CN110016008B - Fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol - Google Patents
Fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol Download PDFInfo
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Abstract
The invention discloses a fluorescent probe for specifically recognizing hydrogen polysulfide and biological mercaptan, which has the following structural formula:
a preparation method of a fluorescent probe for specifically identifying hydrogen polysulfide and biological mercaptan comprises the following steps of dissolving 3-aldehyde-4-chloro-7-diethylamino coumarin in anhydrous acetonitrile, sequentially adding 4-dimethylaminophenol hydrochloride, triethylamine and inert gas for protection, and performing reflux reaction and spin-drying on the solvent to obtain a compound A; and step two, dissolving the compound A and malononitrile in anhydrous dichloromethane, adding triethylamine as an alkali, protecting with inert gas, carrying out ice bath reaction, and separating to obtain the fluorescent probe. The invention has the beneficial effect of specifically recognizing hydrogen polysulfide and biological mercaptan.
Description
Technical Field
The invention relates to the technical field of fluorescent probe preparation. More particularly, the present invention relates to a fluorescent probe that specifically recognizes hydrogen polysulfide and biological thiol.
Background
Biological thiols (Cysteine, Cys), Homocysteine (Hcy), and Glutathione (GSH) are important components of many proteins and small molecules, and play an important role in the process of cell life activities. Among them, cysteine is not only a precursor of glutathione, acetyl coenzyme and taurine, but also a provider of sulfur ligand in a sulfur-iron complex of an organism, and lack of cysteine in a human body causes symptoms such as slow growth, hair discoloration, edema, lethargy, liver function damage, muscle relaxation, and physical weakness. Abnormal cysteine concentrations may also cause alzheimer's disease, cardiovascular disease, cancer. Glutathione is the most abundant non-protein biological sulfhydryl compound in cells and is closely related to a plurality of functions in cell bodies: including intracellular redox activity, xenobiotic metabolism, intracellular signal transduction and gene regulation. Cysteine and glutathione can be interconverted under the action of in vivo biological enzymes, and the contents of the cysteine and the glutathione are changed in organisms and are closely related to a plurality of diseases. Therefore, the detection of their biological thiols in cells is of great importance.
In regulating various physiological processes, H2SnHas high efficacy, including activating ion channels, transcription factors and tumor suppressors; how to detect biological thiol and hydrogen polysulfide is of great interest, and in recent years, a large number of biological thiols and hydrogen polysulfide have been detectedFluorescent probes, including fluorescent proteins and dyes, are used for detecting a certain object in a living body, but most of the existing probes do not distinguish H simultaneously2SnAnd biological thiol, so it is very important to develop a probe capable of distinguishing them at the same time.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to provide a fluorescent probe that specifically recognizes hydrogen polysulfide and biological thiol, and can simultaneously distinguish H2SnAnd biological thiols.
The invention also aims to provide a preparation method of the fluorescent probe for specifically identifying hydrogen polysulfide and biological mercaptan, which has the advantages of easily obtained raw materials, capability of directly preparing the fluorescent probe in the next step without separation and purification of intermediate products, and simple preparation method.
To achieve these objects and other advantages in accordance with the present invention, there is provided a fluorescent probe that specifically recognizes hydrogen polysulfide and bio-thiol, the fluorescent probe having a structural formula as follows:
a preparation method of a fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol is provided, which comprises the following steps:
dissolving 3-aldehyde-4-chloro-7-diethylamino coumarin in anhydrous acetonitrile, sequentially adding 4-dimethylaminophenol hydrochloride and triethylamine under the protection of inert gas, performing reflux reaction, then spin-drying the solvent, and purifying to obtain a compound A, wherein the structural formula of the compound A is as follows:
and step two, dissolving the compound A and malononitrile in anhydrous dichloromethane, adding triethylamine as an alkali, protecting with inert gas, carrying out ice bath reaction, and separating to obtain the fluorescent probe.
Preferably, the inert gas in step one and step two is argon.
Preferably, in the first step, the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the 4-dimethylaminophenol hydrochloride is 1:1-3, the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the triethylamine is 1:1-3, the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the anhydrous acetonitrile is 1mmol:5-15mL, and the reflux reaction is carried out specifically at 80-100 ℃ for 4-5 h.
Preferably, in the step one, the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the 4-dimethylaminophenol hydrochloride is 1:1, the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the triethylamine is 1:1, and the molar ratio of the 3-aldehyde-4-chloro-7-diethylaminocoumarin to the anhydrous acetonitrile is 1mmol:5mL, wherein the refluxing reaction is carried out for 4 hours at 80 ℃.
Preferably, in the second step, the dosage ratio of the compound A to the malononitrile is 1mmol:1-3mmol, the dosage ratio of the compound A to the triethylamine is 1mmol:0.36-1mmol, the molar volume ratio of the compound A to the anhydrous dichloromethane is 1mmol:3-9mL, and the ice-bath reaction time is 50-120 s.
Preferably, in the second step, the dosage ratio of the compound A to the malononitrile is 1mmol:1mmol, the dosage ratio of the compound A to the triethylamine is 1mmol:0.36mmol, the molar volume ratio of the compound A to the anhydrous dichloromethane is 1mmol:3mL, and the ice-bath reaction time is 60 s.
The invention at least comprises the following beneficial effects:
first, the fluorescent probe of the present invention emits blue light after reacting with biological thiol, and hydrogen polysulfide (H)2Sn) After the action, the yellow light emits yellow light, and can specifically recognize hydrogen polysulfide and biological thiol.
Secondly, the raw materials are easy to obtain, and the preparation of the intermediate product can be directly carried out the next step of preparing the fluorescent probe without separation and purification, so that the preparation method is simple.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a nuclear magnetic hydrogen spectrum of the fluorescent probe MCP1 of the invention;
FIG. 2 is a nuclear magnetic carbon spectrum of the fluorescent probe MCP 1;
FIG. 3 is a graph showing the UV-absorbance of the interaction between the fluorescent probe MCP1 and biological thiol Cys according to the present invention;
FIG. 4 is a graph showing the UV-absorption of the interaction of the fluorescent probe MCP1 and the biological thiol GSH;
FIG. 5 is a graph showing the UV-absorption of the interaction between the fluorescent probe MCP1 and hydrogen polysulfide according to the present invention;
FIG. 6 is a graph showing the fluorescence emission of the interaction between the fluorescent probe MCP1 and biological thiol Cys according to the invention;
FIG. 7 is a graph showing the fluorescence emission of the interaction between the fluorescent probe MCP1 and biological thiol Cys according to the invention;
FIG. 8 is a graph showing the fluorescence emission of the interaction of the fluorescent probe MCP1 and the biological thiol GSH;
FIG. 9 is a graph showing the fluorescence emission of the interaction of the fluorescent probe MCP1 and the biological thiol GSH;
FIG. 10 is a graph showing the fluorescence emission of the interaction between the fluorescent probe MCP1 and hydrogen polysulfide according to the present invention;
FIG. 11 is a graph showing the fluorescence emission of the interaction between the fluorescent probe MCP1 and hydrogen polysulfide according to the present invention;
FIG. 12 is a graph showing the fluorescence emission of the fluorescent probe MCP1 of the invention under the excitation condition of 386nm for selective test;
FIG. 13 is a graph showing the fluorescence emission of the fluorescent probe MCP1 of the invention under the excitation condition of 511nm for selective test;
FIG. 14 is a schematic diagram of the kinetic detection of the fluorescent probe MCP1 and biological thiol Cys according to the invention;
FIG. 15 is a schematic diagram of the kinetic detection of the fluorescent probe MCP1 and the biological thiol GSH in the invention;
FIG. 16 is a schematic diagram of the kinetic detection of the fluorescent probe MCP1 and hydrogen polysulfide according to the present invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
< example 1>
A fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol has the following structural formula:
the preparation method of the fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol comprises the following steps:
step one, 3-aldehyde-4-chloro-7-diethylaminocoumarin (label 1, 1mmol, 279mg) was dissolved in 5mL anhydrous acetonitrile (acetonitrile), 4-dimethylaminophenol hydrochloride (1mmol, 173mg) was added, and triethylamine (NEt) was added3) (1mmol, 139 mu L) is used as a base, argon is used for protection, after reflux reaction is carried out for 4h at 80 ℃, the solvent is dried in a spinning mode, a mixture is obtained, and the mixture is purified to obtain a compound A, wherein the mixture is a reddish brown solid, the purification is carried out to obtain a dark brown compound A, the total amount is 304mg, and the yield is 80%;
the main product in the mixture is compound A;
the reaction equation is:
step two, dissolving the compound A (304mg, 0.8mmol) and malononitrile (the label is 3, 0.8mmol, 52.8 μ L) in 2.4mL of anhydrous Dichloromethane (DCM), adding triethylamine (0.288mmol, 40 μ L) as a base, reacting for 1min under the protection of Ice Bath (Ice Bath) and argon, and separating to obtain a fluorescent probe (the label is MCP1), wherein the fluorescent probe is a red solid, and the total amount is 171.2mg and the yield is 50%;
the reaction equation is:
according to the point plate, the raw materials in the first step are basically completely reacted, the mixture is not purified, the second step reaction can be directly carried out, the purification effect on the second step reaction is basically negligible, and when the mixture is directly carried out in the second step reaction without purification, the amount of the mixture is converted according to the yield of 80%.
< example 2>
A fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol has the following structural formula:
the preparation method of the fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol comprises the following steps:
step one, 3-aldehyde-4-chloro-7-diethylaminocoumarin (label 1, 1mmol, 279mg) was dissolved in 10mL anhydrous acetonitrile (acetonitrile), 4-dimethylaminophenol hydrochloride (2mmol, 346mg) was added, and triethylamine (NEt) was added3) (2mmol, 278 μ L) as a base, performing reflux reaction at 100 ℃ for 4.5h under the protection of argon, drying the solvent by spinning to obtain a mixture, and purifying the mixture to obtain a compound A, wherein the mixture is a reddish brown solid, and the mixture is purified to obtain a dark brown compound A, wherein the total amount of 291.8mg and the yield is 76.8%;
the main product in the mixture is compound A;
the reaction equation is:
step two, dissolving the compound A (291.8mg, 0.77mmol) and malononitrile (the label is 3, 1.54mmol, 101.6 μ L) in 4.62mL of anhydrous Dichloromethane (DCM), adding triethylamine (0.55mmol, 76.4 μ L) as a base, reacting for 50min under the protection of Ice Bath (Ice Bath) and argon, and separating to obtain a fluorescent probe (the label is MCP1), wherein the fluorescent probe is a red solid, the total amount is 161mg, and the yield is 49%;
the reaction equation is:
< example 3>
A fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol has the following structural formula:
the preparation method of the fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol comprises the following steps:
step one, 3-aldehyde-4-chloro-7-diethylaminocoumarin (label 1, 1mmol, 279mg) is dissolved in 15mL anhydrous acetonitrile (acetonitrile), 4-dimethylaminophenol hydrochloride (3mmol, 519mg) is added, and finally triethylamine (NEt) is added3) (3mmol, 417 mu L) serving as a base, performing reflux reaction at 90 ℃ for 5 hours under the protection of argon, drying the solvent in a spinning mode to obtain a mixture, and purifying the mixture to obtain a compound A, wherein the mixture is a red brown solid, the dark brown compound A is obtained through purification, the total amount is 297.2mg, and the yield is 78.2%;
the main product in the mixture is compound A;
the reaction equation is:
step two, dissolving the compound A (297.2304mg, 0.78mmol) and malononitrile (3, 2.350.8mmol, 155.1 μ L of the label) in 7.02mL of anhydrous Dichloromethane (DCM), adding triethylamine (0.78288mmol, 108.3 μ L) as a base, reacting for 120s under the protection of Ice Bath (Ice Bath) and argon, and separating to obtain a fluorescent probe (MCP 1 of the label), wherein the fluorescent probe is a red solid, the total amount is 160.7mg, and the yield is 48%;
the reaction equation is:
1. probe hydrogen spectrometry
As shown in FIG. 1, the nuclear magnetic hydrogen spectrum of the fluorescent probe MCP1,1H NMR(300MHz,CDCl3)δ7.67(s,1H),7.35(d,J＝9.9Hz,1H),6.93(d,J＝9.3Hz,2H),6.71(d,J＝9.0Hz,2H),6.50(d,J＝2.1Hz,1H),6.47(s,1H),3.46(q,J＝7.2Hz,4H),1.32–1.17(m,6H)。
2. probe carbon spectrometry
As shown in fig. 2, the nuclear magnetic hydrogen spectrum of the fluorescent probe MCP1,13C NMR(75MHz,CDCl3)δ166.8,159.9,157.6,153.3,150.2,127.7,117.9,115.2,113.7,112.8,109.9,103.3,101.9,97.1,83.3,45.2,41.0,12.5。
3. probes MCP1 and H2SnUV-absorption detection of interaction with RSH
3.1 solution preparation
Blank solution: adding DMSO into the PBS solution, and uniformly mixing, wherein the volume ratio of the DMSO to the PBS solution is 2: 8;
probe-PBS solution containing the analyte to be detected: and sequentially adding DMSO, a fluorescent probe MCP1 and the to-be-detected object into the PBS solution, and uniformly mixing, wherein the volume ratio of the DMSO to the PBS solution is 2:8, the concentration of the fluorescent probe MCP1 is 10 mu M, the mass ratio of the to-be-detected object to the fluorescent probe MCP1 is 100:1, and the to-be-detected object is one of biological thiol Cys, biological thiol GSH and hydrogen polysulfide.
3.2 testing
Adjusting the baseline of the ultraviolet-absorbing visible spectrophotometer by using the blank solution;
and respectively measuring ultraviolet-absorption curves of the probe-PBS solution containing the substance to be detected along with the change of time, wherein the ultraviolet-absorption curve of the probe-PBS solution containing the biological thiol Cys along with the change of time is shown in figure 3, the ultraviolet-absorption curve of the probe-PBS solution containing the biological thiol GSH along with the change of time is shown in figure 4, and the ultraviolet-absorption curve of the probe-PBS solution containing hydrogen polysulfide along with the change of time is shown in figure 5.
3.3 results
As can be seen from FIGS. 3-4, the fluorescence excitation wavelength of the bio-thiol is 386 nm.
As can be seen from FIG. 4, the fluorescence excitation wavelength of hydrogen polysulfide is 511 nm.
4. Probes MCP1 and H2SnFluorescence emission detection of interaction with RSH
4.1 solution preparation
Preparing a blank solution, namely preparing a hollow white solution by using the blank solution and the 3.1 solution;
probe-PBS solution containing the analyte to be detected: preparing a probe-PBS solution containing the substance to be detected in the step of preparing the 3.1 solution;
4.2 testing
Adjusting the baseline of the ultraviolet-absorbing visible spectrophotometer by using the blank solution;
respectively measuring the fluorescence emission curves of the probe-PBS solution containing the substance to be detected along with the change of time under the excitation conditions of 386nm and 511nm,
FIG. 6: a fluorescence emission curve of the Cys-containing probe-PBS solution changing along with time under the excitation of 386nm excitation wavelength;
FIG. 7: a fluorescence emission curve of the Cys-containing probe-PBS solution changing along with time under the excitation of 511nm excitation wavelength;
FIG. 8: fluorescence emission curve of probe-PBS solution containing GSH along with time change under excitation of 386nm excitation wavelength;
FIG. 9: fluorescence emission curve of probe-PBS solution containing GSH along with time change under 511nm excitation wavelength;
FIG. 10: containing H2SnThe fluorescence emission curve of the probe-PBS solution changes along with time under the excitation of 386nm excitation wavelength;
FIG. 11: containing H2SnThe fluorescence emission curve of the probe-PBS solution changes along with time under the excitation of 511nm excitation wavelength;
4.3 results
From FIGS. 6-9, it is clear that the fluorescence emission wavelength of the biological thiol Cys and the biological thiol GSH is 486 nm.
From FIGS. 10 to 11, it can be seen that H2SnThe fluorescence emission wavelength of (2) is 576 nm.
5. Selective testing of Probe MCP1
5.1 solution preparation
Mother liquor: adding DMSO and a fluorescent probe MCP1 into a PBS solution in sequence, wherein the volume ratio of the DMSO to the PBS solution is 2:8, and the concentration of the fluorescent probe MCP1 is 10 mu M;
probe-PBS solution containing the analyte to be detected: adding DMSO, a fluorescent probe MCP1 and a related substance to be detected into a PBS solution in sequence, and mixing uniformly, wherein the volume ratio of the DMSO to the PBS solution is 2:8, the concentration of the fluorescent probe MCP1 is 10 mu M, the mass ratio of the substance to be detected to the fluorescent probe MCP1 is 100:1, and the substance to be detected comprises: various common anions and cations, amino acids, reactive oxygen and nitrogen species, i.e. H2Sn、H2O2、S2O3 2-、SO3 2-、SO4 2-、HSO3 -、ClO-、O2-、NO、NO2 -、NO3 -、Ca2+、K+、Mg2+、Zn2+Cysteine Cys, glutathione GSH, alanine Ala, arginine Arg, aspartic acid Asp, lysine Lys, methionine Met, tyrosine Tyl, glycine Gly, glutamic acid Glu, histidine His, isoleucine Iso, phenylalanine Phe, proline Pro, serine Ser, threonine Thr, valine Val.
5.2, testing
Under the excitation conditions of 386nm and 511nm, the fluorescence emission curves of the probe-PBS solutions containing the substances to be detected along with the change of time are tested by a fluorescence spectrometer, wherein, the fluorescence emission spectrums of the probe-PBS solutions containing the substances to be detected under the excitation condition of 386nm of the excitation wavelength are shown in FIG. 12; FIG. 13 shows fluorescence emission spectra of probe-PBS solutions each containing an analyte under 511nm excitation conditions;
5.3, results
As shown in FIG. 12, under the excitation condition of 386nm, when the substance to be detected is H2O2、S2O3 2-、SO3 2-、SO4 2-、HSO3 -、ClO-、O2-、NO、NO2 -、NO3 -、Ca2+、K+、Mg2+、Zn2+No obvious change exists in the fluorescence of the system when alanine Ala, arginine Arg, aspartic acid Asp, lysine Lys, methionine Met, tyrosine Tyl, glycine Gly, glutamic acid Glu, histidine His, isoleucine Iso, phenylalanine Phe, proline Pro, serine Ser, threonine Thr and valine Val are adopted, specifically shown as 0thers in figure 12, the biological thiol cysteine Cys and the biological thiol glutathione GSH cause the change of the fluorescence intensity at 486nm (blue light region) to different degrees, and H is H2SnCausing a change in fluorescence intensity at 576nm (yellow region);
as shown in FIG. 13, under the excitation condition of 386nm, when the substance to be detected is H2O2、S2O3 2-、SO3 2-、SO4 2-、HSO3 -、ClO-、O2-、NO、NO2 -、NO3 -、Ca2+、K+、Mg2+、Zn2+No obvious change is caused to the fluorescence of the system when alanine Ala, arginine Arg, aspartic acid Asp, lysine Lys, methionine Met, tyrosine Tyl, glycine Gly, glutamic acid Glu, histidine His, isoleucine Iso, phenylalanine Phe, proline Pro, serine Ser, threonine Thr, valine Val, biological thiol cysteine Cys and biological thiol glutathione GSH are adopted, specifically shown as 0thers in figure 12, and only H is adopted2SnCausing a change in fluorescence intensity at 576nm (yellow region);
that is, probe MCP1 can specifically recognize hydrogen polysulfide (H)2Sn) And biological thiols.
6. Probes MCP1 and H2SnAnd acting with RSHKinetic detection
The probe MCP1 itself did not fluoresce, and as can be seen from FIGS. 6, 8, and 11, the addition of Cys, GSH, and H, which are biological thiol cysteine, was performed2SnThe fluorescence intensity of the latter system gradually increased with time, wherein, according to FIGS. 14-16, the equilibrium time of the biological thiol cysteine Cys and the biological thiol glutathione GSH is 15min, H2SnThe equilibrium time of (3) was 10min, indicating that probe MCP1 is for H2SnAnd RSH have a faster response.
7. Probe mechanism study
Studies have shown that the response mechanism of the probe to biological thiols (biothiols), hydrogen polysulfide is as follows:
the hydrogen polysulfide and the biological thiol are subjected to the action with the probe to generate compounds with different structures and emit different fluorescence by utilizing the differences in the structure and chemical properties of the hydrogen polysulfide and the biological thiol, so that the hydrogen polysulfide and the biological thiol are specifically identified.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (7)
1. A fluorescent probe for specifically recognizing hydrogen polysulfide and biological thiol is characterized in that the structural formula of the fluorescent probe is as follows:
2. the method for preparing a fluorescent probe that specifically recognizes hydrogen polysulfide and biological thiol according to claim 1, comprising the steps of:
dissolving 3-aldehyde-4-chloro-7-diethylamino coumarin in anhydrous acetonitrile, sequentially adding 4-dimethylaminophenol hydrochloride and triethylamine under the protection of inert gas, performing reflux reaction, then spin-drying the solvent, and purifying to obtain a compound A, wherein the structural formula of the compound A is as follows:
and step two, dissolving the compound A and malononitrile in anhydrous dichloromethane, adding triethylamine as an alkali, protecting with inert gas, carrying out ice bath reaction, and separating to obtain the fluorescent probe.
3. The method of claim 2, wherein the inert gas used in the first and second steps is argon.
4. The method for preparing a fluorescent probe capable of specifically recognizing hydrogen polysulfide and biological thiol as claimed in claim 2, wherein in the first step, the molar ratio of 3-aldehyde-4-chloro-7-diethylaminocoumarin to 4-dimethylaminophenol hydrochloride is 1:1-3, 3-aldehyde-4-chloro-7-diethylaminocoumarin to triethylamine is 1:1-3, 3-aldehyde-4-chloro-7-diethylaminocoumarin to anhydrous acetonitrile, and wherein the specific conditions for performing the reflux reaction are that the reflux reaction is performed at 80-100 ℃ for 4-5 h.
5. The method for preparing the fluorescent probe capable of specifically recognizing hydrogen polysulfide and biological thiol as claimed in claim 4, wherein in the first step, the molar ratio of 3-aldehyde-4-chloro-7-diethylaminocoumarin to 4-dimethylaminophenol hydrochloride is 1:1, the molar ratio of 3-aldehyde-4-chloro-7-diethylaminocoumarin to triethylamine is 1:1, the molar ratio of 3-aldehyde-4-chloro-7-diethylaminocoumarin to anhydrous acetonitrile is 1mmol:5mL, and wherein the specific condition for carrying out the reflux reaction is that the reflux reaction is carried out at 80 ℃ for 4 h.
6. The method for preparing a fluorescent probe capable of specifically recognizing hydrogen polysulfide and biological thiol according to claim 2, wherein in step two, the dosage ratio of compound A to malononitrile is 1mmol:1-3mmol, the dosage ratio of compound A to triethylamine is 1mmol:0.36-1mmol, the molar volume ratio of compound A to anhydrous dichloromethane is 1mmol:3-9mL, and the ice bath reaction time is 50-120 s.
7. The method for preparing a fluorescent probe capable of specifically recognizing hydrogen polysulfide and biological thiol according to claim 6, wherein in step two, the dosage ratio of compound A to malononitrile is 1mmol:1mmol, the dosage ratio of compound A to triethylamine is 1mmol:0.36mmol, the molar volume ratio of compound A to anhydrous dichloromethane is 1mmol:3mL, and the ice bath reaction time is 60 s.
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