Viscosity and polarity fluorescent probe, and preparation method and application thereof
By synthesizing DHBP fluorescent probes, the problem of simultaneously detecting intracellular viscosity and polarity changes in existing technologies has been solved, enabling highly sensitive bioimaging and disease monitoring with good biocompatibility and stability.
Patent Information
- Application Number
- CN202311005576.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-10
- Publication Date
- 2025-11-25
- Estimated Expiration
- 2043-08-10
AI Technical Summary
Current technologies lack fluorescent probes capable of simultaneously detecting changes in intracellular viscosity and polarity, making it difficult to achieve sensitive and accurate diagnosis of diseases in vivo.
A fluorescent probe, DHBP, was designed and synthesized. Its D-π-A structure makes it highly sensitive to polarity changes, and it responds to viscosity changes by utilizing the carbon-carbon single bond rotational inhibition mechanism. The synthesis route is simple, with good selectivity and high sensitivity.
It achieves highly sensitive detection of viscosity and polarity, has good biocompatibility and photostability, and can monitor changes in viscosity and polarity of cells and tissues in real time in vivo, which can be used for early diagnosis and treatment assessment of diseases.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of analytical chemistry, and relates to a preparation method of a fluorescent probe capable of simultaneously detecting viscosity and polarity and application in biological imaging of inflammation and liver damage. BACKGROUND
[0002] Viscosity, as an important parameter of physiological microenvironment, plays an important role in various cell activities, including diffusion of metabolites and signal transduction; abnormal viscosity is related to diseases such as malignant tumors, hypertension and diabetes;
[0003] The fluorescent imaging method has high selectivity, intuitiveness and accuracy of information acquisition, and provides an effective tool for studying the relationship between viscosity and related diseases.
[0004] In addition, in a biological system, polarity, as a parameter of cell microenvironment, plays an important role in related cell physiological processes such as cell differentiation, proliferation, cell migration and regulation of the immune system; changes in intracellular polarity under different cell states can reflect physiological and pathological processes, and researches show that abnormal changes in polarity are related to diseases such as diabetes and cancer, so monitoring abnormal changes in cell polarity has important significance for diagnosis and pathogenesis research of related diseases.
[0005] So far, many fluorescent probes for detecting a single analyte of polarity or viscosity have been developed, and probes capable of simultaneously detecting polarity and viscosity are still few; in view of the above, we hope to design a dual-functional fluorescent probe capable of simultaneously responding to changes in viscosity and polarity in cells and biological bodies, which can give a signal when an abnormal condition occurs in vivo, and more sensitively and accurately perceive the occurrence of diseases, thereby providing help in preventing, diagnosing and treating diseases. SUMMARY
[0006] Based on the above background, the purpose of the present application is to provide a preparation method of a fluorescent probe capable of simultaneously detecting viscosity and polarity and application in biological imaging, which has the characteristics of simple synthesis route, good selectivity and high sensitivity, and can effectively detect viscosity and polarity at a physiological level.
[0007] The present application provides a fluorescent probe, and the structure of the fluorescent probe is as follows:
[0008]
[0009] The fluorescent probe in the present application is synthesized as shown below:
[0010]
[0011] The preparation steps of the fluorescent probe DHBP are as follows:
[0012] Step 1: Preparation of intermediate A1
[0013] Accurately pipette 4-methylpyridine and iodomethane into a round-bottom flask, add acetonitrile as solvent, reflux at 45℃ overnight; after the reaction is completed, the solution is cooled to room temperature, 30 mL of ether is added, and a precipitate is precipitated, which is filtered to obtain white solid A1 (53%).
[0014] Step 2: Preparation of intermediate A2
[0015] Accurately weigh 4-boronic acid triphenyl acid and 4-bromo-2-hydroxybenzaldehyde into a round-bottom flask, dissolve in tetrahydrofuran, then add potassium carbonate aqueous solution and tetrabutylammonium bromide, stir at room temperature for 30 minutes, add tetrakis(triphenylphosphine)palladium to the above solution, adjust the temperature to 68℃ under argon protection, and reflux for about 7 hours; after cooling to room temperature, extract with dichloromethane and wash the organic phase with saturated brine, spin dry and purify with petroleum ether and ethyl acetate as eluent to obtain compound A2 (43.8%).
[0016] Step 3: Preparation of fluorescent probe DHBP
[0017] Accurately weigh A1 and A2 into a round-bottom flask, ultrasonically dissolve in ethanol, heat to reflux for 8 hours, precipitate is generated, cool and filter, wash with ether solution several times to obtain brown-red solid DHBP (37%).
[0018] The response mechanism of the fluorescent probe of the present application to viscosity is as follows:
[0019]
[0020] The response mechanism of the fluorescent probe DHBP to viscosity is as shown above. In low viscosity solvents, the free rotation of carbon-carbon single bond consumes energy, resulting in a decrease in the fluorescence intensity of the probe, while in high viscosity solvents, the rotation of carbon-carbon single bond is hindered, the probability of non-radiative transition is reduced, resulting in an increase in the fluorescence intensity of the probe.
[0021] The detection mechanism of the fluorescent probe of the present application to polarity is as follows:
[0022]
[0023] DHBP takes quinoline as an electron acceptor and triphenylamine group as an electron donor to form a typical D-π-A structure, making the fluorescent probe DHBP highly sensitive to changes in polarity.
[0024] Figure 4 The UV-visible absorption spectrum of the probe DHBP in different proportions of PBS buffer solution and glycerol mixed system shows a regular change in the absorption spectrum with the change in the proportion of glycerol in the mixed solution.
[0025] Figure 5 The fluorescence emission spectra of the probe DHBP in different proportions of PBS buffer solution and glycerol mixed system, with the increase of the proportion of the viscosity of the mixed system, the fluorescence gradually enhanced; the insert is the color of the solution under the irradiation of 365 nm portable ultraviolet lamp.
[0026] Figure 6 The linear relationship between the logarithmic value of the fluorescence intensity of the probe DHBP at 660 nm and the logarithmic value of the system viscosity, the viscosity coefficient is in the range of 0.83-2.38, and the two have a good linear relationship, the linear correlation coefficient reaches 0.997, indicating that the probe DHBP can be used to detect the change of viscosity.
[0027] Figure 7 The fluorescence intensity of the probe DHBP in pure PBS and 95% glycerol and 5% PBS buffer solution within 60 minutes at different temperatures, indicating that the probe DHBP has good light stability at different temperatures.
[0028] Figure 8 The ultraviolet absorption spectra of the probe DHBP in different proportions of dimethyl sulfoxide and chloroform mixed solution, with the increase of the content of chloroform, the absorption peak has a 50 nanometer shift.
[0029] Figure 9 The fluorescence emission spectra of the probe DHBP in different proportions of dimethyl sulfoxide and chloroform mixed solution (λ ex =405nm), with the increase of the content of chloroform, the fluorescence at 560 nm gradually enhanced, the insert is the color of the solution under the irradiation of 365 nm portable ultraviolet lamp, and the obvious solvatochromic phenomenon can be seen.
[0030] Figure 10 The fluorescence emission spectra of the probe DHBP at 480 nm excitation, with the increase of the content of chloroform, the fluorescence at 660 nm gradually enhanced.
[0031] Figure 11 The linear relationship between the fluorescence intensity of the probe DHBP in different volumes of chloroform solution and the volume of chloroform, indicating a good linear relationship.
[0032] Figure 12 The cytotoxicity diagram of the probe DHBP, the results show that the probe DHBP has low cytotoxicity and good biocompatibility.
[0033] Figure 13 The fluorescence imaging diagram of the probe DHBP in HeLa cells with time, the fluorescence intensity has no obvious change within 30 minutes, indicating that the probe has certain fluorescence stability in cells.
[0034] Figure 14 For the co-localization effect of probe DHBP with mitochondria, there is a good overlap between the blue dye of mitochondria and the green channel and the red channel of probe DHBP, indicating that DHBP has a good co-localization ability with mitochondria.
[0035] Figure 15 For the confocal imaging of probe DHBP in human normal liver cells and cervical cancer cells, in human normal liver cells, the fluorescence intensity of the green channel and the red channel is weak; in cervical cancer cells, the fluorescence intensity of the green channel and the red channel is enhanced.
[0036] Figure 16 For the fluorescence intensity of each channel of probe DHBP after co-incubation with human normal liver cells and cervical cancer cells, it directly shows the change of fluorescence intensity of the green channel and the red channel.
[0037] Figure 17 For the confocal imaging of probe DHBP co-incubated with inflammatory cells, lipopolysaccharide causes cell inflammation, leading to increased cell viscosity and decreased polarity, so compared with the control group A, the fluorescence intensity of the green channel and the red channel of group B is gradually enhanced, and the addition of Robustine can alleviate the inflammatory symptoms of cells to some extent, so compared with group B, the fluorescence intensity of the green channel and the red channel of group C is weakened.
[0038] Figure 18 For the fluorescence intensity of each channel of probe DHBP co-incubated with inflammatory cells, it directly shows the change of fluorescence intensity of the green channel and the red channel.
[0039] Figure 19 For the confocal imaging of probe DHBP in the body of inflammation-induced nude mice, compared with the control group A mice, the fluorescence intensity of the green channel and the red channel is enhanced after lipopolysaccharide induces inflammation in the body of nude mice, and after the addition of Robustine, the inflammation is alleviated, as shown in group C, the fluorescence intensity of the green channel and the red channel is weakened to some extent.
[0040] Figure 20 For the fluorescence intensity of each channel of probe DHBP in the body of inflammation-induced nude mice, it directly shows the change of fluorescence intensity of the green channel and the red channel.
[0041] Figure 21 For the confocal imaging of probe DHBP with diabetic mice A (continuous seven days of injection of normal saline) and different doses of metformin treatment group (group B is continuous seven days of injection of small dose of metformin, group C is continuous seven days of injection of excessive metformin), the results show that a certain amount of metformin has a therapeutic effect on diabetic symptoms, while taking excessive metformin can induce the formation of liver damage symptoms in mice; and probe DHBP has monitoring ability for this symptom.
[0042] Figure 22 The fluorescence intensity of each channel for the co-incubation of probe DHBP with liver tissue sections of the three groups of mice directly shows the change in fluorescence intensity of the green and red channels.
[0043] Figure 23 The confocal imaging of probe DHBP with liver tissue sections of the three groups of mice also shows that excessive use of the hypoglycemic drug metformin can cause the formation of liver damage symptoms.
[0044] Figure 24 The fluorescence intensity of each channel for the co-incubation of probe DHBP with liver sections directly shows the change in fluorescence intensity of the green and red channels.
[0045] Figure 25 The confocal imaging of probe DHBP with the main organs of mice (including liver, kidney, heart, lung, and spleen).
[0046] Figure 26 The hematoxylin-eosin (H&E) staining of liver tissue sections of different mice. BRIEF DESCRIPTION OF DRAWINGS
[0047] Figure 1 Preparation and design route of probe DHBP.
[0048] Figure 2 Mechanism of probe DHBP for detecting viscosity.
[0049] Figure 3 Mechanism of probe DHBP for detecting polarity.
[0050] Figure 4 UV-Vis absorption spectra of probe DHBP in different proportions of PBS buffer solution and glycerol mixed systems.
[0051] Figure 5 Fluorescence emission spectra (λ ex = 450 nm) of probe DHBP in different proportions of PBS buffer solution and glycerol mixed systems.
[0052] Figure 6 Linear relationship diagram between the logarithmic value of the fluorescence intensity of probe DHBP at 660 nm and the logarithmic value of the viscosity of the system.
[0053] Figure 7 Changes in fluorescence intensity of probe DHBP in pure PBS and 95% glycerol and 5% PBS buffer solution over 60 minutes at different temperatures.
[0054] Figure 8UV absorption spectra of probe DHBP in different ratios of dimethyl sulfoxide and chloroform.
[0055] Figure 9 Fluorescence emission spectra of probe DHBP in different ratios of dimethyl sulfoxide and chloroform (λ ex = 405 nm). Inset: color of the solution under 365 nm hand-held UV lamp.
[0056] Figure 10 Fluorescence emission spectra of probe DHBP in different ratios of dimethyl sulfoxide and chloroform (λ ex = 480 nm).
[0057] Figure 11 Linear relationship between fluorescence intensity of probe DHBP and volume of chloroform.
[0058] Figure 12 Cytotoxicity of probe DHBP.
[0059] Figure 13 Fluorescence imaging of probe DHBP in HeLa cells over time.
[0060] Figure 14 Colocalization of probe DHBP with mitochondria in HeLa cells.
[0061] Figure 15 Confocal imaging of probe DHBP in normal human liver cells and cervical cancer cells.
[0062] Figure 16 Fluorescence intensity of each channel after incubation of probe DHBP with normal human liver cells and cervical cancer cells.
[0063] Figure 17 Confocal imaging of probe DHBP with inflammatory cells.
[0064] Figure 18 Fluorescence intensity of each channel after incubation of probe DHBP with inflammatory cells.
[0065] Figure 19 Confocal imaging of probe DHBP in a diabetic mouse model.
[0066] Figure 20 Fluorescence intensity of each channel in a diabetic mouse model treated with probe DHBP.
[0067] Figure 21 Confocal imaging of probe DHBP in diabetic mice and mice treated with different levels of probe DHBP.
[0068] Figure 22The fluorescence intensity of probe DHBP in each channel of diabetic mice and mice with different treatment levels.
[0069] Figure 23 Confocal imaging of probe DHBP in liver tissues of diabetic mice and mice with different treatment levels.
[0070] Figure 24 Column chart of relative fluorescence intensity of probe DHBP in liver tissues of diabetic mice and mice with different treatment levels.
[0071] Figure 25 Confocal imaging of probe DHBP in different organs (liver, kidney, heart, lung, spleen) of mice.
[0072] Figure 26 Confocal imaging of probe in hematoxylin-eosin (H&E) stained liver tissues of different mice.
[0073] DETAILED DESCRIPTION
[0074] Example 1: Synthesis of compound A1
[0075] Accurately pipette 4-methylpyridine (194 μL, 2 mmol) and iodomethane (6 mL, 96 mmol) into 5 mL of acetonitrile, and reflux at 45 °C overnight; after the reaction is completed, the solution is cooled to room temperature, 30 mL of ether is added to it, and a precipitate is precipitated, which is extracted by suction filtration to obtain white solid A1 (250 mg, 1.06 mmol) with a yield of 53%.
[0076] Example 2: Synthesis of compound A2
[0077] Accurately weigh 4-boronic acid triphenyl acid (346.8 mg, 1.2 mmol) and 4-bromo-2-hydroxybenzaldehyde (201.2 mg, 1 mmol) into a round-bottom flask, dissolve them in 6 mL of tetrahydrofuran, and then add 1.6 mL of potassium carbonate aqueous solution (2 mol / L) and tetrabutylammonium bromide (262.2 mg, 0.5 mmol) to it. Stir at room temperature for 30 minutes, add tetrakis(triphenylphosphine)palladium (2.4 mg, 0.002 mmol) to the above solution, and adjust the temperature to 68 °C under argon protection to reflux for about 7 hours. After cooling to room temperature, extract with dichloromethane and wash the organic phase with saturated brine, spin dry, and purify with eluent petroleum ether and ethyl acetate to obtain compound A2 (160 mg, 0.43 mmol) with a yield of 45.0%.
[0078] Example 3: Synthesis of probe DHBP
[0079] A1 (94 mg, 0.57 mmol) and A2 (125 mg, 0.34 mmol) were weighed in a round bottom flask, about 5 mL of ethanol was added, ultrasonic dissolution, heated to reflux for 8 hours, precipitate was generated, cooled and suction filtration, washed with ether solution several times, to get brown red solid DHBP (215 mg, yield 37%); 1 H NMR (600 MHz, DMSO) δ 8.77 (d, J = 6.6 Hz, 2H), 8.17 (d, J = 6.6 Hz, 2H), 7.72 (d, J = 8.1 Hz, 1H), 7.58 (t, J = 8.6 Hz, 3H), 7.35 (t, J = 7.8 Hz, 4H), 7.19 (s, J = 11.8 Hz, 2H), 7.04 (d, J = 8.6 Hz, 8H), 4.23 (s, 3H). 13 C NMR (151 MHz, DMSO) δ 158.63 (s), 153.70 (s), 147.82 (s), 147.36 (s), 145.24 (s), 143.31 (s), 136.95 (s), 133.17 (s), 130.14 (s), 130.03 (s), 127.93 (s), 125.19-125.08 (m), 124.91 (s), 123.99 (s), 123.51 (s), 123.27 (s), 122.56 (s), 121.26 (s), 117.68 (s), 114.07 (s), 47.16 (s).
[0080] Example 4: The probe DHBP monitors the viscosity and polarity of the solution and its application in biological imaging
[0081] Viscosity and polarity detection in solution: Figure 4 is the ultraviolet-visible absorption spectrum of the probe DHBP in different proportion of PBS buffer solution and glycerol mixed solution, with the change of the proportion of the mixed solution, the absorption spectrum presents regular change; Figure 5 is the fluorescence emission spectrum of the probe DHBP in different proportion of PBS buffer solution and glycerol mixed solution (λ ex = 450 nm), the fluorescence intensity value will increase with the increase of the proportion of glycerol content; the insert is the color photo of the solution under the irradiation of 365 nm portable ultraviolet lamp; Figure 6 is the linear relationship between the logarithmic value of the fluorescence intensity of the probe DHBP at 660 nm and the logarithmic value of the viscosity of the system, the results show that they present good linear relationship, indicating that the probe DHBP can detect the change of viscosity in a certain range; Figure 7 is the light stability of the probe DHBP under different temperature conditions within 60 minutes, the results show that the probe DHBP has good light stability in different viscosity value systems under different temperatures;Figure 8 For the UV absorption spectrum of the probe in the mixed solution of dimethyl sulfoxide and chloroform with different proportions, with the increase of the content of chloroform, i.e. the decrease of the polarity of the solution, the UV shows regular changes, with a wavelength shift of about 50 nm; Figure 9 and Figure 10 For the fluorescence emission spectrum (λ ex =405nm, 480nm) of the probe DHBP in the mixed solution of dimethyl sulfoxide and chloroform with different proportions, the results show that under different excitation wavelengths, the corresponding fluorescence intensity values will increase with the increase of the proportion of chloroform, and when the excitation is 480, the fluorescence intensity value at 660nm shows a good linear relationship with the volume of chloroform, as shown in Figure 11 .
[0082] Viscosity and polarity detection biological imaging application: Figure 12 For the cytotoxicity of the probe DHBP, it can be seen from the figure that HeLa cells have a high cell survival rate under the incubation of different concentrations (0μM, 2μM, 5μM, 8μM, 10μM, 20μM, 30μM, 40μM) of DHBP; It shows that the probe DHBP has low cytotoxicity and good biocompatibility.
[0083] Figure 13 For the confocal imaging of the probe DHBP in HeLa cells within 30 minutes, the results show that there is no obvious change in fluorescence intensity within 30 minutes, and it has certain fluorescence stability in cells.
[0084] Figure 14 For the co-localization imaging experiment of the probe DHBP and commercial mitochondrial dye, the results show that the probe DHBP has good mitochondrial localization ability.
[0085] Normal cell and cancer cell cell imaging: Figure 15 , 16 For the confocal fluorescence imaging and relative fluorescence intensity column chart of the probe DHBP in different cell lines (human normal liver cells and cervical cancer cells); Compared with normal cells, cancer cells have lower polarity and higher viscosity, which can be used as a marker for cancer diagnosis; After incubation with the probe DHBP, the fluorescence intensity of the green channel and the red channel in human normal liver cells is weak; while in cervical cancer cells, the fluorescence intensity of the green channel and the red channel is strong; This shows that the probe DHBP has great potential in distinguishing normal cells and cancer cells.
[0086] Inflammatory cell model imaging: Figure 17For the relative fluorescence intensity column chart of the confocal imaging of the probe DHBP co-incubated with inflammatory cells, the invasion of lipopolysaccharide can cause a significant inflammatory effect on the cells, and the intracellular viscosity level is increased and the polarity level is decreased accordingly; compared with the control group A, the fluorescence of the green channel and the red channel of the lipopolysaccharide-induced group B is increased accordingly; Robustine is an anti-inflammatory drug, and its addition can weaken the fluorescence intensity of the green channel and the red channel; Figure 18 For the fluorescence intensity of each channel of the probe DHBP co-incubated with inflammatory cells, the change of the fluorescence intensity of the green channel and the red channel is directly shown; this shows that the probe DHBP can respond to the cell inflammation caused by the intervention of lipopolysaccharide.
[0087] Inflammatory nude mouse model imaging: Figure 19 、 20 For the confocal imaging of the probe DHBP in the induced inflammatory nude mice and the relative fluorescence intensity column chart of each channel; after the induction of lipopolysaccharide, the fluorescence intensity of the green channel and the red channel is increased, and after the addition of Robustine, the fluorescence intensity of the green channel and the red channel is weakened; this shows that the probe DHBP can image and detect the fluctuation of the extreme viscosity and the polarity level in the inflammatory nude mice in real time.
[0088] Liver injury mouse model imaging: studies have shown that the use of the hypoglycemic drug metformin can treat diabetes (increased viscosity and decreased polarity) to some extent, but excessive use can cause liver injury symptoms; a diabetic mouse model is established, which is divided into a control group A, a treatment group B and an over-treatment group C, and is co-incubated with the probe DHBP before imaging, and the confocal imaging graph over time is taken, and the results are shown in Figure 21 Compared with the control group A, the treatment group B taking a small amount of hypoglycemic drugs has weakened fluorescence in the green channel and the red channel, indicating that the viscosity in the mouse body is increased and the polarity is decreased after drug treatment, and the over-treatment group C can observe that the fluorescence of the green channel and the red channel is increased accordingly, indicating the deterioration of the disease, Figure 22 For the relative fluorescence intensity column chart of each channel of the corresponding three groups of mice, the corresponding change of each channel can be more directly observed; this shows that the probe DHBP can monitor the viscosity change caused by liver injury due to diabetes in or in vivo, and monitor the treatment effect in response to the drug; Figure 23 、 24 For the fluorescence imaging of the liver sections of the three groups of mice by the probe DHBP and the relative fluorescence intensity column chart of each channel; the results are consistent with the in vivo imaging results of DHBP; the liver tissue of the over-treatment group of mice has a stronger fluorescence signal, indicating that the probe DHBP has good tissue penetration and can confirm that the viscosity of the liver injury tissue is higher than that of the normal liver tissue and the polarity is lower than that of the normal liver tissue.
[0089] Mouse different organ model imaging: the main organs (liver, kidney, heart, spleen, lung) of three groups of mice were imaged to evaluate the damage level of different organs, and the results are shown in Figure 25 As shown in Figure 8, strong fluorescence can be observed in the liver of the over-treatment group of mice, while only weak fluorescence is shown in other organs; in order to verify whether the establishment of the mouse liver injury model is as expected, hematoxylin-eosin staining (H&E) was performed, as shown in Figure 26 As shown in Figure 8, strong fluorescence can be observed in the liver of the over-treatment group of mice, while only weak fluorescence is shown in other organs; in order to verify whether the establishment of the mouse liver injury model is as expected, hematoxylin-eosin staining (H&E) was performed, as shown in
[0090] In summary, by simple organic synthesis method, we obtained a fluorescent probe DHBP which can detect viscosity and polarity simultaneously, and successfully applied to the in vivo; the prepared probe DHBP has the advantages of high sensitivity, good biocompatibility and good stability; cell experiments show that the prepared probe has low cytotoxicity, good biocompatibility, good mitochondrial targeting ability, and can be used to distinguish cancer cells and normal cells; in addition, it can also realize the real-time monitoring of liver injury symptoms caused by inflammation and diabetes over-treatment of cells and nude mice, which has important significance in early diagnosis of diseases and drug intervention treatment dosage.
Claims
1. A fluorescent probe DHBP capable of detecting viscosity and polarity simultaneously, having the structure of:
2. A process for the preparation of fluorescent probe DHBP capable of detecting viscosity and polarity simultaneously according to claim 1, characterized in that The steps are as follows: Transfer 4-methylpyridine and iodomethane into a round-bottom flask, add acetonitrile as a solvent, and reflux overnight; after the reaction is completed, the solution is cooled to room temperature, ether is added to it, a precipitate is separated out, and white solid A1 is obtained by suction filtration; Weigh 4-boronic acid triphenyl acid and 4-bromo-2-hydroxybenzaldehyde into a round-bottom flask, add tetrahydrofuran to dissolve them, then add potassium carbonate aqueous solution and tetrabutylammonium bromide, stir at room temperature for half an hour, add tetrakis(triphenylphosphine)palladium to the above solution, and reflux under argon protection; after cooling to room temperature, extract with dichloromethane, wash the organic phase with saturated brine, spin dry, and purify with an eluent, wherein the eluent is petroleum ether and ethyl acetate, to obtain compound A2; Weigh A1 and A2 into a round-bottom flask, add ethanol to ultrasonically dissolve, heat to reflux, cool, and suction filter, wash with ether solution several times, and obtain brown-red solid DHBP, The synthesis path is as follows: