1. A small molecular fluorescent probe targeting a mast cell MrgX2 receptor is characterized in that the structural formula of the probe is shown as follows;

wherein R represents a fluorescent chromophore containing carboxyl, and n represents 2-5.

2. The small molecule fluorescent probe for targeting the receptor of mast cells MrgX2 according to claim 1, wherein the fluorescent chromophore containing a carboxyl group is a coumarin-based fluorescent chromophore, a fluorescein-based fluorescent chromophore, or borondipyrolylic acid.

3. The small molecule fluorescent probe for targeting receptors of mast cells MrgX2 according to claim 1, wherein coumarin-based fluorescent chromophores, fluorescein-based fluorescent chromophores and borondipyrrololic acid have the following structural formula:

wherein R is₁Is a hydrogen atom, a methoxy group or an aminoalkyl group; r₂、R₃Is chlorine atom, bromine atomA proton or a fluorine atom.

4. A preparation method of a small molecular fluorescent probe targeting a mastocyte MrgX2 receptor is characterized in that according to the molar ratio of amino-substituted phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths to a fluorescent chromophore to propyl phosphoric anhydride being 1: 1, the amino-substituted phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths, the carboxyl-containing fluorescent chromophore and the propyl phosphoric anhydride are added into DCM for reaction to obtain the fluorescent probe; the structural formula of the fluorescent probe is as follows:

wherein R represents a fluorescent chromophore containing carboxyl, and n represents 2-5.

5. The method for preparing a small molecular fluorescent probe targeting a mast cell MrgX2 receptor according to claim 4, wherein the amino-substituted phenyl-substituted pyrazolopyrimidine derivative with 2-5 different carbon chain lengths is prepared by the following steps:

1) dissolving a methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative in DDM, adding boron tribromide, and demethylating to obtain (R) -4- (7- (3- (dimethylamino) pyrrolidine-1-yl) pyrazolo [1,5-a ] pyrimidine-5-yl) phenol;

2) adding tert-butyl (bromoethyl) carbamate, tert-butyl (bromopropyl) carbamate, tert-butyl (bromobutyl) carbamate or tert-butyl (bromopentyl) carbamate into (R) -4- (7- (3- (dimethylamino) pyrrolidine-1-yl) pyrazolo [1,5-a ] pyrimidine-5-yl) phenol for reaction to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and tert-butyl carbamate substitution at the tail end;

3) removing amino protection from phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and with the terminal substituted by tert-butyl carbamate in hydrochloric acid by using ethyl acetate as a solvent to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and substituted by amino.

6. The method for preparing a small molecule fluorescent probe targeting a mast cell MrgX2 receptor according to claim 5, wherein the methoxy substituted phenyl substituted pyrazolopyrimidine derivative is prepared by the following steps:

1) reacting 4-methoxybenzoyl ethyl acetate with 3-aminopyrazole in an acetic acid solution to obtain 5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7 (4H) -one;

2) with POCl₃Substituted 5- (4-methoxyphenyl) pyrazolo [1,5-a as chlorinating reagent]Carbonyl of pyrimidin-7 (4H) -one to give 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a]A pyrimidine;

3) dissolving 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidine in dioxane, adding DIEA, and reacting with (R) -3- (dimethylamino) pyrrolidine to obtain the methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative.

7. The method for preparing a small-molecule fluorescent probe targeting a mast cell MrgX2 receptor according to claim 4, wherein the fluorescent chromophore containing carboxyl is coumarin fluorescent chromophore, fluorescein fluorescent chromophore or borondipyrrololic acid.

8. The small molecule fluorescent probe for targeting receptors of mast cells MrgX2 according to claim 7, wherein coumarin-based fluorescent chromophores, fluorescein-based fluorescent chromophores and borondipyrrololic acid have the following structural formula:

wherein R is₁Is a hydrogen atom, a methoxy group or an aminoalkyl group; r₂、R₃Is a chlorine atom, a bromine atom or a fluorine atom.

9. Use of a small molecule fluorescent probe targeting a mast cell MrgX2 receptor according to claim 1 in the field of targeted identification of mast cells MrgX 2.

10. Use according to claim 9, characterized in that the use of small molecule fluorescent probes targeting the mast cell MrgX2 receptor for use in MrgX2 competitive ligand screening.

Background

Drug allergy is one of the clinically common adverse drug reactions, and can cause shock or death in severe cases, wherein the anaphylactoid reaction accounts for more than 70% of the drug allergy. The G protein-coupled receptor MrgX2 expressed in mast cells was shown to be closely related to the development of anaphylactoid reactions. Multiple endogenous bioactive peptides can activate MrgX2 in organisms, so that complex physiological regulation or allergic diseases in the organisms are caused; exogenous peptide drugs, micromolecular drugs, components in traditional Chinese medicine injection and the like can also activate mast cells through MrgX2 in clinical application, so that drug anaphylactic reaction is initiated, and the incidence rate of adverse reaction in clinical application is increased. Therefore, the MrgX2 and the ligand thereof have important meanings in allergic diseases and adverse drug reactions.

In pharmaceutical research, gas chromatography, liquid chromatography, and ultraviolet spectroscopy are basic techniques for performing pharmaceutical analysis. Various highly sensitive fluorescence assays, which have been developed in succession in particular in recent years, have been used effectively in the analysis of drugs in pharmaceutical preparations and in biological fluids since the 70 s of the 20 th century. For example, the synchronous scanning fluorescence technology can be used for measuring the amphetamine and the morphine plus quinine in contraband; the fluorescence polarization immunoassay method can be used for detecting various medicaments such as gentamicin, dibenzein, theophylline, pentobarbital, phenobarbital and the like in serum, and can also be used for carrying out pharmacokinetic research on the netilmicin in a burn patient; specific fluorescence polarization immunoassay can be used to monitor the whole blood concentration of cyclosporin a after kidney transplantation; the fluorescence quenching method is used for measuring the content of matrine and oxymatrine; the solid surface fluorometry is used for clinical fluorometric analysis of theophylline in serum and other antibacterial drugs, antiasthmatic drugs, anti-convulsion drugs and the like; measuring the content of carbamazepine in the tablet by an electrolytic fluorescence photometry method, and the like. Among them, the fluorescent probe technology has been developed as an important means for performing real-time detection at a molecular level by means of a fluorescent microscope. The technology has high sensitivity and strong visibility, and has little interference to the studied biological macromolecules or cells, thereby being widely applied.

The response of the molecular fluorescent probe to metal ions, biological micromolecules and biological macromolecules enables people to detect the concentration of molecules or ions in living cells and the change process of the structure of the biological macromolecules in real time by using a fluorescence microscope and a fluorescence spectrum, particularly a fluorescence imaging technology. Over the years, GPCR ligands with fluorescent properties have been widely used to localize receptor distribution and enable real-time monitoring of processes triggered by ligand-receptor interactions (e.g., internalization, transport, sequestration, and recovery). Since the ligand binding domain of GPCR is usually a high viscosity and low polarity environment, the development of environment-sensitive GPCR fluorescent ligand is more important. Fluorescent ligands with environmental sensitivity are very sensitive to changes in the physicochemical properties of the surrounding environment, showing weak fluorescence in aqueous solution, but can release bright fluorescence at low polarity or when bound to hydrophobic domains in cells. However, there have been no reports to date on the detection of small molecule fluorescent probes that detect the distribution of MrgX2 on the cell surface. In view of the advantages of high sensitivity, high selectivity, and rapid response of small molecule fluorescent probes, there is an urgent need to develop a convenient fluorescent ligand to track MrgX2 for further understanding its physiological and pathological functions.

Disclosure of Invention

The invention aims to provide a targeted mast cell MrgX2 small-molecule fluorescent probe and a preparation method and application thereof.

In order to realize the purpose, the invention is realized by the following technical scheme:

a small molecular fluorescent probe targeting a mast cell MrgX2 receptor, wherein the structural formula of the probe is shown as follows;

wherein R represents a fluorescent chromophore containing carboxyl, and n represents 2-5.

The invention further improves that the fluorescent chromophore containing carboxyl is coumarin fluorescent chromophore, fluorescein fluorescent chromophore or boron dipyrrolol acid fluoride.

The invention further improves the structural formula of coumarin fluorescent chromophore, fluorescein fluorescent chromophore and boron fluoride dipyrrolol acid as follows:

wherein R is₁Is a hydrogen atom, a methoxy group or an aminoalkyl group; r₂、R₃Is a chlorine atom, a bromine atom or a fluorine atom.

According to the mol ratio of amino-substituted phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths, a fluorescent chromophore and propyl phosphoric anhydride of 1: 1, adding the amino-substituted phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths, the carboxyl-containing fluorescent chromophore and the propyl phosphoric anhydride into DCM for reaction to obtain the fluorescent probe; the structural formula of the fluorescent probe is as follows:

wherein R represents a fluorescent chromophore containing carboxyl, and n represents 2-5.

The invention further improves that the amino-substituted phenyl-substituted pyrazolopyrimidine derivative with 2-5 different carbon chain lengths is prepared by the following steps:

1) dissolving a methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative in DDM, adding boron tribromide, and demethylating to obtain (R) -4- (7- (3- (dimethylamino) pyrrolidine-1-yl) pyrazolo [1,5-a ] pyrimidine-5-yl) phenol;

2) adding tert-butyl (bromoethyl) carbamate, tert-butyl (bromopropyl) carbamate, tert-butyl (bromobutyl) carbamate or tert-butyl (bromopentyl) carbamate into (R) -4- (7- (3- (dimethylamino) pyrrolidine-1-yl) pyrazolo [1,5-a ] pyrimidine-5-yl) phenol for reaction to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and tert-butyl carbamate substitution at the tail end;

3) removing amino protection from phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and with the terminal substituted by tert-butyl carbamate in hydrochloric acid by using ethyl acetate as a solvent to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and substituted by amino.

In a further development of the invention, methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative is prepared by the following process:

1) reacting 4-methoxybenzoyl ethyl acetate with 3-aminopyrazole in an acetic acid solution to obtain 5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7 (4H) -one;

2) with POCl₃Substituted 5- (4-methoxyphenyl) pyrazolo [1,5-a as chlorinating reagent]Carbonyl of pyrimidin-7 (4H) -one to give 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a]A pyrimidine;

3) dissolving 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidine in dioxane, adding DIEA, and reacting with (R) -3- (dimethylamino) pyrrolidine to obtain the methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative.

The invention is further improved in that the fluorescent chromophore containing carboxyl is coumarin fluorescent chromophore, fluorescein fluorescent chromophore or boron-dipyrrolol acid.

The invention further improves the structural formula of coumarin fluorescent chromophore, fluorescein fluorescent chromophore and boron fluoride dipyrrolol acid as follows:

wherein R is₁Is a hydrogen atom, a methoxy group or an aminoalkyl group; r₂、R₃Is a chlorine atom, a bromine atom or a fluorine atom.

The application of the small-molecule fluorescent probe targeting the receptor MrgX2 of the mast cell in the field of targeted recognition of the MrgX2 of the mast cell.

A further improvement of the invention resides in the use of a small molecule fluorescent probe targeting the mast cell MrgX2 receptor in a method for the competitive ligand screening of MrgX 2.

Compared with the prior art, the invention has the following beneficial technical effects:

the fluorescent probe targeting the mast cell MrgX2, which is constructed by the invention, takes (R) -ZINC-3573 as a basic molecular structure, introduces methoxy at the para-position of a benzene ring, introduces linear alkyl with the carbon atom number of 2-5 and an amino end through substitution reaction, and is connected with a fluorescent chromophore through amidation. The preparation method of the fluorescent probe is simple, easy to realize and high in yield.

The prepared fluorescent probe targeting the mast cell MrgX2 can be used as a targeting tool molecule, can be specifically combined with MrgX2 of the mast cell, and can be used for the identification and function research of MrgX2, the screening of ligands, the research of interaction mechanisms and the research of action mechanisms and action effects of drugs to be tested through the change of fluorescence properties.

Further, unlike conventional fluorescent chromophores, the fluorescent chromophore selected for use in the present invention is an environmentally sensitive coumarin chromophore that has low fluorescence intensity in aqueous solutions and releases bright fluorescence when bound to hydrophobic domains in cell membranes. Due to low fluorescence background interference and low toxicity, the real-time monitoring of living cells can be realized, so that the method is applied to functional research of MrgX 2.

Drawings

FIG. 1 is a fluorescence excitation spectrum and an emission spectrum of a fluorescent probe ZX1 with different concentrations; wherein, (a) is fluorescence excitation spectrum, and (b) is emission spectrum.

FIG. 2 shows the result of the agonist activity of fluorescent probe ZX1 on MrgX2 receptor; wherein, (a) is fluorescence excitation spectrum, and (b) is emission spectrum.

FIG. 3 is an imaging plot of the MrgX2 receptor in human primary mast cells, MrgX2-HEK293 cells and HMC-1 cells with fluorescent probe ZX 1.

Detailed Description

The invention will now be described in further detail with particular reference to the synthesis of fluorescent probes targeting mast cells, their fluorescent properties and their recognition by the mast cell MrgX2 in the examples which are given by way of illustration and not by way of limitation.

The invention provides a micromolecule fluorescent probe targeting a mast cell MrgX2 receptor, which is characterized in that a phenyl ring para-methoxy side chain in a phenyl-substituted pyrazolopyrimidine derivative is connected with fluorescent chromophores substituted by different carbon chain lengths, and the structural general formula of the fluorescent probe is shown as follows:

wherein, R represents a synthetic or commercially available fluorescent chromophore containing carboxyl, and is connected with phenyl-substituted pyrazolopyrimidine derivatives through condensation reaction with amino groups with different carbon chain lengths. n represents a carbon chain having 2 to 5 carbon atoms.

The fluorescent chromophore can be coumarins, fluorescein or Bodipy FL acid (borondipyrollic acid), and the structural formulas are respectively as follows:

wherein R is₁Is a hydrogen atom, a methoxy group or an aminoalkyl group; r₂、R₃Is a chlorine atom, a bromine atom or a fluorine atom.

The carboxyl-containing fluorescent chromophore can be connected with phenyl-substituted pyrazolopyrimidine derivatives through linear alkane with 2-5 carbon atoms and amino at the tail end.

A preparation method of a small-molecule fluorescent probe targeting a mast cell MrgX2 receptor comprises the following steps:

a. synthesis of phenyl-substituted pyrazolopyrimidine derivatives:

1) reacting 4-methoxybenzoyl ethyl acetate with 3-aminopyrazole in an acetic acid solution to obtain 5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7 (4H) -one;

2) with POCl₃Substituted 5- (4-methoxyphenyl) pyrazolo [1,5-a as chlorinating reagent]Carbonyl of pyrimidin-7 (4H) -one to give 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a]A pyrimidine;

3) dissolving 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidine in dioxane, adding DIEA, and reacting with (R) -3- (dimethylamino) pyrrolidine to obtain a methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative;

b. synthesis of phenyl-substituted pyrazolopyrimidine derivatives substituted with different carbon chain lengths:

1) dissolving the methoxy-substituted phenyl-substituted pyrazolopyrimidine derivative in DDM, adding boron tribromide, and demethylating to obtain (R) -4- (7- (3- (dimethylamino) pyrrolidine-1-yl) pyrazolo [1,5-a ] pyrimidine-5-yl) phenol;

2) adding tert-butyl (bromoethyl) carbamate, or tert-butyl (bromopropyl) carbamate, or tert-butyl (bromobutyl) carbamate, or tert-butyl (bromopentyl) carbamate into the product to react to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 carbon chain lengths and tert-butyl carbamate substituted at the tail end;

3) removing amino protection from phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and with tert-butyl carbamate as a terminal in hydrochloric acid by using ethyl acetate as a solvent to obtain phenyl-substituted pyrazolopyrimidine derivatives with 2-5 different carbon chain lengths and amino substitution;

c. synthesis of fluorescent probe:

phenyl-substituted pyrazolopyrimidine derivative, fluorescent chromophore and T, which are substituted by amino in DCM according to 2-5 different carbon chain lengths₃Adding phenyl-substituted pyrazolopyrimidine derivative with 2-5 different carbon chain lengths substituted by amino, fluorescent chromophore and propyl phosphoric anhydride (T) according to the molar ratio of 1: 1₃P) reacting at room temperature for 3h to obtain the fluorescent materialAn optical probe.

Example 1 Synthesis of fluorescent Probe ZX1 targeting mast cells MrgX2

1) The synthesis of phenyl substituted pyrazolopyrimidine derivative comprises the following synthetic route:

the specific synthesis steps are as follows:

synthesis of compound 5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7 (4H) -one (intermediate 2):

1.0g (12mmol) of 3-aminopyrazole is placed in a 50mL round-bottom flask, dissolved in 10mL of acetic acid and 2.7g (13mmol) of ethyl 4-methoxybenzoylacetate are added. After dissolution with stirring, the resulting reaction mixture was refluxed for 3 h. Cooled to room temperature, the solvent was evaporated, and the solid residue was diluted with EtOAc and filtered to give a white solid, i.e. 5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7 (4H) -one. The yield was 69%.

(ii) Synthesis of the Compound 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidine (intermediate 3):

1.0g (4.1mmol) of intermediate 2 are introduced into a 50mL round-bottom flask using 5mL (55mmol) of POCl₃The resulting solution was dissolved, and 0.25mL (3.1mmol) of pyridine was added thereto at room temperature. After stirring the reaction at room temperature for 3 days, the reaction mixture was taken up in 50mL Et₂O dilution and filtration. The solid obtained is again used in Et₂And (4) flushing. Then Et is added₂The O solutions were combined and cooled to 0 ℃ and poured into a mixture of ice and water. After separation, the organic layer was washed with water and brine, respectively. After the washing was completed, the organic layer was dried over sodium sulfate and the solvent was evaporated to give a pale yellow solid, i.e., 7-chloro-5- (4-methoxyphenyl) pyrazolo [1,5-a]A pyrimidine. The yield was 66%.

Synthesis of compound (R) -1- (5- (4-methoxyphenyl) pyrazolo [1,5-a ] pyrimidin-7-yl) -N, N-dimethylpyrrolidin-3-amine (intermediate 4):

100mg of intermediate 3(0.38mmol) were dissolved in 2mL of dioxane solution, and 0.19mL of DIEA was added(0.6mmol) and then 44mg (0.38mmol) of (R) -3- (dimethylamino) pyrrolidine were added. The resulting solution was stirred at room temperature for 16h, concentrated and purified by silica gel column to give intermediate 4. The yield was 61%. ESI-MS (M/z):338.19[ M]⁺.

2) The synthesis of 4- (3-aminopropoxy) phenyl pyrazole derivatives comprises the following synthetic route:

synthesis of compound (R) -4- (7- (3- (dimethylamino) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-5-yl) phenol (intermediate 5):

to a 100mL single-neck flask were added intermediate 4(150mg,0.45mmol) and 30mL of DCM, boron tribromide (550mg,2.3mmol) was added dropwise at 0 deg.C, and after 10 minutes of reaction, the temperature was raised to room temperature, and the reaction was stirred for 8 hours. The reaction mixture was added to 100ml of methanol at-78 ℃ in portions, 1ml of water was added, the solvent was removed under reduced pressure (water bath: 45 ℃), and the residue was purified by FLASH (MeOH: DCM (0.1% NH3) ═ 0-20%, 1h) to give (R) -4- (7- (3- (dimethylamino) pyrrolidin-1-yl) pyrazolo [1, 5-a) as a white solid product]Pyrimidin-5-yl) phenol. The yield was 83%. ESI-MS (M/z):324.30[ M]⁺.

(II) Synthesis of Compound (R) - (3- (4- (7- (3- (dimethylamino) pyrrolidin-1-yl) pyrazolo [1,5-a ] pyrimidin-5-yl) phenoxy) propyl) carbamic acid tert-butyl ester (intermediate 6):

to a 100mL single-necked flask were added intermediate 5(120mg, 0.37mmol), cesium carbonate (360mg, 1mmol), tert-butyl (bromopropyl) carbamate (180mg, 0.8mmol) and 10mL of DMF, and the reaction was stirred at room temperature for 6 hours. After the reaction was complete, 20mL of water was added for dilution. Extracted with ethyl acetate (50 mL. times.3) and the organic phases combined. The organic phase was dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure (water bath: 45 ℃), and the residue was purified by FLASH (MeOH: DCM (0.1% NH3) ═ 0-5%, 1h) to give the product as a white solid, i.e., (R) - (3- (4- (7- (3- (dimethylamino) pyrrolidin-1-yl) pyrazolo [1, 5-a)]Pyrimidin-5-yl) phenoxy) propyl) carbamic acid tert-butyl ester. The yield was 63%. ESI-MS (m/z) 481.45[ sic ], [ solution (c) ]M]⁺.

Synthesis of Compound (R) -1- (5- (4- (4- (3-aminopropoxy) phenyl) pyrazolo [1,5-a ] pyrimidin-7-yl) -N, N-dimethylpyrrolidin-3-amine (intermediate 7)

To a 100mL single-necked flask were added intermediate 6(110mg, 0.23mmol) and 10mL hydrochloric acid to ethyl acetate. The reaction was stirred at room temperature for 3 hours. After the reaction, the solvent was removed under reduced pressure (water bath: 45 ℃ C.) to obtain a white solid product, i.e., (R) -1- (5- (4- (4- (3-aminopropoxy) phenyl) pyrazolo [1,5-a]Pyrimidin-7-yl) -N, N-dimethylpyrrolidin-3-amine. The yield was 91%. ESI-MS (M/z):381.25[ M]⁺.

3) The synthesis of the small molecular fluorescent probe ZX1 is as follows:

to a 100mL single-necked flask was added intermediate 7(80mg, 0.22mmol), 7-diethylaminocoumarin-3-carboxylic acid (60mg,0.22mmol), DIEA (N, N-diisopropylethylamine, 120mg, 1.1mmol), and T₃P (50% EA) (140mg, 0.22mmol) to give a mixture, and the above mixture was mixed and added to 10ml of DCM, and the reaction was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure (water bath: 45 ℃) and the residue was purified by FLASH (MeOH: DCM (0.1% NH3) ═ 0-5%, 1h) to give the product as a yellow solid. The yield was 19%.

ESI-MS(m/z):647.31[M]⁺.¹H NMR(400MHz,MeOD)δ(ppm):8.59(s,1H),7.93-7.91(m,3H),7.52-7.50(d,1H),7.09-7.06(d,2H),6.79-6.76(dd,1H),6.55(s,1H),6.35(s,1H),6.22(s,1H),4.39-4.34(m,1H),4.19-4.16(m,3H),4.04-3.94(m,2H),3.65-3.62(m,2H),3.54-3.48(m,4H),3.12-3.08(m,1H),2.46(s,6H),2.37-2.34(m,1H)，2.14-2.09(m,2H),2.05-1.97(m,1H),1.24-1.20(m,6H).

The specific steps of the fluorescence spectrometry of the small molecular fluorescent probe with the receptor targeting the mast cell MrgX2 are as follows:

1) preparation of stock solution of substance to be tested

Respectively and accurately weighing the ZX1 as the test substance, dissolving in DMSODecomposing to obtain 1.0 × 10^-2mol/L stock solution.

2) Measurement of fluorescence spectrum of analyte

The fluorescence excitation and emission spectra of the above stock solutions were measured using a spectrophotometer model F-4500. The concentration of the analyte was adjusted to 4.0 × 10 before the test using PBS (pH 7.4) as a solvent according to the concentration of the stock solution^-6mol/L、2.0×10^{- 6}mol/L and 1.0X 10^-6mol/L. And (3) testing conditions are as follows: the sample cell is a 1cm × 1cm × 4cm quartz cuvette at room temperature, the slit width is 10nm, and the sensitivity is 2. The fluorescence excitation spectrum (left) and emission spectrum (right) of ZX1 are shown in FIGS. 1 (a) and (b), in which the abscissa is the wavelength and the ordinate is the relative fluorescence intensity. As can be seen from FIG. 1, the fluorescence excitation intensity and emission intensity of ZX1 increased with the increase of concentration. The maximum excitation wavelength is 437nm, and the maximum emission wavelength is 482 nm.

Calcium mobilization activity studies with small molecule fluorescent probes targeting mast cell MrgX2 receptors the specific steps were as follows:

1) a suspension of MrgX2-HEK293 cells at an appropriate concentration was mixed and seeded into 96-well plates at 100. mu.L per well. The inoculated cells were incubated at 37 ℃ with 5% CO₂Culturing for 24h under saturated humidity condition to make it adhere to the wall.

2) Before measurement, CIB buffer solution is preheated in advance in 37 ℃ water bath, the culture medium is discarded from the cells after adherence, and the cells are washed for 2 times by the preheated CIB. Adding 100 mu L (5 mu M) Fluo-3 calcium ion probe dye into each hole under the condition of keeping out of the sun, and placing the mixture in an incubator for incubation for 30 min;

3) after the incubation is finished, discarding the incubation liquid, adding a CIB buffer solution to wash for 2 times, and adding 50 mu L of CIB into each hole;

6) standing for 5min, sequentially adding 50 μ L of ZX1 with different concentrations into each well under a fluorescence microscope, and measuring the difference of fluorescence intensity of cells before and after addition.

The activity difference of ZX1 and the positive drug for activating the MrgX2 receptor is examined by a calcium mobilization experiment by taking (R) -ZINC-3573 as a positive control. Results as in fig. 2 (a) and (b), both positive compounds and ZX1 can cause activation of MrgX2 receptor to trigger an increase in intracellular calcium ion concentration. However, fromStructural differences, leading to differences in the activating abilities of the two compounds, (R) -ZINC-3573 and ZX1 activate MrgX2 calcium mobilised EC₅₀The values were 0.622. + -. 0.12. mu.M and 2.609. + -. 0.588. mu.M, respectively. It was shown that the introduction of a fluorophore in the (R) -ZINC-3573 structure resulted in a reduction of about 4-fold in its activity.

The specific steps of a cell imaging study with a fluorescent probe targeting the mast cell MrgX2 receptor are as follows:

1) centrifuging human primary mast cells, MrgX2-HEK293 cells and HMC-1 cells, diluting with PBS, mixing, inoculating into confocal imaging cell culture dish (20000 cells/dish), standing at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator overnight.

2) Before photographing, ZX1 was diluted to the corresponding concentration with PBS, added to a cell culture dish, and placed at 37 ℃ in 5% CO₂Culturing in constant temperature incubator for 5 min. After the incubation, the cells were examined for image formation under a confocal laser microscope (63 × objective).

The human primary mast cell MrgX2 has higher expression, while the mast cell line HMC-1 cell has no MrgX2 expression basically. To examine the targeting effect of ZX1 on MrgX2 receptors in mast cells, imaging experiments of ZX1 on human primary mast cells and MrgX2 transfected HEK293 cells were performed with HMC-1 cells as negative controls. As a result, as shown in FIG. 3, it can be seen that the background fluorescence of the ZX1 fluorescent probe is very weak, and that the living cells can be imaged without washing. When the probe is combined with human primary mast cells and MrgX2-HEK293 cells, bright green fluorescence is displayed, and HMC-1 cells cannot be stained, so that the selectivity of the probe on an MrgX2 receptor is proved. Cell imaging experiment results show that ZX1 can be used as an environment-sensitive small-molecule fluorescent probe to realize real-time dynamic monitoring of MrgX2 receptors, so that the probe is used for the visual research of the receptors.

The application of the small-molecule fluorescent probe targeting the MrgX2 receptor of the mast cell in MrgX2 competitive ligand screening is demonstrated.

The coumarin fluorescent chromophore, the fluorescein fluorescent chromophore and R in boron fluoride dipyrrolol acid in the invention₁Is a hydrogen atom, a methoxy group or an aminoalkyl group；R₂、R₃And a chlorine atom, a bromine atom or a fluorine atom, all of which can maintain agonistic activity to the MrgX2 receptor.

The phenyl-substituted pyrazolopyrimidine derivative of the invention retains agonistic activity on the MrgX2 receptor, while the indicative fluorescence is emitted by a fluorescent chromophore. The fluorescent probe disclosed by the invention not only keeps the activation activity on the MrgX2 receptor, but also has certain fluorescence property. As a targeting tool molecule, the fluorescent probe can directly act with mast cell cells, and can be used for the visualization research and the drug screening research of a receptor through the change of the fluorescence property.