystallized with n-hexane to obtain intermediate 2 as a red solid at 201 mg with a yield of about 69%. 1H NMR (400 MHz, Chloroform-d) δ 7.09 (s, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.27 (d, J = 4.0 Hz, 1H), 6.12 (s, 1H), 3.71 (t, J = 7.6 Hz, 3H), 3.31 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H), 2.58 (s, 3H), 2.25 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 173.00, 160.49, 157.00, 143.91, 135.25, 133.30, 128.05, 123.85, 120.47, 116.66, 51.76, 33.27, 23.98, 14.97, 11.31.

[0097] (3) Step c: synthesis of intermediate 3

[0098] Take 100 mL single neck flask, add 201 mg of intermediate 2, add 32 mL of tetrahydrofuran, after dissolving, add 20 mL of water and 10 mL of concentrated hydrochloric acid, carry out the reaction at room temperature, stir for 36 hours, after the reaction is completed, add dichloromethane, separate the organic phase, collect the organic phase, vacuum concentration and drying, fast column chromatography (DCM:MeOH=10:1) separation and purification, then recrystallized with dichloromethane and n-hexane mixture solution, get intermediate 3 red solid about 147 mg, yield about 76%. 1 H NMR (400 MHz, Chloroform-d) δ 7.43 (s, 1H), 7.01 (d, J = 4.0 Hz, 1H), 6.35 (d, J = 4.0 Hz, 1H), 6.22 (s, 1H), 3.22 (t, J = 7.7 Hz, 2H), 2.73 (dd, J = 8.5, 6.9 Hz, 2H), 2.53 (s, 3H), 2.29 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 174.83, 159.95, 157.00, 144.42, 133.52, 128.17, 124.38, 119.94, 116.12, 53.39, 32.62, 23.56, 13.45, 9.77.

[0099] (4) Step d: synthesis of BDP-A

[0100] Take 25 mL of a two-necked flask, add 147 mg of intermediate 3, 82 mg of p-hydroxyphenethylamine (first dissolved in 5 mL of N,N-dimethylformamide), 209 μL of triethylamine, 81 mg of 1-hydroxybenzotriazole and 115 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir under nitrogen protection at room temperature for 12 hours. After the reaction is completed, add a mixed solution (ethyl acetate: water = 1:1), separate and collect the organic phase, then successively wash the organic phase with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine, collect the organic phase, vacuum concentrate and dry, and separate and purify by flash column chromatography (PE: EA = 1:1), then recrystallize from n-hexane to obtain about 120 mg of BDP-A red solid with a yield of about 58%.

[0101]

[0102] 1 H NMR (400 MHz, Chloroform-d) δ 7.28 (s, 1H), 7.10 (s, 1H), 6.98 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 4.0 Hz, 1H), 6.75 (dd, J = 8.8, 2.5 Hz, 2H), 6.26 (d, J = 4.0 Hz, 1H), 6.15 (s, 1H), 5.82 (t, J = 5.7 Hz, 1H), 3.44 (q, J = 6.7 Hz, 2H), 3.26 (t, J = 7.4 Hz, 2H), 2.64 (dt, J = 17.9, 7.2 Hz, 4H), 2.58 (s, 3H), 2.28 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 172.03, 160.29, 157.16, 154.60, 143.98, 133.39, 130.42, 129.76, 128.33, 123.86, 120.46, 117.55, 115.50, 40.92, 36.05, 34.73, 24.91, 14.96, 11.35. HRMS (ESI): calcd. For C 22 H 24 BF2N3O2[M+Na]+434.1898; found 434.1832.

[0103] Example 2: 3-(8-ethyl-5,5-difluoro-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[l,2-c:2',l'- f][l,3,2]diazaborinine-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-B)

[0104] The synthetic route of the fluorescent substrate BDP-B is shown below.

[0105]

[0106] (1) Step e: synthesis of intermediate 4

[0107] Take 25 ml of single mouth bottle, add 36 mg of DMF, 0 ℃ stirring, add 62 mg of oxalyl chloride, wait for white crystal to appear, transfer to room temperature stirring for 15 minutes, add 5 ml of dichloromethane, stir evenly. 68 mg of intermediate 1 was dissolved in 10 ml of dichloromethane, and was added dropwise to the above reaction bottle at room temperature, and the reaction was stirred. After the reaction was completed, 183 mg of sodium acetate was dissolved in tap water, and then added to the above reaction bottle, and stirred at room temperature for 30 minutes. The reaction liquid was transferred to a separatory funnel, separated, and the organic phase was collected. The organic phase was washed with chloroform, and the organic phase was collected. The organic phase was successively washed with saturated sodium bicarbonate solution and saturated brine, dried over anhydrous magnesium sulfate, and the organic phase was collected, vacuum concentrated and dried, and separated and purified by flash column chromatography (PE:EA = 3:1) to obtain about 64 mg of intermediate 4 solid, with a yield of about 79%. 1 H NMR (400 MHz, Chloroform-d) δ 10.24 (s, 2H), 9.35 (s, 1H), 6.86 (dd, J = 3.8, 2.5 Hz, 1H), 6.06 (dd, J = 3.8, 2.5 Hz, 1H), 3.68 (s, 3H), 2.98 (t, J = 7.2 Hz, 2H), 2.68 (t, J = 7.2 Hz, 2H).

[0108] (2) Step f: synthesis of intermediate 5

[0109] Take 50 ml of double mouth bottle, add 64 mg of intermediate 4 and 48 mg of 2,4-dimethyl-3-ethyl pyrrole (first dissolved in 30 ml of dichloromethane), nitrogen protection, 0 ℃ stirring, add 60 mg of phosphorus oxychloride with a syringe, 0 ℃ stirring for 30 minutes, transfer to room temperature for reaction for 6 hours. The reaction liquid was cooled to 0 ℃, and then 201 mg of boron trifluoride ether and 193 mg of N,N-diisopropyl ethylamine were added in sequence with a syringe, and stirred at room temperature for 12 hours. After the reaction was completed, tap water was added, mixed evenly, filtered with diatomite, and the organic phase was collected by separation. The aqueous phase was washed with dichloromethane, and the organic phase was collected, concentrated under reduced pressure, separated and purified by flash column chromatography (PE:EA = 5:1), and recrystallized with n-hexane to obtain about 75 mg of intermediate 5 red solid, with a yield of about 63%. 1H NMR (400 MHz, Chloroform-d) δ 7.03 (s, 1H), 6.82 (d, J = 4.0 Hz, 1H), 6.22 (d, J = 4.0 Hz, 1H), 4.13 (q, J = 7.1 Hz, 1H), 3.71 (s, 3H), 3.29 (t, J = 7.7 Hz, 2H), 2.78 (dd, J = 8.4, 6.9 Hz, 2H), 2.56 (s, 3H), 2.40 (q, J = 7.6 Hz, 2H), 2.17 (s, 3H), 1.08 (t, J = 7.6 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 173.07, 160.54, 155.43, 139.67, 134.93, 133.98, 132.88, 126.78, 122.78, 115.71, 33.37 (d, J = 1.7 Hz), 23.90 (d, J = 1.9 Hz), 21.03, 17.25, 14.24, (d, J = 9.3 Hz), 13.00 (t, J = 2.2 Hz), 9.36.

[0110] (3) Step g: synthesis of intermediate 6

[0111] Take 100 ml single mouth bottle, add 75 mg of intermediate 5, dissolved in 9.5 ml of tetrahydrofuran, add 6.0 ml of water and 4.0 ml of concentrated hydrochloric acid, stir at room temperature for 36 hours, after the reaction is completed, dichloromethane is added, the organic phase is collected, vacuum concentration and drying, flash column chromatography (DCM:MeOH = 10:1) separation and purification, n-hexane recrystallization, to get intermediate 6 as a red solid about 37 mg, the yield is about 52%. 1 H NMR (400 MHz, Chloroform-d) δ 7.03 (s, 1H), 6.82 (d, J = 4.0 Hz, 1H), 6.22 (d, J = 4.0 Hz, 1H), 4.13 (q, J = 7.1 Hz, 1H), 3.71 (s, 3H), 3.29 (t, J = 7.7 Hz, 2H), 2.78 (dd, J = 8.4, 6.9 Hz, 2H), 2.56 (s, 3H), 2.40 (q, J = 7.6 Hz, 2H), 2.17 (s, 3H), 1.08 (t, J = 7.6 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 178.49, 160.78, 154.93, 139.79, 135.01, 134.08, 132.89, 126.76, 122.82, 115.71 (d, J = 3.6 Hz), 33.21, 23.57, 17.27, 14.31, 13.06, 9.41.

[0112] (4) Step h: synthesis of BDP-B

[0113] Into a 25 ml two-necked flask, 37 mg of intermediate 6 and 20 mg of p-hydroxyphenethylamine (dissolved in 3 ml of N,N-dimethylformamide) were added, 49 μl of triethylamine, 19 mg of 1-hydroxybenzotriazole and 27 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added, and the reaction was stirred at room temperature under nitrogen. After the reaction was completed, a mixed solution (ethyl acetate: water = 1; 1) was added, the organic phase was separated, and the organic phase was collected. The organic phase was washed with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in turn, and the organic phase was collected. The organic phase was concentrated and dried under vacuum, and purified by flash column chromatography (PE: EA = 1:1) and recrystallized from n-hexane to obtain about 40 mg of BDP-B red solid with a yield of about 77%.

[0114] 1 H NMR (400 MHz, Chloroform-d) δ 7.04 (s, 1H), 6.96 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 4.0 Hz, 1H), 6.75 (d, J = 8.4 Hz, 2H), 6.20 (d, J = 3.9 Hz, 1H), 3.42 (q, J = 7.0 Hz, 2H), 3.24 (t, J = 7.5 Hz, 2H), 2.62 (dt, J = 12.1, 7.3 Hz, 4H), 2.56 (s, 3H), 2.41 (q, J = 7.6 Hz, 2H), 1.92 (s, 3H), 1.09 (t, J = 7.6 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 172.42, 160.41, 155.39, 154.90, 139.89, 134.85, 134.05, 132.95, 130.10, 127.11, 122.84, 116.58, 115.55, 41.02, 36.11, 34.70, 24.84, 17.27, 14.33, 13.03, 9.44.

[0115] Example 3: 3-(5,5-difluoro-8-iodo-7,9-dimethyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinine-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-C)

[0116] The synthesis route of the fluorescent substrate BDP-C is shown below.

[0117]

[0118] (1) Step i: synthesis of intermediate 7

[0119] Take 100 ml of a two-necked flask, add 60 mg of 3,5-dimethyl-2-pyrrole carboxaldehyde, 100 g of potassium carbonate and 10 ml of methanol to it in turn, protect it with argon, add 134 mg of iodine, and after 12 hours of stirring at 0°C, add ether to the reaction flask, transfer it to a separatory funnel, and separate the liquid. Wash the liquid with saturated sodium thiosulfate solution, dry it with anhydrous magnesium sulfate, and concentrate it under vacuum to obtain a crude product. Recrystallize the crude product with dichloromethane at room temperature, filter it, and dry the filter cake at 40°C overnight to obtain about 91 mg of intermediate 7 as a solid, with a yield of about 75%. 1 H NMR (400 MHz, Chloroform-d) δ 9.51 (s, 1H), 2.37 (s, 3H), 2.30 (s, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 176.23, 140.06, 136.81, 129.11, 72.66, 14.35, 12.69.

[0120] (2) Step j: synthesis of intermediate 8

[0121] Take a 50 ml two-necked flask, add 62 mg of intermediate 2 and 91 mg of intermediate 7 (first dissolved in 50 ml of dichloromethane), protect it with nitrogen, and stir it at 0°C for 5 minutes. Add 62 mg of phosphorus oxychloride through a syringe, stir it at 0°C for 30 minutes, and then transfer it to room temperature for 6 hours of stirring. Cool the reaction to 0°C, add 208 mg of boron trifluoride etherate and 200 mg of N,N-diisopropylethylamine through a syringe in turn, and stir the reaction at room temperature for 12 hours. After the reaction is complete, add tap water, mix it well, filter it with diatomite, collect the organic phase by liquid separation, wash the aqueous phase with dichloromethane, collect the organic phase, concentrate it under reduced pressure, separate and purify it by fast column chromatography (PE:EA = 5:1), and recrystallize it with n-hexane to obtain about 77 mg of intermediate 8 as a red solid, with a yield of about 49%. 1 H NMR (400 MHz, Chloroform-d) δ 7.13 (s, 1H), 6.96 (d, J = 4.1 Hz, 1H), 6.34 (d, J = 4.1 Hz, 1H), 3.72 (s, 3H), 3.31 (t, J = 7.6 Hz, 2H), 2.79 (t, J = 7.6 Hz, 2H), 2.63 (s, 3H), 2.22 (s, 3H). 13C NMR (101 MHz, Chloroform-d) δ 172.78, 159.15 (d, J = 32.7 Hz), 145.17, 133.84 (d, J = 23.2 Hz), 129.96 (d, J = 2.5 Hz), 124.10, 118.02 (d, J = 4.5 Hz), 82.52, 51.83, 33.02, 29.71, 24.08, 15.78, 13.72, 11.31.

[0122] (3) Step k: synthesis of intermediate 9

[0123] Take 100 ml single mouth bottle, add 77 mg of intermediate 8 (first dissolved in 8.3 ml of tetrahydrofuran), add 5.5 ml of water and 3.3 ml of concentrated hydrochloric acid, stir at room temperature for 36 hours, after the reaction is completed, add dichloromethane, separate the liquid, collect the organic phase, vacuum concentration and drying, fast column chromatography (DCM:MeOH = 10:1) separation and purification, n-hexane recrystallization, get intermediate 9 about 39 mg of red solid, yield about 51%. 1 H NMR (400 MHz, DMSO-d6) δ 7.84 (s, 1H), 7.22 (d, J = 4.2 Hz, 1H), 6.50 (d, J = 4.1 Hz, 1H), 3.36 (s, 3H), 3.10 (t, J = 7.7 Hz, 2H), 2.67 (dd, J = 9.0, 6.7 Hz, 2H), 2.22 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 173.78, 160.17, 157.82, 145.70, 133.94 (d, J = 29.4 Hz), 131.51, 126.50, 118.59, 83.69, 32.49, 24.15, 15.89, 13.85, 11.47.

[0124] (4) Step l: synthesis of BDP-C

[0125] Take 25 ml of double mouth bottle, add 39 mg of intermediate 9 and 15 mg of p-hydroxyphenethylamine (first dissolved in 2 ml of N,N-dimethylformamide) to it, add 38 ul of triethylamine, 15 mg of 1-hydroxybenzotriazole and 21 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, argon protection, stirring reaction at room temperature, after the reaction is completed, add mixed solution (ethyl acetate:water = 1:1), separate the liquid, collect the organic phase, wash the organic phase with dilute hydrochloric acid, saturated sodium bicarbonate solution and saturated brine in turn, collect the organic phase, vacuum concentration and drying, fast column chromatography (PE:EA = 1:1) separation and purification, n-hexane recrystallization, get BDP-C red solid about 38 mg, yield about 78%.

[0126]

[0127] 1 H NMR (600 MHz, Methanol-d4) δ 7.40 (s, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.26 (d, J = 4.0 Hz, 1H), 6.20 (s, 1H), 3.34 (t, J = 7.4 Hz, 2H), 3.20 (t, J = 7.7 Hz, 2H), 2.66 (t, J = 7.3 Hz, 2H), 2.56 (t, 3H), 2.51 (s, 3H), 2.27 (s, 3H). 13 C NMR (151 MHz, Methanol-d4) δ 173.17, 159.86, 157.16, 155.46, 144.36, 135.10, 133.50, 129.87, 129.35, 128.24, 124.33, 119.92, 116.31, 114.85, 40.95, 34.28, 31.67, 26.71, 24.23, 22.34, 13.47, 9.79.

[0128] Example 4 BDP-D, E, F

[0129] The synthetic route of the fluorescent substrate BDP-D, E, F is shown below.

[0130]

[0131] (1) Synthesis of fluorescent substrate (E)-3-(7-(2,4-dimethoxystyryl)-5,5-difluoro-9-methyl-5H-5l4,6l4-dipyrrolo[l,2-c:2',l'- f][l,3,2]diazaborinine-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-D)

[0132] Take 25 ml of double mouth bottle, add 60 mg of BDP-A and p-326 mg dimethylaminobenzaldehyde, argon protection, add 2 ml of anhydrous ethanol with syringe, stir uniformly at room temperature. Add 131 mg of acetic acid and 647 mg of pyrrolidine in sequence with syringe, stir for 15 minutes, transfer the reaction solution to a single mouth bottle, vacuum concentration and drying, separate and purify by flash column chromatography (DCM:MeOH = 50:1), recrystallize with petroleum ether to obtain BDP-D solid about 47 mg, yield about 61%.

[0133]

[0134] 1H NMR (600 MHz, Acetonitrile-d3) δ 7.51 - 7.47 (m, 2H), 7.21 (s, 1H), 7.06 - 7.01 (m, 2H), 6.88 (d, J = 3.9 Hz, 1H), 6.84 (s, 1H), 6.76 (d, J = 8.9 Hz, 2H), 6.72 (d, J = 8.4 Hz, 1H), 6.23 (d, J = 3.9 Hz, 1H), 3.33 (td, J = 7.2, 5.8 Hz, 2H), 3.15 (t, J = 7.7 Hz, 2H), 3.04 (s, 1H), 3.01 (s, 6H), 2.96 (s, 1H), 2.65 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 7.7 Hz, 2H), 2.28 (s, 3H). 13 CNMR (151 MHz, Acetonitrile-d3) δ 171.91, 159.04, 155.86, 155.48, 152.53, 144.62, 141.43, 137.52, 133.72, 131.28, 130.71, 130.40, 130.06, 126.42, 124.21, 121.50, 117.39, 116.14, 115.67, 113.15, 112.73, 112.67, 41.26, 39.94, 35.08, 31.90, 28.33, 24.80, 11.09.

[0135] (2) Synthesis of fluorescent substrate (E)-3-(7-(4-(dimethylamino)styryl)-5,5-difluoro-9-methyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'-f][1,3,2]

[0136] Bispidine-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-E)

[0137] Take 25 ml of double mouth bottle, add 60 mg BDP-A and 364 mg of 2,4-dimethoxybenzaldehyde, argon protection, add 2 ml of anhydrous ethanol with a syringe, stir uniformly at room temperature. Add 131 mg of acetic acid and 647 mg of pyrrolidine in turn with a syringe, stir for 15 minutes, transfer the reaction solution to a single mouth bottle, vacuum concentration and drying, separate and purify by flash column chromatography (DCM:MeOH=50:1), recrystallize with petroleum ether to obtain BDP-E solid about 40 mg, yield about 49%.

[0138]

[0139] 1H NMR (600 MHz, Methanol-d4) δ 7.72 (d, J = 16.5 Hz, 1H), 7.59 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 16.3 Hz, 1H), 7.30 (s, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.91 (d, J = 4.0 Hz, 1H), 6.84 (s, 1H), 6.70 (d, J = 8.5 Hz, 1H), 6.60-6.56 (m, 2H), 6.25 (d, J = 4.0 Hz, 1H), 3.91 (s, 2H), 3.84 (s, 3H), 3.35 (t, J = 7.4 Hz, 2H), 3.23 (t, J = 7.7 Hz, 2H), 2.68 (t, J = 7.3 Hz, 2H), 2.59 (t, J = 7.7 Hz, 2H), 2.31 (s, 3H). 13 C NMR (151 MHz, Methanol-d4) δ 174.74, 164.31, 160.84, 156.90, 135.30, 131.30, 130.77, 129.93, 122.85, 119.33, 117.40, 117.06, 116.25, 107.21, 99.27, 56.20, 56.00, 42.38, 36.19, 35.73, 25.75, 11.25.

[0140] (3) Synthesis of fluorescent substrate (E)-3-(7-(2-(1H-indol-3-yl)vinyl)-5,5-difluoro-9-methyl-5H-5l4,6l4-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-3-yl)-N-(4-hydroxyphenethyl)propanamide (BDP-F)

[0141] Take 25 ml of double mouth bottle, add 60 mg of BDP-A and 318 mg of 3-indole formaldehyde, protect with argon, add 2 ml of anhydrous ethanol with syringe, stir uniformly at room temperature. Add 131 mg of acetic acid and 647 mg of pyrrolidine with syringe in turn, stir for 15 minutes, transfer the reaction solution to a single mouth bottle, vacuum concentration and drying, separate and purify by flash column chromatography (DCM:MeOH = 50:1), recrystallize with petroleum ether to obtain BDP-F solid about 38 mg, yield about 48%.

[0142]

[0143] 1H NMR (600 MHz, Methanol-d4) δ 8.02 (dd, J = 6.7, 1.9 Hz, 1H), 7.81 (d, J = 16.2 Hz, 1H), 7.68 (s, 1H), 7.62 (d, J = 16.2 Hz, 1H), 7.45 - 7.43 (m, 1H), 7.25 - 7.19 (m, 3H), 7.03 (d, J = 8.4 Hz, 2H), 6.93 (s, 1H), 6.84 (d, J = 3.9 Hz, 1H), 6.70 (d, J = 8.5 Hz, 2H), 6.22 (d, J = 3.9 Hz, 1H), 3.37 (t, J = 7.4 Hz, 2H), 3.27 (t, J = 7.7 Hz, 2H), 2.96 (t, J = 7.3 Hz, 2H), 2.62 (t, J = 7.7 Hz, 2H), 2.32 (s, 3H). 13 C NMR (151 MHz, Methanol-d4) δ 175.20, 161.52, 157.10, 139.64, 136.65, 131.81, 131.55, 131.01, 126.73, 126.28, 124.28, 122.56, 121.38, 117.85, 116.63, 116.48, 116.30, 114.96, 113.40, 49.79, 42.61, 36.58, 35.99, 25.92, 11.50.

[0144] Optical properties of BDP-A, BDP-B, BDP-C

[0145] (1) Relative fluorescence quantum yield of BDP-A, BDP-B and BDP-C

[0146] The UV maximum absorption wavelength (λ abs ), fluorescence maximum emission wavelength (λ em ), Stoke’s shift and fluorescence quantum yield (Φ) were evaluated. A certain amount of fluorescent substrate BDP-A, BDP-B and BDP-C was accurately weighed using an analytical balance, dissolved with dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution, and stored in a -7 °C refrigerator in the dark. 3 μL of the above stock solution was diluted to 3 mL with H2O and dimethyl sulfoxide (DMSO) to obtain a 10 μM solution, and its UV spectrum was determined using a UV spectrophotometer. The 10 μM fluorescent substrate solution was diluted to obtain a 1 μM fluorescent substrate solution, and its fluorescence spectrum was determined using a fluorescence spectrometer. The fluorescence quantum yield was determined by reference method, and calculated according to the following formula:

[0147]

[0148] Φ probe and Φ standard represent the fluorescence quantum yield of the test fluorescent substrate molecule and the reference standard, respectively; F porbe and F standard represent the integrated fluorescence intensity of the test fluorescent substrate molecule and the reference standard, respectively; A probe and A standard represent the absorbance of the test substance and the reference substance at the excitation wavelength, respectively; η represents the refractive index of the solvent. The results show that the excitation wavelength of BDP-A and BDP-B in aqueous solvent is 503 nm, and the emission wavelength is 512 nm and 513 nm, respectively. The fluorescence quantum yield of BDP-A is 0.97, the fluorescence quantum yield of BDP-B is 0.07, and the fluorescence quantum yield of BDP-C is 0.04. BDP-A has a relatively high fluorescence quantum yield (Table 1). Therefore, BDP-A is selected for subsequent verification.

[0149] Table 1 Basic spectral properties of fluorescent substrates

[0150]

[0151] (2) Light stability test of BDP-A, BDP-B and BDP-C

[0152] Take 96-well plates, add 1 μM fluorescent substrate solution to each plate, 6 replicates, numbered 0, 1, 2, 3, 4, 5, and irradiate with 520 nm green light for 0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours and 4 hours, respectively. After irradiation, the fluorescence intensity was measured using a multifunctional microplate analysis system. The related fluorescence intensity changes of compound A, B and C PBS solution (1 μM) under green light irradiation for 0 to 150 minutes are shown in Figure 1 After 150 minutes of green light irradiation, their related fluorescence intensity is still higher than 0.95, and almost does not decrease with the increase of irradiation time, indicating that compound A, B and C have good light stability.

[0153] (3) Fluorescence intensity response of BDP-A, BDP-B and BDP-C to different amino acids and metal ion solutions

[0154] In order to exclude the interference of some biological factors, the fluorescence response of compound A, B and C (1 μM) to different metal ions (10 μM) and amino acids (10 μM) in solution state was determined.

[0155] The results are shown in Figure 2The fluorescence intensity of compounds A, B and C after interacting with different biological factors from top to bottom, and the probe represents the addition of only fluorescent substrate dye, metal ions or amino acids in the solution. The three compounds do not have fluorescence response to glutamic acid, methionine, cysteine, arginine, serine, aspartic acid, alanine and cuprous chloride, ferric chloride, potassium chloride, palladium chloride, sodium sulfate, aluminum chloride, zinc chloride, cadmium chloride and magnesium sulfate, excluding the non-specificity of BDP-A, BDP-B and BDP-C in biological reaction. It can be seen that there is no fluorescence response phenomenon for the different biological factors tested for compounds A, B and C, that is, the fluorescence intensity of the three compounds does not change significantly, neither enhanced nor quenched, thus excluding the influence of different biological factors on fluorescence imaging in the whole tyramide signal amplification process.

[0156] Example 6 Optical properties of BDP-D, BDP-E and BDP-F

[0157] (1) Relative fluorescence quantum yield of BDP-D, BDP-E and BDP-F

[0158] The ultraviolet maximum absorption wavelength (λ abs ), fluorescence maximum emission wavelength (λ em ), stoke shift and fluorescence quantum yield (Φ) were evaluated. Using an analytical balance, a certain amount of compound BDP-D, BDP-E and BDP-F was accurately weighed and dissolved with dimethyl sulfoxide (DMSO) to obtain a 10 mM stock solution, which was stored in a -7 °C refrigerator in the dark. Take 3 μL of the above stock solution and dilute to 3 mL with different organic solvents acetone (Acetone), N,N-dimethylformamide (DMF), acetonitrile (ACN), tetrahydrofuran (THF), PBS, ethanol (EtOH), dimethyl sulfoxide (DMSO) and dichloromethane (DCM) to obtain a 10 μM solution. The ultraviolet spectrum was determined by ultraviolet spectrophotometer. The 10 μM fluorescent substrate solution was diluted to obtain a 1 μM fluorescent substrate solution, and the fluorescence spectrum was determined by fluorescence spectrophotometer. The fluorescence quantum yield was determined by reference method, and calculated according to the following formula:

[0159]

[0160] Φ probe and Φ standard represent the fluorescence quantum yield of the fluorescent substrate molecule to be tested and the reference standard substance, respectively; F porbe and F standard represent the integral fluorescence intensity of the fluorescent substrate molecule to be tested and the reference standard substance, respectively; A probe and Astandard respectively represent the absorbance of the incident light at the excitation wavelength of the test substance and the reference substance; η represents the refractive index of the solvent; in the actual experiment, rhodamine 6G was used as a standard, and its fluorescence quantum yield (Φ) in ethanol was 0.94. The results showed that in the PBS solvent, the excitation light wavelengths of BDP-D, BDP-E and BDP-F were 594 nm, 580 nm and 594 nm, respectively, and the emission wavelengths were 750 nm, 604 nm and 633 nm, respectively. The fluorescence quantum yield of BDP-D was <0.01, the fluorescence quantum yield of BDP-E was 0.07, and the fluorescence quantum yield of BDP-F was 0.26. BDP-F had a relatively high fluorescence quantum yield (Table 2). From the applicability of fluorescence microscopy, when using a Red filter to detect, it is more suitable to use BDP-F, and its excitation light wavelength and emission light wavelength are more suitable. Therefore, for the Red fluorescence channel, BDP-F is selected for subsequent verification.

[0161] Table 2 Basic spectral properties of fluorescent substrates

[0162]

[0163]

[0164] (2) Light stability test of BDP-D, BDP-E and BDP-F

[0165] Take 96-well plates, add 1 μM fluorescent substrate solution to each plate, 6 replicates, numbered 0, 1, 2, 3, 4, 5, and irradiate with 520 nm green light for 0 hours, 0.5 hours, 1 hour, 2 hours, 3 hours and 4 hours, respectively. After irradiation, the fluorescence intensity was measured by using a multifunctional microplate analysis system. The related fluorescence intensity changes of compounds D, E and F were as shown in Figure 3 After irradiation with green light for 150 minutes, their related fluorescence intensity was still higher than 0.95, and almost did not decrease with the increase of irradiation time, indicating that compounds D, E and F had good light stability.

[0166] (3) Investigation of the fluorescence intensity changes of BDP-D, BDP-E and BDP-F in different amino acid solutions and metal solutions

[0167] To exclude the interference of some biological factors (Glu, Met, Cys, Arg, Ser, Asp, Ile, Ala, CuCl, FeCl3, KCl, PdCl2, NaSO4, AlCl3, ZnCl2, MgSO4), the fluorescence response of compounds D, E and F (1 μM) to different metal ions (10 μM) and amino acids (10 μM) in solution was determined, and the results are shown in Figure 2. Figure 4 From top to bottom, the relevant fluorescence intensities of compounds D, E and F after interaction with different biological factors are shown, and probe indicates the addition of only fluorescent substrate dye, metal ions or amino acids in solution. No fluorescence response was observed for different biological factors tested for compounds D, E and F, that is, the fluorescence intensity of the three compounds did not change significantly for the different biological factors tested, neither enhanced nor quenched, thus excluding the influence of different biological factors on fluorescence imaging in the entire tyramide signal amplification process.

[0168] Example 7 Reliability of fluorescent substrate molecules in tyramide signal amplification

[0169] This example studies the feasibility of implementing staining of fluorescent substrate molecules in tyramide signal amplification technology.

[0170] Experimental method:

[0171] Experimental group:

[0172] (1) Sample sealing: Take SKBR3 (human breast adenocarcinoma cells, cell concentration 3 x 10 5 6 / ml, 1 ml / portion) cell samples, add blocking solution, mix well, and incubate the cell samples at 37°C for 10-30 minutes; remove the sample, centrifuge to remove the supernatant and collect the cells, resuspend the cells in 1% FBS PBS solution, centrifuge to remove the supernatant and collect the cells, resuspend the cells in 1% FBS PBS solution, centrifuge to remove the supernatant and collect the cells, resuspend the cells in 1% FBS PBS solution, and divide into 100 μl / portion for standby.

[0173] (2) Antibody incubation: add 100 μl / portion of incubation antibody to the cell solution obtained in step (1) according to a certain use concentration, incubate overnight at 2-8°C, resuspend the cells in 1% FBS PBS solution, centrifuge to remove the supernatant and collect the cells, resuspend the cells in 1% FBS PBS solution, centrifuge to remove the supernatant and collect the cells, obtain the antibody-cell complex sample, and resuspend the cells in 100 μl of 1% FBS PBS solution for standby.

[0174] (3) Fluorescent substrate incubation: add 100 μl of fluorescent substrate solution to the cell solution obtained in step (2) at a certain use concentration, mix well, and incubate at 37°C for a period of time. Add 1 mL of 1% FBS PBS solution to resuspend the cells, centrifuge to remove the supernatant and collect the cells, add 1 mL of 1% FBS PBS solution to resuspend the cells, centrifuge to remove the supernatant and collect the cells, obtain the cell complex sample, add 100 μl of 1% FBS PBS solution to resuspend the cells, take samples, add DAPI staining, and then use a fluorescence microscope to detect and observe the results.

[0175] Control group 1: (SKBR3-fluorescent secondary antibody):

[0176] Steps (1) and (2) are the same as the experimental group.

[0177] (3) Add 100 μl of fluorescent secondary antibody solution to the cell solution obtained in step (2) at a certain use concentration, mix well, and incubate at 37°C for a period of time. Add 1 mL of 1% FBS PBS solution to resuspend the cells, centrifuge to remove the supernatant and collect the cells, add 1 mL of 1% FBS PBS solution to resuspend the cells, centrifuge to remove the supernatant and collect the cells, remove the supernatant and collect the cells, and use a fluorescence microscope to detect and observe the results.

[0178] Control group 2: (WBC-fluorescent substrate):

[0179] Replace the SKBR3 cells in step (1) of the experimental group with white blood cells (cell concentration 3 x 10 5 cells / ml, 1 ml per portion), and the other steps remain unchanged.

[0180] Control group 3: (WBC-fluorescent secondary antibody)

[0181] Replace the SKBR3 cells in step (1) of the control group 1 with white blood cells (cell concentration 3 x 10 5 cells / ml, 1 ml per portion), and the other steps remain unchanged.

[0182] I. Detection and verification of BDP-A

[0183] After the cells were blocked with 0.5% H2O2, HRP-labeled HER2 antibody was added for incubation (using a concentration of 4 μg / ml), and then BDP-A or fluorescent secondary antibody (anti-HRP IgG Alexa Fluor 488, Jackson ImmunoResearch, item number 123-545-021) was added for incubation (using concentrations of 0 μg / ml, 0.15 μg / ml, 0.3 μg / ml, 0.6 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, respectively), and the corresponding detection results were obtained according to the steps (1)-(3) of the experimental group and control experimental groups 1-3 (Table 3).

[0184] Table 3 Detection verification of BDP-A fluorescent substrate

[0185]

[0186] The results (Table 3) show that, using the BDP-A fluorescent substrate test, the average fluorescence intensity value of the fluorescent substrate in the test group is higher than that in the secondary antibody test group under the same concentration; when the concentration is 0.15-10 μg / mL, the difference between the test group and the control group is relatively optimal, and when the concentration is 5 μg / mL, the difference between the test group and the control group is the largest, but when the concentration is greater than 1.25 μg / mL, the control group also has a certain detection signal (fluorescence value greater than 30), therefore, the preferred concentration of BDP-A is 0.6 μg / mL.

[0187] II. Detection verification of BDP-F

[0188] After the cells were blocked with 0.5% H2O2, HRP-labeled HER2 antibody was added for incubation (using a concentration of 4 μg / ml), and then BDP-F or fluorescent secondary antibody (anti-HRP IgG Alexa Fluor 594, Jackson ImmunoResearch, item number 123-585-021) was added for incubation (using concentrations of 0 μg / ml, 0.15 μg / ml, 0.3 μg / ml, 0.6 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, respectively), and the corresponding detection results were obtained according to the steps (1)-(3) of the experimental group and control experimental groups 1-3 (Table 4).

[0189] Table 4 Detection verification of BDP-F fluorescent substrate

[0190]

[0191] The results show (Table 4) that, using the BDP-F fluorescent substrate test, the average fluorescence intensity value of the fluorescent substrate in the test group is higher than that in the secondary antibody test group under the same concentration; when the concentration is 0.6-10 μg / mL, it has a higher signal-to-noise ratio, and when the concentration is 5 μg / mL, the difference between the test group and the control group is the largest, but when the concentration is greater than 2.5 μg / mL, the control group also has a certain detection signal. According to the fluorescence microscope used in this embodiment, the fluorescence value is lower than 30, and the naked eye cannot observe the fluorescence signal, therefore, the preferred concentration of BDP-F is 1.25 μg / mL.

[0192] III. Verification of fluorescent substrate in cells with different protein expression levels

[0193] (1) Verification of BDP-A in cells with different protein expression levels

[0194] FITCT, BDP-A and fluorescent secondary antibody groups were used to detect and verify the tyrosine signal amplification of cell samples with different protein expression levels, including SKBR3 (human breast cancer cells, HER2 high expression), ZR-75-1 (human breast cancer cells, HER2 low expression), MCF7 (human breast cancer cells, HER2 low expression), A549 (human lung adenocarcinoma cells, HER2 low expression), and WBC cells; the fluorescence intensity expression of different cell samples under three (two) different methods was compared.

[0195] FITCT control experiment group:

[0196] After the cells were blocked with 0.5% H2O2, HRP-labeled HER2 antibody was added for incubation (the concentration used was 4 μg / ml), then fluorescent substrate FITCT was added for incubation (the concentration used was 5 μg / ml (the concentration of fluorescent substrate BDP-A used was 0.6 μg / ml)), and SKBR3, ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells were used as cell sample replacement groups instead of SKBR3 cells in step (1), and the corresponding detection results were obtained according to the above steps (1)-(3).

[0197] Fluorescent secondary antibody experiment group:

[0198] After the cells are blocked with 0.5% H2O2, HRP-labeled HER2 antibody is added for incubation (a concentration of 4 μg / ml is used), and then a secondary antibody is added for incubation (a concentration of 5 μg / ml (anti-HRP IgG Alexa Fluor 488, Jackson ImmunoResearch, item number 123-545-021) is used), and the SKBR3 cells in step (1) of the control experiment group are replaced with SKBR3, ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells; the remaining steps remain unchanged.

[0199] Table 5 Experimental settings and results of detection of different expression levels of protein markers

[0200]

[0201] The results show (Table 5 and Figure 5 ), in the low expression cell lines of the protein marker, the detection signal of the fluorescent substrate BDP-A detection system is much higher than that of the fluorescent secondary antibody detection system and the FITC fluorescent substrate group, and in the non-expressing cell line (WBC), both are negative. Therefore, using the fluorescent substrate detection system of the present disclosure (especially the BDP-A fluorescent substrate) has higher detection sensitivity while not affecting specificity.

[0202] (2) Verification of BDP-D, BDP-E, and BDP-F in different protein expression level cell lines

[0203] The tyrosine signal amplification detection verification of different protein expression level cell samples is performed using BDP-D, BDP-E, BDP-F, and the fluorescent secondary antibody group, respectively, and the cell samples include SKBR3, ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells; the fluorescence intensity expressions of different cell samples under three (two) different methods are compared.

[0204] Experimental group:

[0205] After the cells are blocked with 0.5% H2O2, HRP-labeled HER2 antibody is added for incubation (a concentration of 4 μg / ml is used), and then a secondary antibody is added for incubation (a concentration of 5 μg / ml (anti-HRP IgG Alexa Fluor 488, Jackson ImmunoResearch, item number 123-545-021) is used), and the SKBR3 cells in step (1) of the control experiment group are replaced with SKBR3, ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells; the remaining steps remain unchanged.

[0206] Fluorescent secondary antibody experimental group:

[0207] After the cells are blocked with 0.5% H2O2, HRP-labeled HER2 antibody is added for incubation (a concentration of 4 μg / ml is used), and a secondary antibody is added for incubation (a concentration of 5 μg / ml (anti-HRP Alexa Fluor 594, from Jackson ImmunoResearch, item number: 123-585-021) is used), and the SKBR3 cells in step (1) of the control experiment group 1 are replaced with SKBR3, ZR-75-1 (human breast cancer cells), MCF7 (human breast cancer cells), A549 (human lung adenocarcinoma cells), and WBC cells; and the remaining steps remain unchanged.

[0208] Table 6: Experimental settings and results of detection of different expression levels of protein markers

[0209]

[0210] The results show (Table 6 and Figure 6 ), in the low expression cell lines of the protein marker, the detection signal of the fluorescent substrate detection system (BDP-D, BDP-E, BDP-F) is much higher than that of the fluorescent secondary antibody detection system, and in the non-expressing cell line (WBC), both are negative. Therefore, using the fluorescent substrate detection system has higher detection sensitivity while not affecting the specificity.

[0211] FITCT, secondary antibody, and the fluorescent substrate provided by the present application are all based on the fluorescent staining detection scheme of the HRP-labeled antibody. The antibody combination used in the present scheme is HRP-labeled HER2 antibody as the incubation antibody, in which SKBR3 is a HER2 high expression cell, ZR-75-1, MCF7, and A549 are HER2 low expression cells, and WBC white blood cells are HER2 negative cells. As can be seen from the figure, in the HER2 high expression SKBR3, the fluorescent substrates BDP-A, BDP-D, BDP-E, and BDP-F are uniformly dyed, and the fluorescence intensity is high; at the same time, in the HER2 low expression cell lines ZR-75-1, MCF7, and A549, the amplification detection signal of the fluorescent substrate is obviously stronger than that of FITCT and the secondary antibody, and has higher sensitivity.

[0212] Four, comparison of the fluorescent detection stability of FITCT, BDP-A, and BDP-F

[0213] SKBR3 cell samples were detected by tyrosine signal amplification using FITCT, DBP-A, and BDP-F, respectively. After the cells were blocked with 0.5% H2O2, HRP-labeled HER2 antibody was added for incubation (at a concentration of 4 μg / ml), and then fluorescent substrate FITCT was added for incubation (at a concentration of 5 μg / ml (the concentration of fluorescent substrate BDP-A was 0.6 μg / ml, and the concentration of fluorescent substrate BDP-F was 1.25 μg / ml)). The labeled cell samples were obtained according to the above steps (1)-(3), and 20 repeated scans were performed using a fluorescence microscope to observe the fluorescence signal decay of the samples (). Figure 7 The results showed that BDP-A and BDP-F had less fluorescence signal intensity decay than FITCT, and the fluorescence signal intensity value of BDP-A was the most stable. Therefore, BDP-A and BDP-F have obvious advantages over FITCT.

[0214] Comparative Example

[0215] FITCT fluorescent substrate detection

[0216] The cells were blocked with 0.5% H2O2, HRP-labeled HER2 antibody was added for incubation (at a concentration of 4 μg / ml), and then FITCT (manufacturer: AAT bioquest, product number: 11062, CAS number: 210236-90-1, absorption wavelength near 488 nm; the concentration of FITCT was 0.6 μg / ml, 1.25 μg / ml, 2.5 μg / ml, 5 μg / ml, and 10 μg / ml, respectively) was added. The corresponding detection results were obtained according to the above experimental group and control experimental group 2 steps (1)-(3) (Table 7).

[0217] Table 7 FITCT fluorescent substrate detection verification

[0218]

[0219]

[0220] The results show that the fluorescence intensity of the control group is less than 30 when the concentration of FITCT is 0.6 μg / mL-5 μg / mL, and the background interference caused by FITCT is small at this concentration. Meanwhile, in the test group, the fluorescence intensity is obviously different from that of the control group, which is 54.10 at 0.6 μg / mL, 74.15 at 1.25 μg / mL, and 118.40 at 2.5 μg / mL, respectively, and the TSA amplification detection can be realized. While the fluorescence substrate with the same absorption wavelength near 488 nm, the fluorescence intensity of BDP-A provided by the application is 100.25 when the concentration is 0.15 μg / mL (Table 3-test group), which is equivalent to the fluorescence intensity of FITCT when the concentration is 2.5 μg / mL. Therefore, the compound provided by the application can realize TSA amplification detection at a lower concentration, which has a significant advantage.

Claims

1. A compound represented by formula (I), (I) in, R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogens, C1-C9 alkyl groups, and C1-C9 alkoxy groups; R3 is selected from hydrogen, halogen, C1-C9 alkyl, C1-C9 alkoxy, -C1-C9 alkylene-unsubstituted aryl or substituted aryl, -C1-C9 alkylene-unsubstituted heteroaryl or substituted heteroaryl, -enyl-unsubstituted aryl or substituted aryl, -enyl-unsubstituted heteroaryl or substituted heteroaryl; the enyl group is selected from C2-C9 enyl groups; the aryl group is selected from phenyl, naphthyl, 2,3-dihydro-1H-indenyl and biphenyl; the heteroaryl group is selected from 5- or 6-membered monocyclic aromatic rings or 7- to 12-membered bicyclic aromatic rings; wherein the substituted aryl or heteroaryl group is substituted by 1 to 3 substituents independently selected from the following groups: halogen, C1-C3 alkyl, C1-C3 alkoxy, halo-C1-C3 alkyl, halo-C1-C3 alkoxy.

2. The compound according to claim 1, wherein, R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogens, C1-C3 alkyl groups, and C1-C3 alkoxy groups.

3. The compound according to claim 1 or 2, wherein, R3 is selected from C1-C9 alkyl, C1-C9 alkoxy, -eneyl-unsubstituted aryl or substituted aryl, -eneyl-unsubstituted heteroaryl or substituted heteroaryl.

4. The compound according to claim 1 or 2, wherein, R3 is a C1-C3 alkyl or C1-C3 alkoxy group.

5. The compound according to claim 1 or 2, wherein, The subalkenyl group is a C2-C5 subalkenyl group.

6. The compound according to claim 5, wherein, The imenoyl group is selected from -CH=CH-CH=CH-CH2-, -CH=CH-CH2- and -CH=CH-.

7. The compound according to claim 1 or 2, wherein, The compound is one of compounds shown in A, B, or C. 。 8. A compound represented by formula (II), (II) in, R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogens, C1-C9 alkyl groups, and C1-C9 alkoxy groups; A is selected from unsubstituted heteroaryl or substituted heteroaryl and unsubstituted aryl or substituted aryl; the aryl is selected from phenyl, naphthyl, 2,3-dihydro-1H-indenyl and biphenyl; the heteroaryl is selected from 5- or 6-membered monocyclic aromatic ring or 7- to 12-membered bicyclic aromatic ring; the substituted aryl or heteroaryl is substituted by 1 to 3 substituents independently selected from the following groups: halogen, C1-C3 alkyl, C1-C3 alkoxy, halo-C1-C3 alkyl, halo-C1-C3 alkoxy.

9. The compound according to claim 8, wherein, R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogens, C1-C3 alkyl groups, and C1-C3 alkoxy groups.

10. A compound represented by formula (II), (II) in, R1, R2, R4, R5, and R6 are independently selected from hydrogen, halogens, C1-C9 alkyl groups, and C1-C9 alkoxy groups; A is 2,4-dimethoxy-phenyl, 4-N,N-dimethylaminophenyl, or indole.

11. The compound according to claim 10, wherein the compound is a compound represented by D, E, or F. 。 12. A reagent combination comprising any one of claims 1-11.

13. The reagent combination according to claim 12, wherein, The reagent combination also includes enzyme-labeled antibodies.

14. The reagent combination according to claim 13, wherein, The enzyme is selected from any one of dehydrogenase, oxidase, peroxidase, and oxygenase.

15. The reagent combination according to claim 13, wherein, The enzyme is any one of horseradish peroxidase, catalase, or superoxide dismutase.

16. The reagent combination according to claim 15, wherein, The enzyme in question is horseradish peroxidase.

17. The reagent combination according to any one of claims 13-16, wherein, The antibody is one or more of the following: anti-HER2 antibody, anti-Trop2 antibody, anti-Claudin18.2 antibody, anti-HER3 antibody, anti-MUC1 antibody, anti-PD-L1 antibody, anti-EpCAM antibody, anti-EGFR antibody, anti-c-met antibody, anti-folate receptor antibody, anti-CEA antibody, anti-PMSA antibody, anti-AR-V7 antibody, anti-CK antibody, anti-PLAP antibody, anti-GPC3 antibody, anti-CD31 antibody, anti-CD44 antibody, anti-Vimentin antibody, and anti-cadherin antibody.

18. The reagent combination according to claim 12 or 13, wherein, The reagent combination also includes a sealing agent.

19. The reagent combination according to claim 18, wherein, The sealing agent is a peroxide.

20. The reagent combination according to claim 19, wherein, The peroxide is H2O2.

21. The reagent combination according to claim 18, wherein, The reagent combination also includes a buffer solution.

22. The reagent combination according to claim 21, wherein, The buffer solution is a PBS solution, a Tris-HCl solution, or a Hepes solution.

23. The reagent combination according to claim 22, wherein, The buffer solution is a PBS solution containing FBS or BSA, or a Tris-HCl solution or a Hepes solution.

24. A kit comprising a compound according to any one of claims 1-11 or a combination of reagents according to any one of claims 12-23.

25. A method for labeling a molecular target in vitro for non-disease diagnostic and therapeutic purposes, the method comprising the step of contacting the molecular target with a compound of any one of claims 1-11 or a combination of reagents of any one of claims 12-23.

26. The method according to claim 25, wherein, The concentration of the compound is at least 0.05 μg / mL.

27. The method according to claim 25, wherein, The concentration of the compound is at least 0.1 μg / mL.

28. The method according to claim 25, wherein, The concentration of the compound is at least 0.15 μg / mL.

29. The method according to claim 25, wherein, The concentration of the compound is 0.15 μg / mL to 2.5 μg / mL.

30. The method according to claim 25, wherein, The concentration of the compound is 0.3 μg / mL to 1.25 μg / mL.

31. The method according to claim 25, wherein, The concentration of the compound is 0.6 μg / mL to 1.25 μg / mL.

32. A method for imaging a sample in vitro for non-disease diagnostic and therapeutic purposes, the method comprising contacting the sample with a compound of any one of claims 1-11 or a combination of reagents of any one of claims 12-23.

33. The method according to claim 32, wherein, The sample is a sample containing cells or cell fragments.

34. The method according to claim 33, wherein, The samples are blood, plasma, urine, saliva, pleural effusion, ascites, cerebrospinal fluid, or tissue digestion samples.

35. The method according to any one of claims 32-34, comprising the following steps: (1) Sample sealing: Take a sample, add sealing agent, incubate, and obtain a sealed sample; (2) Antibody incubation: Mix the enzyme-labeled antibody with the blocked sample obtained in step (1) and incubate to obtain an incubated antibody-sample complex solution; (3) Substrate incubation: Contact the compound of any one of claims 1-22 or the reagent combination of any one of claims 23-23 with the incubation antibody-sample complex solution obtained in step (2) and incubate to obtain a fluorescent substrate-incubation antibody-sample complex solution; (4) Sample imaging: The fluorescent substrate-incubated antibody-sample complex was dropped onto a glass slide, dried, stained with DAPI, and then scanned and analyzed using a fluorescence microscope.

36. The method according to any one of claims 32-34, wherein, The concentration of the compound is at least 0.05 μg / mL.

37. The method according to any one of claims 32-34, wherein, The concentration of the compound is at least 0.1 μg / mL.

38. The method according to any one of claims 32-34, wherein, The concentration of the compound is at least 0.15 μg / mL.

39. The method according to any one of claims 32-34, wherein, The concentration of the compound is 0.15 μg / mL to 2.5 μg / mL.

40. The method according to any one of claims 32-34, wherein, The concentration of the compound is 0.3 μg / mL to 1.25 μg / mL.

41. The method according to any one of claims 32-34, wherein, The concentration of the compound is 0.6 μg / mL to 1.25 μg / mL.

42. Use of the compound of any one of claims 1-11, the reagent combination of any one of claims 12-23, or the kit of claim 24 in the preparation of a fluorescence detection reagent or a cell imaging reagent.

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