Small molecule probe for detecting p53 dysfunction and preparation method and application thereof

Abstract

The application belongs to the field of biological fluorescent probes, and particularly relates to a small-molecule probe for detecting abnormal p53 function and a preparation method and application thereof. A benzopyran cation and a quinoline cation are combined to construct a small-molecule probe, the emission wavelength of the probe has reached the near-infrared region (about 745 nm), the probe can detect the NAD(P)H level change, and can be used as a biomarker of p53 functional loss by the influence of p53 on NAD(P)H, thereby providing a powerful tool for diagnosing p53 functional loss in tumors.

Description

Technical Field

[0001] This invention belongs to the field of biological fluorescent probes, specifically relating to a small molecule probe for detecting p53 dysfunction, its preparation method, and its application. Background Technology

[0002] As is well known, p53 is an important tumor suppressor gene, encoding a protein with a molecular weight of 43.7 kDa. p53 not only repairs cellular DNA but also induces controlled cell death in abnormal cells, thereby preventing the occurrence and development of cancer. However, p53 can lose its function due to factors such as base deletions and mutations, making it susceptible to drug resistance in cancer cells. Furthermore, the pentose phosphate pathway (PPP) is closely related to cancer occurrence and progression, and PPP is the primary source of NAD(P)H. Recent studies have shown that p53 protein can regulate PPP by inactivating glucose-6-phosphate dehydrogenase (G6PD). Therefore, NAD(P)H levels can serve as a biomarker for p53 functionalization. However, NAD(P)H levels are easily affected by various factors, making accurate and real-time monitoring of NAD(P)H levels challenging. Therefore, diagnosing p53 functional defects in tumors is crucial, as it helps predict tumor growth and interventional treatments.

[0003] Currently, widely used methods for detecting p53 functional deficiencies include immunohistochemical analysis, single-strand conformational polymorphism (SSCP) analysis, and polymerase chain reaction (PCR). However, these methods have drawbacks: they require tissue samples and are time-consuming. Meanwhile, the availability of biomarkers for detecting p53 functional deficiencies remains limited. Small-molecule-based fluorescent probes, due to their advantages such as simple preparation, ease of use, good cell permeability, low toxicity, and suitability for in vivo experiments, are considered indispensable tools for real-time study of NAD(P)H dynamics and serve as biomarkers for p53 functionalization.

[0004] Therefore, there is an urgent need to develop a small-molecule fluorescent probe that can be used to detect p53 functional defects. Summary of the Invention

[0005] Based on the above background, the present invention aims to construct an NIR binary cation probe by combining benzopyran cation and quinoline cation. The emission wavelength of the probe is around 745 nm, which can detect changes in NAD(P)H level and serve as a biomarker for the functional loss of p53 by observing the effect of p53 on NAD(P)H. This can provide a powerful tool for diagnosing the loss of p53 function in tumors.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a small molecule probe for detecting p53 dysfunction, wherein the small molecule probe is compound 1 having the following structural formula:

[0008]

[0009] Another aspect of the present invention provides a method for preparing the above-mentioned small molecule probe, comprising: reacting 4-diethylaminoketo acid with cyclohexanone, concentrated sulfuric acid, and perchloric acid to prepare compound 2; reacting compound 2 with 3-quinoline carboxaldehyde to prepare compound 3; reacting compound 3 with methyl trifluoromethanesulfonate to prepare compound 4; and reacting compound 4 with 5-amino-2-hydroxymethylphenyl borate salt to prepare compound 1.

[0010]

[0011]

[0012] Furthermore, the specific preparation process is as follows:

[0013] (1) Preparation of compound 2

[0014] Cyclohexanone was added dropwise to concentrated sulfuric acid at -4 to 0°C with constant stirring. Then 4-diethylaminoketo acid was slowly added. The reaction was carried out at 90 to 100°C for 3 to 4 hours. After the reaction was completed, the final reaction mixture was poured into ice water and perchloric acid was slowly added to precipitate the precipitate. The precipitate was filtered under reduced pressure, washed with cold water, and dried to obtain an orange-red solid compound 2.

[0015] (2) Preparation of compound 3

[0016] 3-quinoline carboxaldehyde and piperidine were added to compound 2 in sequence, and then anhydrous ethanol was added to dissolve the mixture. The mixture was heated to reflux at 98-100°C overnight. After the reaction was completed, the temperature was restored to room temperature and then frozen. A purple solid precipitated out. The solid was washed with tert-butyl methyl ether and dried to obtain compound 3.

[0017] (3) Preparation of compound 4

[0018] Add methyl trifluoromethanesulfonate and compound 3 to dichloromethane, stir at room temperature for 3-4 hours, stop the reaction, freeze, precipitate a purple solid, filter under reduced pressure, wash with dichloromethane, and dry to obtain compound 4;

[0019] (4) Preparation of compound 1

[0020] N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine and compound 4 were mixed, and dichloromethane was added to dissolve the mixture. Then, 5-amino-2-hydroxymethylphenyl borate salt was added to the mixture, and the mixture was stirred overnight at room temperature. After the reaction was completed, compound 1 was obtained by column chromatography.

[0021] Furthermore, the molar ratio of compound 2 to 3-quinoline carboxaldehyde is 1:1.5 to 1.8.

[0022] Furthermore, the molar ratio of methyl trifluoromethanesulfonate to compound 3 is 4.08 to 6:1.

[0023] Further, the molar ratio of N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine and compound 4 is (0.32-0.35):(0.13-0.16):(0.13-0.16):(0.13-0.16).

[0024] Furthermore, the concentrated sulfuric acid is 98% concentrated sulfuric acid.

[0025] Furthermore, the perchloric acid is 70% perchloric acid.

[0026] In another aspect, the present invention also provides the application of the above-mentioned small molecule probe as a real-time detection tool for p53 functionalization abnormalities.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] (1) This invention provides a simple synthetic route for small molecule probes based on the combination of benzopyran salt and quinoline. The raw materials for the probes are readily available and have low cost.

[0029] (2) This invention provides a tool for detecting p53 dysfunction by detecting NAD(P)H levels;

[0030] (3) The small molecule probe provided by the present invention has a long emission wavelength, which enables it to distinguish between normal cells and cancer cells in cells, and to target tumors and distinguish HCT-116 and HCT-116p53- / - cell transplanted tumors in mouse experiments.

[0031] (4) This invention provides a powerful tool for the later clinical diagnosis and selection of tumor treatment methods by detecting tumor heterogeneity using small molecule probes. Attached Figure Description

[0032] Figure 1 This is the proton NMR spectrum of compound 1 of the present invention;

[0033] Figure 2 This is the carbon NMR spectrum of compound 1 of the present invention;

[0034] Figure 3 Here are the spectral test results for Compound 1 of this invention: [(a) Change in UV absorbance over time after adding 100 μM NADH to Compound 1 (10 μM); (b) Change in fluorescence intensity over time after Compound 1 (10 μM) and 100 μM NADH bind; (c) Change in fluorescence intensity of Compound 1 (10 μM) with varying NADH concentrations (10-100 μM); (d) Working curves of Compound 1 (10 μM) in the presence of different concentrations of NADH; (e) Kinetic curve of Compound 1 after adding 100 μM NADH; (f) Fluorescence intensity of Compound 1 (10 μM) at 745 nm in the presence of various anions and cations, amino acids, enzymes, etc. (200 μM): 1. K + , 2.Na + 3.Ca 2+ 4.Mg 2+ 5.Fe 2+ 6.SO3 2- 7.OH - 8.HSO3 - ,9.Br - 10.SCN - 11.NO2 - 12.F - , 13.Cys, 14.Hcy, 15.GSH, 16.Asn, 17.Lys, 18.VC, 19.H2O2, 20.Lipase, 21.Trypsin, 22.Pepsin, 23.NADPH, 24.NADH; (g) Effect of different pH values ​​on fluorescence intensity in the presence or absence of NADH (100 μM); (All tests were performed in a PBS system with the temperature controlled at 37 °C, λex = 680 nm, slit: 9 nm / 9 nm)];

[0035] Figure 4 The cell viability graphs were determined using the SRB method: [(a) HeLa cells were cultured for 5 hours and 10 hours in the presence of 0-50 μM compound 1, respectively; (b) HCT-116, HCT-116p53- / -, HT-29 and NCM-460 cells were cultured for 72 hours in the presence of 0-50 μM compound 1];

[0036] Figure 5Here are the colocalization fluorescence imaging images of mitochondria in cells: [(ac) After pre-incubating HeLa cells with compound 1 (10 μM) for 15 minutes, they were then treated with mitochondrial green dye (1 μM) for 20 minutes. Fluorescence imaging was performed on different channels: (a) Compound 1 channel, λex = 633 nm and λem = 715-755 nm; (b) Mitochondrial green dye channel, λex = 488 nm and λem = 500-540 nm; (c) Bright field channel; (d) Combined channel of compound 1 and mitochondrial green; (e) Colocalization map of probe and mitochondrial dye, scale bar: 10 μm];

[0037] Figure 6 Here are confocal images of cellular glycolysis: [(a) HeLa cells were pre-incubated with compound 1 (10 μM) for 15 minutes and then imaged; (b) HeLa cells were pre-incubated with 2 mM glucose for 15 minutes and then incubated with compound 1 (10 μM) for another 15 minutes and then imaged (λex = 633 nm, λem = 715-755 nm)];

[0038] Figure 7 These are confocal fluorescence images of normal cells and cancer cells: [Fluorescence imaging was performed after incubating different cells with compound 1 for 15 minutes. (a) Normal colon cells NCM-460; (b) Colon cancer cells HCT-116; (c) Fluorescence intensity images of the two cell types. Data are expressed as mean ± standard deviation (n=3). The excitation wavelength was 633 nm, and the red channel was 715-755 nm];

[0039] Figure 8 The following were screened for different modulators of the PPP pathway: [(a) using different compounds 1. 6PGD-IN-S3 (10 μM, 10 h); 2. Aspirin (250 μM, 4 h); 3. Deguelin (100 nM, 24 h); 4. H-89dihydrochloride (4 μM, 30 min); 5. KU-55933 (1 μM, 8 h); 6. ML385 (10 μM, 30 min); 7. RRX-001 (0.5 μM, 8 h); 8. SrcInhibitor 1 (30 μM, 6 h); 9. Torkinib (200 nM, 4 h); 10. Kevetrin (a) Pretreatment of HCT-116 cells with hydrochloride (2 μM, 10 h); 11. Confocal imaging of cells after incubation with Pifithrin-α hydrobromide (20 μM, 1 h) and incubation at 37 °C, followed by incubation with compound 1 (10 μM) for 15 min; (b) Changes in NAD(P)H levels in HCT-116 cells, with mean intensity data in the fluorescence intensity map expressed as mean ± standard deviation (n = 3);

[0040] Figure 9 These are cell imaging images showing the concentration-dependent effects of p53 agonists and inhibitors: [(a) Fluorescence intensity changes of p53 agonist (kevetrin hydrochloride) concentration-dependent; (b) Fluorescence intensity changes of p53 inhibitor (Pif-α) concentration-dependent; (c) Fluorescence imaging of cells pretreated with different concentrations of p53 agonist (kevetrin hydrochloride) for 10 hours, followed by incubation with compound 1 for 15 minutes; (d) Fluorescence imaging of cells pretreated with different concentrations of p53 inhibitor (Pif-α) for 1 hour, followed by incubation with compound 1 for 15 minutes];

[0041] Figure 10 Confocal imaging of different colon cancer cells: [Images were taken after cells were incubated with compound 1 for 15 minutes: (a) HCT-116; (b) HCT-116p53- / -; (c) HT-29; (d) mean fluorescence intensity of different colon cancer cells. Data are expressed as mean ± standard deviation (n=3). Excitation wavelength was 633 nm, and red channel was 715-755 nm];

[0042] Figure 11 Time-dependent fluorescence imaging of tumor-bearing nude mice after intravenous injection of compound 1: [The stock solution of compound 1 at a concentration of 2 mM was diluted to 500 μM and 20 μl was injected into mice via the tail vein. (a) Changes in mouse imaging over time; (b) Fluorescence imaging of various organs and tumors after dissection of the mice; (c) Changes in fluorescence intensity of tumor sites in mice over time];

[0043] Figure 12 Time-dependent fluorescence imaging of compound 1 (500 μM, 40 μl) injected via tail vein into tumor-bearing mice (HCT-116 tumor on the left and HCT-116p53- / - tumor on the right): [(a) Fluorescence imaging of mice at different times; (b) Changes in fluorescence intensity on the left and right sides of the mice over time];

[0044] Figure 13Fluorescence imaging of tumor heterogeneity: the tumor portion consisted of two different proportions of cells, 70% HCT116 p53-Luc and 30% HCT116 p53- / - cells. Compound 1 was administered to mice via tail vein injection (500 μM, 50 μl), followed by intraperitoneal injection of 25 mg / kg 5-Fu daily, and photographs were taken to record (a) fluorescence imaging of the GFP channel; (b) fluorescence imaging of the compound 1 channel; fluorescence imaging of mouse anatomical organs using (c) GFP channel and (d) compound 1 channel; and fluorescence changes in the mouse tumor site over time using (e) GFP channel and (f) compound 1 channel. Detailed Implementation

[0045] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0046] Example 1: Method for preparing the probe of the present invention

[0047] (1) Preparation of compound 2

[0048]

[0049] 72 ml of 98% concentrated sulfuric acid was added to a 200 ml flask, and 6.6 ml (64 mmol) of cyclohexanone was added dropwise at 0 °C with constant stirring. Then, 9.82 g (32 mmol) of 4-diethylaminoketo acid was slowly added, and the reaction was carried out at 90 °C for 3 h. The reaction mixture was then poured into ice water, and 7.9 ml of 70% perchloric acid was slowly added. The precipitate was filtered through a vacuum pump and washed three times with cold water to give 15 g (15 g) of an orange-red solid, with a yield of 98%.

[0050] (2) Preparation of compound 3

[0051]

[0052] Compound 2 (2 g, 4.2 mmol), 3-quinolinecarbaldehyde (1 g, 6.3 mmol), and piperidine (40 μl) were added sequentially to a 50 mL flask, followed by the addition of anhydrous ethanol (35 mL) to dissolve the mixture. The mixture was heated to reflux at 100 °C overnight. After the reaction was complete and the temperature returned to room temperature, the mixture was frozen for approximately 2 hours, resulting in the precipitation of a purple solid. The solid was then washed three times with tert-butyl methyl ether and dried to give compound 3 (1.5513 g), with a yield of 61%.

[0053] (3) Preparation of compound 4

[0054]

[0055] Methyl trifluoromethanesulfonate (1200 μl, 10.2 mmol) and compound 3 (1.53 g, 2.5 mmol) were added to dichloromethane (108 mL). The mixture was stirred at room temperature for 3 h. The reaction was stopped and the mixture was placed in a refrigerator. A purple solid precipitated. The solid was filtered under reduced pressure and washed three times with dichloromethane. After drying, compound 4 (0.9133 g) was obtained, with a yield of 58%.

[0056] (4) Preparation of compound 1

[0057]

[0058] N,N'-dicyclohexylcarbodiimide (DCC, 66 mg, 0.32 mmol), 1-hydroxybenzotriazole (HOBT, 60.26 mg, 0.13 mmol), 4-dimethylaminopyridine (DMAP, 16 mg, 0.13 mmol), and compound 4 (100 mg, 0.13 mmol) were sequentially added to a vial, and 2 mL of dichloromethane was added to dissolve the mixture. Then, 5-amino-2-hydroxymethylphenylboronic acid (60.26 mg, 0.33 mmol) was added to the mixture and stirred overnight at room temperature. After the reaction was complete, compound 1 was separated by column chromatography (dichloromethane:methanol = 20:1) in 20% yield.

[0059] Nuclear magnetic resonance (NMR) spectral data of compound 1:

[0060] 1H NMR(600MHz,Chloroform-d)δ9.87(s,1H),9.58(s,1H),8.66(d,J=8.0Hz,1H),8.58(s,1H),8.30(d,J=7.9Hz,1H),8.25(d, J=8.8Hz,1H),8.22–8.17(m,1H),8.01–7.97(m,1H),7.83(t,J=7.2Hz,1H),7.73(dq,J=7.0,3.6Hz,3H),7.62(s,1H),7.55( dq,J=7.2,3.9Hz,2H),7.07–6.99(m,2H),4.78(s,2H),4.32(t,J=6.7Hz,3H),4.16–4.08(m,1H),3.81(s,2H),3.51(s,2H), 2.27–2.21(m,2H),2.04(d,J=6.5Hz,1H),2.02(d,J=6.7Hz,1H),1.98(dd,J=12.0,6.1Hz,2H),1.74(dt,J=14.6,6.8Hz,6H).

[0061] 13 C NMR (151MHz, CDCl3) δ165.44(s), 158.86(s), 152.16(s), 136.57(d, J=1.3Hz), 134.56(s), 133.49(s), 131.34(s) ,130.89(s),130.54(t,J=9.1Hz),130.17–129.74(m),129.49(s),128.94(dd,J=13.9,9.3Hz),125.88(d,J=3.1H z),124.26(s),118.75(s),118.36(s),52.89(s),45.71(s),37.11(s),36.29(s),35.93(d,J=2.9Hz),33.71(s), 33.41(s),32.75(s),30.11(d,J=18.4Hz),27.24–26.99(m),26.71(s),25.53(d,J=2.8Hz),21.10(s),19.74(s).

[0062] Example 2 Spectral Testing

[0063] The test was conducted in a pure PBS system. The stock solution concentration of compound 1 was 2 mM, and the stock solutions concentrations of NADH and NADPH were both 20 mM. The test results are shown below. Figure 3As can be seen, the UV absorption peak of compound 1 is at 550 nm. After the addition of NADH, a new absorption peak appears at 725 nm. Furthermore, the UV absorption at 550 nm gradually decreases with increasing time, while the UV absorption at 725 nm gradually increases. Subsequently, using 680 nm as the excitation wavelength, the fluorescence at 745 nm gradually increases with increasing time when 100 μM NADH is added. Similarly, when different concentrations of NADH (0-100 μM) are added, the fluorescence intensity at 745 nm also gradually increases. In addition, compound 1 responds to 100 μM NADH in approximately 1 hour and exhibits good selectivity, linearity, and pH stability.

[0064] Example 3 Cell Experiment

[0065] Prior to the cell experiments, the present invention completed the cytotoxicity experiment of compound 1 using the SRB method. From Figure 4 It can be found that compound 1 has very low toxicity to different types of cells, and even the duration of action is 72 hours.

[0066] In co-localization experiments, it was found that (e.g.) Figure 5 Compound 1 can be localized to mitochondria with a localization coefficient of 0.88. Related studies have shown that in cancer cells, the Warburg effect leads to an abnormal increase in glycolysis, thereby inducing an overproduction of NAD(P)H. Therefore, in glycolysis experiments (such as...), it was found that... Figure 6 In HeLa cells treated with glucose, the fluorescence intensity was significantly higher than that of the control group. Furthermore, compound 1 could distinguish between normal cells and cancer cells. Figure 7 As can be seen, the fluorescence intensity of cancer cells HCT-116 is higher than that of normal cells NCM-460. Subsequently, a series of regulators affecting NAD(P)H levels related to the PPP pathway were screened. Figure 8 As can be seen, only p53 agonists (kevetrin hydrochloride) and p53 inhibitors (Pif-α) can significantly decrease or increase NAD(P)H levels. Furthermore, this invention also investigated the relationship between the concentrations of these two compounds, p53 agonists and p53 inhibitors, and NAD(P)H levels. Figure 9 As can be seen, the fluorescence intensity gradually decreases with increasing p53 agonist concentration, while the fluorescence intensity increases with increasing p53 inhibitor concentration. Subsequently, this invention also performed confocal imaging on different colon cancer cells (e.g., Figure 10The study found that the fluorescence intensity of HCT-116p53- / - and HT-29 cells was higher than that of HCT-116 cells. This is because HCT-116p53- / - cells are p53-deficient cells, while HT-29 cells are p53-mutant cells. This indicates that the level of NAD(P)H detected by compound 1 can serve as a biomarker for the functional loss or mutation of p53.

[0067] Example 4: In vivo experiment

[0068] Compound 1 was injected into nude mice with HeLa cell xenografts via the tail vein, followed by mouse imaging (e.g., Figure 11 It was observed that the fluorescence intensity at the mouse tumor site increased from weak to strong and then slowly decreased over time, indicating that compound 1 can effectively target tumors and detect the level of NAD(P)H in the tumor. Obvious fluorescence was clearly visible on the tumor in the anatomical diagram of the mouse's major organs. Fluorescence was also observed in the liver, likely due to the metabolism of compound 1.

[0069] To further demonstrate that the detection of NAD(P)H levels can serve as a biomarker for the loss of p53 functionalization, this invention transplanted different tumors (e.g., tumors on the left and right sides of the same mouse) into the same mouse. Figure 12 The left image shows an HCT-116 cell xenograft, and the right image shows an HCT-116p53- / - cell xenograft. As can be seen from the image, over time, the fluorescence intensity of the area circled on the right side of the mouse cell is higher than that of the area circled on the left side. This indicates that p53 functional deficiency can be detected in vivo.

[0070] This invention conducted experiments to detect tumor heterogeneity. First, two different cell types were mixed: 70% HCT116 p53-Luc cells labeled with green fluorescent protein and 30% HCT116 p53- / - cells were mixed as xenografts. Then, 25 mg / kg of 5-fluorouracil was injected into the peritoneum of mice daily, and fluorescence imaging was performed on different channels. It was found that in the GFP-labeled channels, the fluorescence intensity gradually decreased with the increase of days, while the fluorescence of compound 1 channel gradually increased. This indicates that 5-fluorouracil can kill the green fluorescent protein-labeled HCT116 p53-Luc cell xenografts to a certain extent, but has little effect on HCT116 p53- / - cells. This provides a powerful tool for the selection and replacement of clinical anticancer treatment methods in the later stages.

[0071] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A small molecule probe for detecting p53 dysfunction, characterized in that: The small molecule probe is compound 1 having the following structural formula: 。 2. A method for preparing a small molecule probe for detecting p53 dysfunction as described in claim 1, characterized in that: 4-Diethylaminoketo acid reacts with cyclohexanone, concentrated sulfuric acid, and perchloric acid to prepare compound 2; compound 2 reacts with 3-quinoline carboxaldehyde to prepare compound 3; compound 3 reacts with methyl trifluoromethanesulfonate to prepare compound 4; compound 4 reacts with 5-amino-2-hydroxymethylphenyl borate salt to prepare compound 1. 。 3. The preparation method according to claim 2, characterized in that: The specific preparation process is as follows: (1) Preparation of compound 2 Cyclohexanone was added dropwise to concentrated sulfuric acid at -4 to 0°C with constant stirring. Then 4-diethylaminoketo acid was slowly added. The reaction was carried out at 90 to 100°C for 3 to 4 hours. After the reaction was completed, the final reaction mixture was poured into ice water and perchloric acid was slowly added to precipitate the precipitate. The precipitate was filtered under reduced pressure, washed with cold water, and dried to obtain an orange-red solid compound 2. (2) Preparation of compound 3 3-quinoline carbaldehyde and piperidine were added to compound 2 in sequence, and then anhydrous ethanol was added to dissolve the mixture. The mixture was heated to reflux at 98-100°C overnight. After the reaction was completed, the temperature was restored to room temperature and then frozen. A purple solid precipitated out. The solid was washed with tert-butyl methyl ether and dried to obtain compound 3. (3) Preparation of compound 4 Add methyl trifluoromethanesulfonate and compound 3 to dichloromethane, stir at room temperature for 3-4 h, stop the reaction, freeze, precipitate a purple solid, filter under reduced pressure, wash with dichloromethane, and dry to obtain compound 4; (4) Preparation of compound 1 N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine and compound 4 were mixed, and dichloromethane was added to dissolve the mixture. Then, 5-amino-2-hydroxymethylphenyl borate salt was added to the mixture, and the mixture was stirred overnight at room temperature. After the reaction was completed, compound 1 was obtained by column chromatography.

4. The preparation method according to claim 3, characterized in that: The molar ratio of compound 2 to 3-quinoline carboxaldehyde is 1:1.5~1.

8.

5. The preparation method according to claim 3, characterized in that: The molar ratio of methyl trifluoromethanesulfonate to compound 3 is 4.08 to 6:

1.

6. The preparation method according to claim 3, characterized in that: The molar ratio of N,N'-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, 4-dimethylaminopyridine and compound 4 is (0.32~0.35): (0.13~0.16): (0.13~0.16): (0.13~0.16).

7. The preparation method according to claim 3, characterized in that: The concentrated sulfuric acid mentioned is 98% concentrated sulfuric acid.

8. The preparation method according to claim 3, characterized in that: The perchloric acid is 70% perchloric acid.

9. The application of the small molecule probe for detecting p53 dysfunction as described in claim 1, characterized in that: Application in the development of a real-time detection tool for p53 functionalization anomalies.

Images