A fluorescent probe for detecting viscosity in different pH environments and its preparation method

Abstract

The present invention discloses a preparation method of a fluorescent probe capable of simultaneously detecting the viscosity of different pH environments, and belongs to the field of chemical analysis detection technology. Probe (E) 2 (2 (5-benzothiazol-2-yl) 2,3-dihydro-1H-oxacyclic-4-vinyl) 1 (2-hydroxyethyl) 3,3-dimethyl-3H-indole-1-ium iodide (BDHV) can detect the change of viscosity of different pH environments. In an acidic environment (pH=5), as the viscosity of the solution increases, the fluorescence intensity of the red channel gradually increases, and the linear relationship diagram between the logarithm of the fluorescence intensity of the probe BDHV at 675nm and the logarithm of the system viscosity is good. The two are in a good linear relationship, and the probe can sensitively monitor the change of the viscosity of the acidic environment. In an alkaline environment (pH=8), as the viscosity of the solution increases, the fluorescence intensity of the green channel gradually decreases, and the fluorescence intensity of the red channel gradually increases, showing different fluorescent signals. Meanwhile, the probe can sensitively monitor the change of the viscosity of the alkaline environment. The probe BDHV is expected to become a powerful tool for detecting viscosity in different environments and has potential application in the diagnosis of viscosity-related diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry and relates to a preparation method of a viscosity fluorescent probe capable of detecting different pH environments. Background Art

[0002] Viscosity is a key factor involved in physiological activities such as interactions between biomolecules, substance transport, and the diffusion of metabolites. Abnormal fluctuations in viscosity can directly affect the diffusion rate of metabolites, causing organelle dysfunction and, in turn, leading to related diseases such as Parkinson's disease, fatty liver disease, diabetes, and malignant tumors. Fluorescence imaging methods have the advantages of high sensitivity and specificity, simple operation, and fast response, and have been widely used in biology, medicine, analytical chemistry, and other fields.

[0003] In addition, intracellular pH is a crucial parameter of cellular metabolism, playing an irreplaceable role in various physiological and pathological processes, including ion transport, energy production and conversion, cell division, and apoptosis. As an indicator of cell health, abnormal changes in intracellular pH are often associated with functional disorders such as cancer, stroke, and Alzheimer's disease. Therefore, sensitive measurement of intracellular pH has become an extremely important scientific research topic in cellular function, physiology, and pathology.

[0004] So far, many fluorescent probes for detecting viscosity have been developed, but many of them are used for viscosity detection at a single pH. We hope to design fluorescent probes that can give different fluorescent signals for viscosity in different pH environments, which is expected to become a powerful tool for detecting viscosity-related diseases. Summary of the Invention

[0005] Based on the above background, the purpose of the present invention is to provide a method for preparing a fluorescent probe capable of detecting viscosity in different pH environments, which has the characteristics of simple synthesis route, good selectivity, high sensitivity, etc. and can effectively detect viscosity in different pH environments.

[0006] The present invention provides a fluorescent probe (E)-2-(2-(5-benzothiazol-2-yl)-2,3-dihydro-1H-oxacyclo-4-vinyl)-1-(2-hydroxyethyl)-3,3-dimethyl-3H-indol-1-ium iodide (BDHV). The structure of the fluorescent probe is as follows:

[0007]

[0008] The fluorescent probe synthesis in the present invention is as follows:

[0009]

[0010] The preparation steps of the fluorescent probe BDHV are as follows:

[0011] Step 1: Preparation of intermediate A1

[0012] Pipette anhydrous N,N-dimethylformamide into a round-bottom flask, then add chloroform and phosphorus tribromide. Stir under argon for 1 hour, and the solution will turn milky white. Dissolve cyclohexanone in chloroform, and add the mixed solution to the above solution. Reflux overnight under argon. During the reaction, the solution gradually turns light yellow. Adjust the pH to neutral with a large amount of saturated sodium bicarbonate solution, extract with dichloromethane to obtain the organic phase, spin dry, and purify with an eluent to obtain Compound A1 (51%) as a yellow oily liquid.

[0013] Step 2: Preparation of intermediate A2

[0014] 2,3,3-Trimethyl-3H-indole and diiodoethanol were dissolved in acetonitrile solvent, sonicated to completely dissolve, heated under reflux, cooled to room temperature, filtered and washed with ether to obtain a pink solid A2 (2.5 g, yield 75%).

[0015] Step 3: Preparation of intermediate B1

[0016] Transfer o-hydroxybenzaldehyde and 2-aminobenzenethiol to a round-bottom flask, add dimethyl sulfoxide, and sonicate to completely dissolve. Heat under reflux. After the reaction is complete, cool to room temperature and add dropwise to cold water. Filter with suction and wash with ether to obtain a bluish-white solid B1 (1.1 g, 80% yield).

[0017] Step 4: Preparation of intermediate B2

[0018] Compound B1 and hexamethylenetetramine were weighed into a round-bottom flask, trifluoroacetic acid was added, and sonication was performed to dissolve the mixture. Under argon protection, the mixture was heated to reflux. After the solution cooled to room temperature, the pH was adjusted to neutral with NaOH solution. The organic phase was extracted with dichloromethane and washed with saturated brine. The solution was spin-dried and purified with petroleum ether and ethyl acetate as eluents to obtain Compound B2 (420 mg, 54.8%).

[0019] Step 5: Preparation of intermediate B3

[0020] Compound A1 and compound B2 were weighed into N,N-dimethylformamide, cesium carbonate was added and sonicated to completely dissolve them. Under argon protection, the mixture was stirred at room temperature. The solution was spin-dried and purified using petroleum ether and ethyl acetate as eluents to obtain product B3 (100.8 mg, yield 51%).

[0021] Step 6: Preparation of fluorescent probe BDHV

[0022] Compound A2 and compound B3 were dissolved in ethanol, heated under reflux, cooled to room temperature and filtered to obtain a dark gray solid BDHV (130.2 mg, yield 43.7%) ((E)-2-(2-(5-benzothiazol-2-yl)-2,3-dihydro-1H-oxacyclo-4-vinyl)-1-(2-hydroxyethyl)-3,3-dimethyl-3H-indol-1-ium iodide).

[0023] The response mechanism of the fluorescent probe of the present invention to pH and viscosity is as follows:

[0024]

[0025] The response mechanism of the fluorescent probe BDHV to pH and viscosity is shown above. The probe exists in an open-ring form in an acidic environment and in a cyclized form in an alkaline environment. When in a closed-ring structure, the conjugation is reduced, resulting in a blue shift in the fluorescence signal, showing different fluorescence signals. In low-viscosity solvents, the fluorescence intensity of the probe is weakened due to the energy consumed by the free rotation of the carbon-carbon single bond, while in high-viscosity solvents, the carbon-carbon single bond is hindered from rotating, and the probability of non-radiative transition is reduced, resulting in an enhancement of the fluorescence intensity of the probe.

[0026] Figure 3 This is the UV absorption spectrum of the probe BDHV in different solvents. There are two absorptions at around 400nm and 590nm.

[0027] Figure 4 is the normalized fluorescence emission spectrum of the probe BDHV in different solvents (λ ex =400nm), the spectrum blue-shifts as the polarity of the solvent decreases.

[0028] Figure 5 Fluorescence emission spectra of probe BDHV in different solvents (λ ex =590 nm), the fluorescence intensity of the probe in glycerol was higher, indicating that the probe might be sensitive to viscosity.

[0029] Figure 6 This is the UV absorption spectrum of the probe BDHV in solutions with different pH values. The absorbance around 420 nm increases with increasing pH, while the absorbance around 590 nm decreases with increasing pH. The spectrum shows regular changes.

[0030] Figure 7 is the fluorescence emission spectrum of the probe BDHV in different pH solutions (λ ex =420 nm), and the fluorescence intensity value around 510 nm increased with the increase of pH.

[0031] Figure 8 is the fluorescence emission spectrum of the probe BDHV in different pH solutions (λ ex=590 nm), and the fluorescence intensity value around 670 nm decreased with the increase of pH.

[0032] Figure 9 This is the UV-visible absorption spectrum of the probe BDHV in a mixed system of PBS (pH=5) buffer solution and glycerol at different ratios. As the glycerol ratio of the mixed solution changes, the absorption spectrum shows regular changes.

[0033] Figure 10 Figure 3 is the fluorescence emission spectrum of the probe BDHV in a mixture of PBS (pH=5) buffer solution and glycerol at different ratios. As the viscosity of the mixed system increases, the fluorescence intensity gradually increases.

[0034] Figure 11 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9948, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an acidic environment.

[0035] Figure 12 The anti-interference results of the probe BDHV in PBS (pH=5) system. When various anti-interference substances were added, there was no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under acidic conditions. (1.Blank, 2.Cu 2+ ,3.Fe 2+ ,4.Ca 2+ ,5.Na + ,6.K + ,7.Cl - ,8.F - ,9.ClO - ,10.CO3 2- ,11.H2O2,12.S 2- ,13.HSO3 - ,14.Cys,15.Hcy,16.GSH).

[0036] Figure 13 This is the UV-visible absorption spectrum of the probe BDHV in a mixed system of PBS (pH=8) buffer solution and glycerol in different proportions. As the proportion of glycerol in the mixed solution increases, the absorbance at 420nm gradually decreases and the absorbance at 590nm gradually increases, and the absorption spectrum shows regular changes.

[0037] Figure 14 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) buffer solution and glycerol at different ratios. As the viscosity ratio of the mixed system increases, the fluorescence intensity gradually decreases (λ ex =420nm).

[0038] Figure 15 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 510 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9935, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an alkaline environment.

[0039] Figure 16 This is the anti-interference result of the probe BDHV in PBS (pH=8) system. When various anti-interference substances are added, there is no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under alkaline conditions. (λ ex =420nm).

[0040] Figure 17 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) buffer solution and glycerol at different ratios. As the viscosity ratio of the mixed system increases, the fluorescence intensity gradually increases (λ ex =590nm).

[0041] Figure 18 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9939, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an alkaline environment.

[0042] Figure 19 This is the anti-interference result of the probe BDHV in PBS (pH=8) system. When various anti-interference substances are added, there is no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under alkaline conditions. (λ ex =590nm). BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Preparation and design route of probe BDHV.

[0044] Figure 2 Mechanism of probe BDHV detecting viscosity under different pH conditions.

[0045] Figure 3 UV absorption spectra of probe BDHV in different solvents.

[0046] Figure 4 is the normalized fluorescence emission spectrum of the probe BDHV in different solvents (λ ex =400nm).

[0047] Figure 5 Fluorescence emission spectra of probe BDHV in different solvents (λex =590nm).

[0048] Figure 6 The UV absorption spectra of the probe BDHV in solutions with different pH values ​​are shown in Figure 2.

[0049] Figure 7 is the fluorescence emission spectrum of the probe BDHV in different pH solutions (λ ex =420nm).

[0050] Figure 8 is the fluorescence emission spectrum of the probe BDHV in different pH solutions (λ ex =590nm)

[0051] Figure 9 The UV-visible absorption spectra of the probe BDHV in a mixture system of PBS (pH=5) buffer solution and glycerol at different ratios.

[0052] Figure 10 The fluorescence emission spectra of the probe BDHV in a mixture system of PBS (pH=5) buffer solution and glycerol at different ratios.

[0053] Figure 11 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity (pH = 5).

[0054] Figure 12 This is the anti-interference result of probe BDHV in PBS (pH=5) system.

[0055] Figure 13 The UV-visible absorption spectra of the probe BDHV in a mixture system of PBS (pH=8) buffer solution and glycerol at different ratios.

[0056] Figure 14 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) and glycerol at different ratios (λ ex =420nm).

[0057] Figure 15 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 510 nm and the logarithm of the system viscosity (pH = 8).

[0058] Figure 16 is the anti-interference result of probe BDHV in PBS (pH=8) system (λ ex =420nm).

[0059] Figure 17 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) and glycerol at different ratios (λex =590nm).

[0060] Figure 18 This is a linear relationship diagram between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity.

[0061] Figure 19 is the anti-interference result of probe BDHV in PBS (pH=8) system (λ ex =590nm).

[0062] Specific implementation examples

[0063] Example 1: Synthesis of Compound A1

[0064] 6 mL of anhydrous N,N-dimethylformamide was transferred to a round-bottom flask, and 25 mL of chloroform and 4.5 mL of phosphorus tribromide (PBr3) were added. Under argon protection, the mixture was stirred at 0°C for 1 hour, and a milky white solution was obtained. 2 mL of cyclohexanone was dissolved in 5 mL of chloroform, and the mixture was added to the above solution. Under argon protection, the mixture was stirred at 25°C for about 14 hours. During the reaction, the solution gradually turned light yellow. The pH was adjusted to neutral with a large amount of saturated sodium bicarbonate solution, and the organic phase was extracted with dichloromethane (DCM). The organic phase was dried and purified with an eluent to obtain Compound A1 as a yellow oily liquid with a yield of 51%.

[0065] Example 2: Synthesis of Compound A2

[0066] Dissolve 2,3,3-trimethyl-3H-indole (1 g, 6.28 mmol) and diiodoethanol (0.58 mL, 7.5 mmol) in 3 mL of acetonitrile and sonicate until completely dissolved. Reflux at 80°C for 8 hours. Cool to room temperature, filter, and wash with diethyl ether to obtain a pink solid A2 (2.5 g, 75% yield).

[0067] Example 3: Synthesis of Compound B1

[0068] o-Hydroxybenzaldehyde (626.3 μL, 6 mmol) and 2-aminobenzenethiol (625.9 μL, 6 mmol) were transferred to a round-bottom flask. 6 mL of dimethyl sulfoxide (DMSO) was added and completely dissolved by sonication. Refluxed at 180°C for 1.5 hours, the mixture was cooled to room temperature and added dropwise to 25 mL of cold water. Filtered and washed with ether to obtain a bluish-white solid B1 (1.1 g, 80% yield).

[0069] Example 4: Synthesis of Compound B2

[0070] Weigh compound B1 (765.8 mg, 3 mmol) and hexamethylenetetramine (HMTA) (420.6 mg, 3 mmol) in a round-bottom flask, add about 5 mL of trifluoroacetic acid (TFA), and sonicate to dissolve. Under argon protection, heat to 110 ° C and reflux for 4 hours. After the solution cools to room temperature, adjust the pH to neutral with NaOH solution. Extract with DCM and wash the organic phase with saturated brine, spin dry and elute with V PE :V EA =6:1 to purify to obtain compound B2 (420 mg, 54.8%)

[0071] Example 5: Synthesis of Compound B3

[0072] Compound A1 (220.9 mg, 1.175 mmol) and compound B2 (150 mg, 0.5875 mmol) were weighed and dissolved in 6 mL of N,N-dimethylformamide. Cesium carbonate (574.32 mg, 1.762 mmol) was added and ultrasonicated to completely dissolve the mixture. The mixture was stirred at room temperature for about 30 hours under argon protection. The solution was then dried and the eluent was V PE :V EA =10:1 to purify the product B3 (100.8 mg, yield 51%).

[0073] Example 6: Synthesis of probe BDHV

[0074] Compound A2 (151 mg, 0.453 mmol) and compound B3 (156 mg, 0.452 mmol) were dissolved in 6 mL of ethanol and refluxed at 80°C for 8 hours. After the solution was cooled to room temperature, it was filtered to obtain a dark gray solid BDHV (130.2 mg, yield 43.7%). 1 H NMR (600MHz, CDCl3) δ8.41(d,J=14.7Hz,1H),8.22(d,J=7.2Hz,1H),8.05(d,J=7.0H z,1H),7.88(d,J=6.2Hz,1H),7.67(s,1H),7.60(s,1H),7.45(s,2H),7.35(d,J=7.6H z,1H),7.12(d,J=6.4Hz,1H),7.06(s,1H),7.00(d,J=14.8Hz,1H),5.23(d,J=14.0Hz ,3H),4.65(s,2H),4.12(s,2H),2.96(s,2H),2.75(s,2H),1.98(s,2H),1.19(s,6H). 13C NMR (151MHz, CDCl3) δ180.4,161.9,158.8,153.4,150.2,146.9,142.3,141.1,136.1,132.5,131.9,129.8,128.0,12 6.8,125.9,124.9,124.2,122.8,122.0,121.8,121.8,117.3,113.7,108.9,58.8,50.7,49.4,29.8,26.6,26.3,20.0.

[0075] Example 4: Detection of viscosity in different pH environments using probe BDHV

[0076] Detection of viscosity in different pH environments of solution: Figure 3 This is the UV absorption spectrum of the probe BDHV in different solvents, with two absorption peaks at around 400nm and 590nm. Figure 4 and Figure 5 The fluorescence normalized spectra and fluorescence emission spectra (λ ex =400nm, 590nm), Figure 4 The results showed that the probe spectrum tended to blue-shift as the polarity of the solvent decreased; under excitation at 590 nm, the fluorescence intensity of the probe in glycerol was stronger, indicating that the probe might be a polarity-sensitive probe. Figure 6 This is the UV absorption spectrum of the probe BDHV in solutions with different pH values. The absorbance around 420 nm increases with increasing pH, while the absorbance around 590 nm decreases with increasing pH, and the spectrum shows regular changes. Figure 7 and Figure 8 is the fluorescence emission spectrum of the probe BDHV in different pH solutions (λ ex =420nm, 590nm), the fluorescence intensity value around 510nm increases with the increase of pH, and the fluorescence intensity value around 670nm decreases with the increase of pH. Figure 9 This is the UV-visible absorption spectrum of the probe BDHV in a mixed system of PBS (pH=5) buffer solution and glycerol at different ratios. As the glycerol ratio of the mixed solution changes, the absorption spectrum shows regular changes. Figure 10 Figure 3 is the fluorescence emission spectrum of the probe BDHV in a mixture of PBS (pH=5) buffer solution and glycerol at different ratios. As the viscosity of the mixed system increases, the fluorescence intensity gradually increases. Figure 11 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9948, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an acidic environment. Figure 12This is the anti-interference result of the probe BDHV in PBS (pH=5) system. When various anti-interference substances are added, there is no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under acidic conditions. Figure 13 This is the UV-visible absorption spectrum of the probe BDHV in a mixed system of PBS (pH=8) buffer solution and glycerol in different proportions. As the proportion of glycerol in the mixed solution increases, the absorbance at 420nm gradually decreases and the absorbance at 590nm gradually increases, and the absorption spectrum shows regular changes. Figure 14 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) buffer solution and glycerol at different ratios. As the viscosity ratio of the mixed system increases, the fluorescence intensity gradually decreases (λ ex =420nm). Figure 15 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 510 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9935, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an alkaline environment. Figure 16 This is the anti-interference result of the probe BDHV in PBS (pH=8) system. When various anti-interference substances are added, there is no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under alkaline conditions. (λ ex =420nm). Figure 17 is the fluorescence emission spectrum of the probe BDHV in the mixed system of PBS (pH=8) buffer solution and glycerol at different ratios. As the viscosity ratio of the mixed system increases, the fluorescence intensity gradually increases (λ ex =590nm). Figure 18 The linear relationship between the logarithm of the fluorescence intensity of the probe BDHV at 675 nm and the logarithm of the system viscosity is shown in FIG. 1 , and the two show a good linear relationship with a linear correlation coefficient of 0.9939, indicating that the probe BDHV can be used to sensitively detect changes in viscosity in an alkaline environment. Figure 19 This is the anti-interference result of the probe BDHV in PBS (pH=8) system. When various anti-interference substances are added, there is no obvious change in fluorescence intensity, indicating that the probe has good anti-interference ability in detecting viscosity under alkaline conditions. (λ ex =590nm).

[0077] In summary, through a simple organic synthesis method, we obtained a fluorescent probe BDHV that can detect viscosity in different pH environments. The prepared probe BDHV has the advantages of simple synthesis and high sensitivity. The spectral test results show that the probe exhibits different fluorescence signals in response to changes in viscosity content in different acidic and alkaline environments, and there is a good linear relationship between the fluorescence intensity and the logarithm of the system viscosity. The probe BDHV is expected to become a powerful tool for detecting viscosity in different environments and has application potential in the diagnosis of viscosity-related diseases.

Claims

1. A fluorescent probe (E)-2-(2-(5-benzothiazol-2-yl)-2,3-dihydro-1H-oxacyclo-4-vinyl)-1-(2-hydroxyethyl)-3,3-dimethyl-3H-indol-1-ium iodide capable of detecting viscosity in different pH environments, having the structural formula:

2. A method for preparing a fluorescent probe BDHV capable of detecting viscosity in different pH environments according to claim 1, comprising the following steps: Place N,N-dimethylformamide and phosphorus tribromide in a round-bottom flask and stir at zero degrees for one hour. Dissolve cyclohexanone in chloroform and add the mixture to the N,N-dimethylformamide and phosphorus tribromide solution, stirring overnight at room temperature. After the reaction, adjust the pH to neutral with saturated sodium bicarbonate solution, extract with dichloromethane to obtain an organic phase, spin-dry, and purify with petroleum ether and ethyl acetate as eluents to obtain a brown-yellow oily liquid A1. Dissolve 2,3,3-trimethyl-3H-indole and diiodoethanol in acetonitrile, sonicate to completely dissolve, reflux overnight, cool the solution to room temperature, filter, and wash with ether to obtain a pink solid A2; Place o-hydroxybenzaldehyde and 2-aminobenzenethiol in a round-bottom flask, add dimethyl sulfoxide, sonicate until completely dissolved, heat under reflux for two hours, cool to room temperature, and add dropwise to cold water. A precipitate forms, which is filtered and washed with ether to obtain a bluish-white solid B1. Compound B1 and hexamethylenetetramine were weighed into a round-bottom flask, trifluoroacetic acid was added, and the mixture was dissolved by sonication. Under argon protection, the mixture was heated to reflux. After the solution was cooled to room temperature, the pH was adjusted to neutral with sodium hydroxide solution. The organic phase was extracted with dichloromethane and washed with saturated brine. The solution was spin-dried and purified with petroleum ether and ethyl acetate as eluents to obtain compound B2. Compound A1 and compound B2 were weighed and dissolved in N,N-dimethylformamide. Cesium carbonate was added and completely dissolved by ultrasonication. The mixture was stirred at room temperature under argon protection. After the reaction was completed, the solution was spin-dried and purified using petroleum ether and ethyl acetate as eluents to obtain product B3. Weigh A2 and B3 in a round-bottom flask, add ethanol and ultrasonically dissolve, heat to reflux, a precipitate is generated, cool and filter to obtain a black-green solid BDHV. The synthesis path is as follows:

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