A fluorescent probe and its preparation method and application

Abstract

The present invention discloses a fluorescent probe and its preparation method and application. The structural formula of the fluorescent probe of the present invention is: ‑ Cl ‑ Br ‑ , I ‑ The fluorescent probe of the present invention can rapidly undergo a bioorthogonal shear reaction to specifically activate fluorescence at the tumor site, thereby improving the imaging specificity of the target site. Simultaneously, the bioorthogonal coupling group of the probe is used to undergo biocoupling with cells, allowing the probe to be anchored on tumor cell membrane glycoproteins for long-term fluorescence imaging. The probe is suitable for non-invasive detection of tumors and surgical resection of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence imaging, and in particular to a fluorescent probe and a preparation method and application thereof. Background Art

[0002] Fluorescence imaging is an important tumor imaging method with advantages such as visualization, non-invasiveness, and real-time imaging, and is widely used. However, the fluorescent probes currently used in clinical practice (such as indocyanine green and methylene blue) are non-specific probes with low signal-to-noise ratio and short retention time in tumor tissue. Although researchers have long been committed to developing fluorescent probes that are specifically activated or coupled to tumor tissue to improve imaging specificity and prolong imaging time, achieving both precise activation and long-term retention of fluorescent probes in complex physiological environments remains a huge challenge.

[0003] Therefore, it is of great significance to develop a fluorescent probe that can achieve tumor-specific activation and retention in complex physiological environments, synergistically improve the fluorescence imaging signal-to-noise ratio and prolong the imaging time. Summary of the Invention

[0004] The purpose of the present invention is to provide a fluorescent probe and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention is:

[0006] A fluorescent probe, the structural formula of which is:

[0007]

[0008] A sort of.

[0009] A method for preparing the fluorescent probe as described above comprises the following steps:

[0010] 1) conducting a nucleophilic addition reaction of 2,3,3-trimethyl-3H-indole and an ethane halide to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, and conducting an amide addition reaction of cyclohexanone and N,N-dimethylformamide to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde;

[0011] 2) reacting 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde to obtain

[0012] 3) Conduct and 3,5-dihydroxybenzoic acid to obtain

[0013]

[0014] 4) Conduct and (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to obtain

[0015]

[0016] 5) Carry out The fluorescent probe is obtained by reacting with cyclopropanecyclooctyne polyethylene glycol.

[0017] Preferably, a method for preparing the fluorescent probe as described above comprises the following steps:

[0018] 1) dispersing 2,3,3-trimethyl-3H-indole and ethane halide in a solvent to carry out a nucleophilic addition reaction, and then separating and purifying the product to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide; dispersing cyclohexanone, N,N-dimethylformamide, and a catalyst in a solvent to carry out an amide addition reaction, and then separating and purifying the product to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde;

[0019] 2) dispersing 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde and a catalyst in a solvent for reaction, and then separating and purifying the product to obtain

[0020] 3) 3,5-Dihydroxybenzoic acid and a catalyst are reacted in a solvent, and then the product is separated and purified to obtain

[0021] 4) (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate and a catalyst are dispersed in a solvent for reaction, and then the product is separated and purified to obtain

[0022] 5) Cyclopropanecyclooctyne polyethylene glycol and a catalyst are dispersed in a solvent to react, and then the product is separated and purified to obtain a fluorescent probe.

[0023] Preferably, in step 1), the molar ratio of 2,3,3-trimethyl-3H-indole to ethane halides is 1:1-2.

[0024] Preferably, in step 1), the usage ratio of the 2,3,3-trimethyl-3H-indole to the solvent is 1 g: 20 mL to 50 mL.

[0025] Preferably, the halogenated ethane in step 1) is one of ethyl chloride, ethyl bromide and ethyl iodide.

[0026] Preferably, the solvent for the nucleophilic addition reaction in step 1) is acetonitrile.

[0027] Preferably, the nucleophilic addition reaction in step 1) is carried out at a temperature of 60° C. to 100° C., and the reaction time is 12 h to 48 h.

[0028] Preferably, the product separation and purification process of the nucleophilic addition reaction in step 1) comprises the following operations: adding the reaction product dropwise into anhydrous ether for precipitation, filtering, and drying the solid.

[0029] Preferably, the molar ratio of cyclohexanone to N,N-dimethylformamide in step 1) is 1:3-4.

[0030] Preferably, the molar ratio of N,N-dimethylformamide to the catalyst in step 1) is 1:1-2.

[0031] Preferably, the catalyst in step 1) is phosphorus oxychloride.

[0032] Preferably, the solvent for the amide addition reaction in step 1) is dichloromethane.

[0033] Preferably, the amide addition reaction in step 1) is carried out at a temperature of 40° C. to 60° C., and the reaction time is 4 h to 6 h.

[0034] Preferably, the separation and purification process of the product of the amide addition reaction in step 1) comprises the following operations: dropping the reaction product onto ice, crystallizing overnight, filtering, and drying the solid.

[0035] Preferably, in step 2), the molar ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde is 1 to 3:1.

[0036] Preferably, in step 2), the molar ratio of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde to the catalyst is 1:1 to 3.

[0037] Preferably, the catalyst in step 2) is sodium acetate.

[0038] Preferably, in step 2), the usage ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide and the solvent is 1 g:4 mL to 15 mL.

[0039] Preferably, the solvent in step 2) is acetic anhydride.

[0040] Preferably, the reaction in step 2) is carried out at a temperature of 120° C. to 1600° C., and the reaction time is 1 h to 2 h.

[0041] Preferably, the product separation and purification process in step 2) comprises the following operations: adding the reaction product dropwise into anhydrous ether for precipitation, filtering, and drying the solid.

[0042] Preferably, step 3) The molar ratio of 3,5-dihydroxybenzoic acid is 1:3-4.

[0043] Preferably, in step 3), the molar ratio of the compound 3,5-dihydroxybenzoic acid to the catalyst is 1:3-4.

[0044] Preferably, the catalyst in step 3) is triethylamine.

[0045] Preferably, step 3) The usage ratio of the solvent is 1g:20mL~50mL.

[0046] Preferably, the solvent in step 3) is acetonitrile.

[0047] Preferably, the reaction in step 3) is carried out at room temperature (20° C. to 25° C.) and the reaction time is 12 h to 24 h.

[0048] Preferably, the product separation and purification process in step 3) comprises the following operations: purifying the reaction product by column chromatography.

[0049] Preferably, step 4) The molar ratio of (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate is 1:1-2.

[0050] Preferably, in step 4), the molar ratio of the (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to the catalyst is 1:1-2.

[0051] Preferably, the catalyst in step 4) is triethylamine.

[0052] Preferably, in step 4), the ratio of the (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to the solvent is 1 g: 100 mL to 150 mL.

[0053] Preferably, the solvent in step 4) is N,N-dimethylformamide.

[0054] Preferably, the reaction in step 4) is carried out at room temperature for 48 to 72 hours.

[0055] Preferably, the product separation and purification process in step 4) comprises the following operations: purifying the reaction product by column chromatography.

[0056] Preferably, step 5) The molar ratio of cyclopropanecyclooctyne polyethylene glycol is 1:1-2.

[0057] Preferably, step 5) The molar ratio of the catalyst is 1:1-3.

[0058] Preferably, the catalyst in step 5) is at least one of N,N-diisopropylethylamine (DIPEA) and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU).

[0059] Preferably, step 5) The usage ratio of the solvent is 1g:150mL~250mL.

[0060] Preferably, the solvent in step 5) is at least one of N,N-dimethylformamide, dimethyl sulfoxide, and dichloromethane.

[0061] Preferably, the reaction in step 5) is carried out at room temperature for 12 to 24 hours.

[0062] Preferably, the product separation and purification process in step 5) comprises the following operations: purifying the reaction product by column chromatography.

[0063] A tumor detection reagent comprises the above fluorescent probe.

[0064] A detection reagent for guiding surgical resection of cancer tissue, comprising the fluorescent probe.

[0065] The beneficial effects of the present invention are: the fluorescent probe of the present invention can quickly undergo a bioorthogonal shear reaction to specifically activate fluorescence at the tumor site, thereby improving the imaging specificity of the target site. At the same time, the bioorthogonal coupling group of the probe is used to undergo a biocoupling effect with the cell, and the probe can be anchored on the tumor cell membrane glycoprotein for long-term fluorescence imaging, which is suitable for non-invasive detection of tumors and surgical resection of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 For compound 1 in the example 1 H NMR spectrum.

[0067] Figure 2 For compound 2 in the example 1 H NMR spectrum.

[0068] Figure 3 For compound 3 in the embodiment 1 H NMR spectrum.

[0069] Figure 4 is the compound CyOH in the examples 1 H NMR spectrum.

[0070] Figure 5 For the compound CyT in the examples 1 H NMR spectrum.

[0071] Figure 6 The fluorescent probe CyTB in the embodiment 1 H NMR spectrum.

[0072] Figure 7 The fluorescent probe CyB in the comparative example 1 H NMR spectrum.

[0073] Figure 8 This is a reaction route diagram of the click reaction between the fluorescent probe CyTB and Tz in the embodiment.

[0074] Figure 9 These are the UV spectra and fluorescence spectra of the fluorescent probe CyTB in the example reacting with different concentrations of Tz.

[0075] Figure 10 Graph showing the selectivity test results of the fluorescent probe CyTB in the examples.

[0076] Figure 11 This is the reaction rate constant test curve of the fluorescent probe CyTB in the example.

[0077] Figure 12 This is a laser confocal image of the fluorescent signal activation and retention of the fluorescent probe CyTB on the cell surface in the example.

[0078] Figure 13 This is a laser confocal image of the specific imaging of the fluorescent probe CyTB on the cell surface in the example.

[0079] Figure 14 This is a laser confocal image of the fluorescent probe CyTB in the example being captured and activated in the mobile phase.

[0080] Figure 15 Specific fluorescence imaging of the fluorescent probe CyTB in the example at the tumor site in living mice.

[0081] Figure 16 The diagram shows the fluorescence imaging and H&E staining results of the fluorescent probe CyTB in the example to guide the resection of isolated mouse organs.

[0082] Figure 17 Graph showing the safety test analysis results of the fluorescent probe CyTB in mice. DETAILED DESCRIPTION

[0083] The present invention will be further explained and illustrated below with reference to specific embodiments.

[0084] Example:

[0085] A fluorescent probe, the preparation method of which is as follows:

[0086] 1) 4.93 g (31 mmol) of 2,3,3-trimethyl-3H-indole and 7.25 g (46.5 mmol) of iodoethane were dissolved in 100 mL of acetonitrile, refluxed at 90 ° C for 24 h, cooled to room temperature naturally, concentrated by rotary evaporation, and the reaction product was added dropwise to anhydrous ether for precipitation. The filter residue was washed with ether three times and then dried in a vacuum drying oven for 24 h to obtain 8.78 g (27.9 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide (off-white block solid, designated as compound 1; yield 90%); 40 mL (324 mmol) of N,N-dimethylformamide was dissolved in 40 mL of anhydrous dichloromethane and precipitated under ice bath. After stirring for 10 minutes, 36 mL of a dichloromethane solution of phosphorus oxychloride (containing 383.4 mmol of phosphorus oxychloride and 30 mL of dichloromethane) was added dropwise under nitrogen protection. After the addition was completed, stirring was continued for 15 hours. Then 10.6 mL (102.4 mmol) of cyclohexanone precooled to 0°C was added and stirring was continued for 30 minutes. The mixture was heated to 55°C and reacted for 5 hours. The mixture was naturally cooled to room temperature, and the reaction solution was dropped onto ice. Crystallization was allowed to proceed overnight, and the residue was filtered. The filter residue was washed with water three times and then dried in a vacuum drying oven for 24 hours to obtain 10.57 g (61.44 mmol) of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde (bright yellow solid, designated as compound 2; yield 60%).

[0087] 2) 2.40 g (13.95 mmol) of 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde was dissolved in 36 mL of acetic anhydride, and 8.78 g (27.9 mmol) of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide and 2.29 g (27.9 mmol) of sodium acetate were added under a nitrogen atmosphere. The mixture was heated to 130 ° C. and reacted for 1 h. The mixture was filtered and the filtrate was added dropwise to anhydrous ether and precipitated with ether 3 times. The solid was filtered and dried in a vacuum drying oven for 24 h to obtain 8.0 g (12.6 mmol) of (a dark green solid with metallic luster, designated as compound 3; yield 90%);

[0088] 3) 1.0 g (1.57 mmol) of Dissolved in 40 mL of acetonitrile, and then slowly added 0.73 g (4.73 mmol) of 3,5-dihydroxybenzoic acid and 1.59 g (15.75 mmol) of triethylamine under a nitrogen atmosphere, stirred at room temperature for 12 h, the reaction was monitored by thin layer chromatography, the solvent was removed by rotary evaporation, and then purified by silica gel column chromatography. The eluent of the column chromatography consisted of dichloromethane and methanol in a volume ratio of 10:1 to obtain 0.27 g (0.47 mmol) of (dark green solid, recorded as compound CyOH; yield 30%);

[0089] 4) Dissolve 40 mg (0.14 mmol) of (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate in 5 mL of N,N-dimethylformamide, and add 14 mg (0.26 mmol) of triethylamine and 40 mg (0.07 mmol) of The reaction was then allowed to proceed at room temperature in the dark for 72 h. The reaction was monitored by thin layer chromatography. The solvent was removed by rotary evaporation and the product was purified by silica gel column chromatography. The eluent for the column chromatography consisted of dichloromethane and methanol in a volume ratio of 15:1 to obtain 29 mg (0.046 mmol) of (dark blue solid, denoted as fluorescent probe CyT; yield 33%);

[0090] 5) 29 mg (0.046 mmol) of The product was dissolved in 5 mL of N,N-dimethylformamide, and then 7.1 mg (0.055 mmol) of DIPEA and 20.9 mg (0.055 mmol) of HATU were added and stirred for 5 min. Then 16.9 mg (0.046 mmol) of cyclopropanecyclooctyne polyethylene glycol was added and the mixture was allowed to react for 24 h in the dark at room temperature. The completion of the reaction was monitored by thin-layer chromatography, and the solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography. The eluent of the column chromatography consisted of dichloromethane and methanol in a volume ratio of 20:1 to obtain 27 mg (0.025 mmol) of the fluorescent probe (dark blue solid, designated as probe CyTB; yield 55%).

[0091] The synthesis reaction of the fluorescent probe CyTB in this example is as follows:

[0092]

[0093] Comparative Example:

[0094] A fluorescent probe, the preparation method of which is as follows:

[0095] 24 mg (0.042 mmol) of The product was dissolved in 5 mL of N,N-dimethylformamide, and then 6.5 mg (0.05 mmol) of DIPEA and 19 mg (0.05 mmol) of HATU were added and stirred for 5 min. Then 15.5 mg (0.042 mmol) of cyclopropanecyclooctyne polyethylene glycol was added and the mixture was allowed to react for 24 h in the dark at room temperature. The completion of the reaction was monitored by thin-layer chromatography, and the solvent was removed by rotary evaporation. The product was purified by silica gel column chromatography. The eluent of the column chromatography consisted of dichloromethane and methanol in a volume ratio of 20:1 to obtain 24 mg (0.026 mmol) of the fluorescent probe (dark blue solid, designated as probe CyB; yield was 63%).

[0096] The synthesis reaction of the fluorescent probe CyB in this comparative example is as follows:

[0097]

[0098] Performance testing:

[0099] 1) Structural characterization of fluorescent probes:

[0100] The nuclear magnetic resonance hydrogen spectrum of compound 1 in the embodiment ( 1 H NMR) images Figure 1 As shown, compound 2 1 H NMR spectrum Figure 2 As shown, compound 3 1 H NMR spectrum Figure 3 As shown, the compound CyOH 1 H NMR spectrum Figure 4 As shown, compound CyT 1 HNMR images Figure 5 As shown, the fluorescent probe CyTB 1 H NMR spectrum Figure 6 shown.

[0101] The fluorescent probe CyB in the comparative example 1 H NMR spectrum Figure 7 shown.

[0102] Depend on Figures 1 to 7 It can be seen that the bioorthogonal fluorescent probe with two click groups, TCO and BCN, was successfully synthesized in the embodiment.

[0103] 2) UV and fluorescence spectra of the fluorescent probe CyTB reacting with different concentrations of Tz:

[0104] The fluorescent probe CyTB was dissolved in PBS buffer containing 10% (volume percentage) DMSO to prepare a solution with a concentration of 100 μM. Then, different concentrations (0-100 μM) of 6-methyl-1,2,4,5-tetrazine-3-mercaptoacetic acid (Tz) were added and incubated in a shaker at 37°C for 30 minutes (the reaction route of the click reaction between the fluorescent probe CyTB and Tz is shown in Figure 2). Figure 8 The UV spectrum and fluorescence spectrum of the reaction solution were measured using an UV spectrophotometer and a fluorescence spectrophotometer. The test results are shown in FIG. Figure 9 As shown (a is the ultraviolet spectrum, b is the fluorescence spectrum).

[0105] Depend on Figure 9 As can be seen from a in the figure, with the addition of Tz to the reaction, the maximum ultraviolet absorption of the fluorescent probe CyTB undergoes an obvious red shift.

[0106] Depend on Figure 9 As shown in b, with the increase of Tz concentration (0-100 μM), the fluorescence signal of the fluorescent probe CyTB showed a 6-fold enhancement, and this fluorescence intensity enhancement was positively correlated with the Tz concentration.

[0107] 3) Selectivity test of fluorescent probe CyTB:

[0108] Different fluorescent probes (control group: fluorescent probe CyT / CyB; experimental group: fluorescent probe CyTB) were reacted with Tz respectively, and the reactivity of fluorescent probes with Tz was detected by high performance liquid chromatography. Fluorescent probe CyTB was mixed with different interfering substances, and the fluorescence intensity was tested by small animal in vivo imaging. The selectivity test results of fluorescent probe CyTB were as follows: Figure 10 (a is a high performance liquid chromatography diagram, b is a fluorescence imaging diagram and a fluorescence intensity test result diagram).

[0109] Depend on Figure 10 From a in the figure, we can see that different fluorescent probes (containing TCO or BCN groups) can undergo click reactions with Tz and have good reactivity.

[0110] Depend on Figure 10 From b in Figure 3, we can see that when the fluorescent probe CyTB is mixed with other interfering substances, other substances except Tz fail to cause changes in the fluorescence intensity of the fluorescent probe CyTB, indicating that the fluorescent probe CyTB has good reaction selectivity.

[0111] 4) Reaction rate constant test of fluorescent probe CyTB:

[0112] The fluorescence intensity of the fluorescent probe CyT and different concentrations of Tz (0-50 μM) incubated for 5 minutes in 10% (volume percentage) DMSO PBS buffer and 10% mass fraction cell lysate was measured by a microplate reader, and its secondary reaction rate constant was calculated based on the fluorescence change. For the bioorthogonal coupling reaction of the fluorescent probe CyB and Tz, Tz was incubated with different concentrations of BCN (0-5 μM) in 10% (volume percentage) DMSO PBS buffer and 10% mass fraction cell lysate, and the consumption of Tz was detected by ultraviolet absorption and its secondary reaction rate constant was calculated. The reaction rate constant curve of the fluorescent probe CyTB is shown in Figure 2. Figure 11 (a is the reaction rate constant curve of the bioorthogonal cleavage reaction between CyT and Tz, and b is the reaction rate constant curve of the bioorthogonal coupling reaction between the fluorescent probe CyB and Tz) as shown (the fluorescent probe CyT and Tz will undergo a bioorthogonal cleavage reaction, and the fluorescent probe CyB and Tz will undergo a bioorthogonal coupling reaction. By detecting the reaction rates of the two reactions, the reaction conditions of the two groups in the fluorescent probe CyTB and Tz can be inferred).

[0113] Depend on Figure 11 It can be seen that in the cell lysate reaction system, the reaction rate of bioorthogonal shearing between fluorescent probe CyT and Tz is 11.8M -1 ·s -1 ±0.45M -1 ·s -1 The bioorthogonal coupling reaction rate of fluorescent probe CyB and Tz is 9.73M -1 ·s -1 ±0.56M -1 ·s -1 The reaction rate constants of the two reactions differ by less than one order of magnitude, which provides a basis for the simultaneous occurrence of the two reactions in the fluorescent probe CyTB.

[0114] 5) Laser confocal imaging test of fluorescence signal activation and retention of the fluorescent probe CyTB on the cell surface:

[0115] KPC cells were seeded in a confocal microplate. After culturing for 12 hours, the adherent KPC cells were incubated with the pre-targeting molecule tetrazine sugar (MTz; 200 nM; prepared by reference to Acta Pharm Sin B. 2023 Jun; 13(6): 2736-2746) for 24 hours to label the cell surface with Tz molecules. The cells were then incubated with different fluorescent probes for 20 minutes, and then stained with Hoechst33342 for nuclear labeling and fluorescence imaging was performed by laser confocal microscopy (channels: DAPI, Cy5). The test results are shown in Figure 2. Figure 12 shown.

[0116] Depend on Figure 12 It can be seen that when only the fluorescent probe CyTB exists in the culture medium, no red fluorescence appears within 72 hours, indicating that the prepared fluorescent probe CyTB has good stability and will not spontaneously activate fluorescence under the cellular metabolic environment; the MTz+CyT group shows obvious red fluorescence after 5 minutes of incubation, which lasts until 24 hours. After 48 hours, the red fluorescence is significantly weakened and is almost invisible after 72 hours; in the MTz+CyTB group, the fluorescence intensity first increases and then slowly decreases. After 72 hours, the red fluorescence still exists, showing good fluorescence activation and retention effects.

[0117] 6) Laser confocal imaging test of specific imaging of fluorescent probe CyTB on the cell surface:

[0118] MTz was coupled to cetuximab (Cet) (which binds to the EGFR receptor) to prepare Cet-MTz as a pre-targeting molecule for high EGFR expression. KPC cells (high EGFR expression) and MEF cells (low EGFR expression) were selected as verification cells. KPC cells were pre-stained with CFSE (green fluorescence) in advance. KPC cells and MEFs were then co-cultured in a confocal microplate. After Cet-MTz pretreatment and replacement of the supernatant, CyTB probe was added and incubated for 30 minutes. After replacement of the supernatant, Hoechst33342 was added to stain the nucleus and the activation of the probe in the cells was detected by confocal microscopy (channels: DAPI, Cy5, FITC). The test results are as follows: Figure 13 shown.

[0119] Depend on Figure 13 It can be seen that the fluorescent probe CyTB can activate imaging in tumor cells with high EGFR expression, but cannot be activated in cells with low EGFR expression, and has specific imaging function.

[0120] 7) Laser confocal imaging test of the fluorescent probe CyTB being captured and activated in the mobile phase:

[0121] The microfluidic chip device of ibidi (μ-Slide I 0.4Luer, 80176, Germany) was used to simulate the process of probe being captured and activated in the blood flow. The chip was 50 mm long, 5.0 mm wide, and 0.4 mm high. 100 μL of cell suspension (2×10 6 mL -1 ) incubated, and after the cells stabilized, the fluorescent probe CyTB (20 μM) or fluorescent probe CyT (20 μM) solution was prepared into a 50 mL syringe as the mobile phase and injected into the chip at a constant speed through a syringe pump, with a flow rate controlled at 1 mL / min. After different time periods, the cells on the chip were nuclear stained and the activation of the probes was detected by confocal microscopy (channels: DAPI, Cy5). The test results are shown in Figure 2. Figure 14 shown.

[0122] Depend on Figure 14 It can be seen that the fluorescent probe CyT, which can only perform bioorthogonal cleavage activation, has a poor retention effect in the simulated vascular environment, while the fluorescent probe CyTB, which has both bioorthogonal activation and coupling functions, has a significantly prolonged retention time.

[0123] 8) Specific fluorescence imaging of the fluorescent probe CyTB at tumor sites in living mice:

[0124] Fifteen C57BL / 6 mice were orthotopically implanted with pancreatic cancer. One week later, the successful implantation was confirmed by bioluminescence. The tumor-bearing mice were randomly divided into five groups, with three mice in each group, and labeled as G1: blank group, G2: CyOH group, G3: Cet-MTz+CyT group, G4: Cet-MTz+CyB group, and G5: Cet-MTz+CyTB group. Groups G3, G4, and G5 were continuously injected with Cet-MTz (20 mg / kg) for 3 days for pre-targeting (once a day). On the 4th day, the Cet-MTz group was injected with CyOH (20 mg / kg). On the first three days, the fluorescent probes CyT, CyB, and CyTB (1 mg / kg) were injected through the tail vein of the mice, respectively. The G1 group was injected with the same volume of normal saline at the same time point each time. The G2 group was injected with the same volume of normal saline for the first three days and CyOH (1 mg / kg) on ​​the fourth day. The distribution of the probes in the mice was observed by small animal in vivo imaging at different time points after injection. The changes in fluorescence intensity and tumor tissue / normal tissue fluorescence intensity ratio (TNR) of each group were further quantified. The test results are shown in Figure 2. Figure 15 (a is a fluorescence imaging diagram, b is the average radiation efficiency of the tumor-injection time relationship curve, and c is the TNR-time relationship curve).

[0125] Depend on Figure 15 It can be seen that: except for the blank group, the fluorescence intensity of the other groups first increased and then decreased with time, but the fluorescence decrease rate of the two groups injected with coupled fluorescent probes (CyB, CyTB) was significantly slower than that of groups G2 and G3, verifying that the probes have a good retention effect; secondly, quantitative analysis showed that group G5 had the highest signal-to-noise ratio at all time points, and the signal-to-noise ratio reached more than 15 as time increased; the above results all indicate that in the mouse in situ pancreatic cancer model, the fluorescent probe CyTB has the advantages of tumor-specific activation, high signal-to-noise ratio (TNR>15) and long-term imaging (>130h).

[0126] 9) Testing the performance of the fluorescent probe CyTB in guiding resection in isolated mouse organs:

[0127] As above, mice were implanted with pancreatic cancer in situ and dosed in groups. 48 hours after dosing, the mice were euthanized and the tumor tissue at the pancreatic cancer site was removed according to fluorescence imaging. The removed tumor tissue was then stained with H&E for observation. At the same time, the main organs (heart, liver, spleen, lung, kidney) and muscle tissue of each group were obtained. The fluorescence intensity of each tissue sample was detected by small animal live imaging. The test results are as follows: Figure 16 shown.

[0128] Depend on Figure 16 It can be seen that after the G5 group probe guided the resection of the tumor, no fluorescence was observed in the tissues surrounding the tumor, and there was almost no fluorescence in other ex vivo organs. Combined with the clean tumor resection margins of the G5 group in H&E staining, this indicates that the fluorescent probe CyTB has excellent tumor specificity and can effectively distinguish tumor tissue from normal tissue.

[0129] 10) Safety testing of the fluorescent probe CyTB in mice:

[0130] Fifteen C57BL / 6 mice were randomly divided into five groups as above. 24 hours after administration, the mice were eyeballed and blood was collected. Blood samples for physiological indicators were collected using EDTA anticoagulant tubes, and blood samples for biochemical indicators were collected using 1.5 mL centrifuge tubes. The physiological and biochemical indicators of each group were measured. The test results are shown in Table 1. Figure 17 shown.

[0131] Depend on Figure 17 It can be seen that after different drug treatments, the mice's alkaline phosphatase (ALP), alanine aminotransferase (ALT), urea nitrogen (BUN), creatinine (CREN), white blood cell (WBC), red blood cell count (RBC), hemoglobin concentration (HGB) and hematocrit (HCT) indicators were all within the normal range, indicating that the fluorescent probe CyTB has almost no effect on the liver and kidney function and routine blood indicators of mice.

[0132] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A fluorescent probe, characterized in that The structural formula is: , where X - Cl - Br - , I - One of them.

2. A method for preparing a fluorescent probe according to claim 1, characterized in that: The following steps are involved: 1) A nucleophilic addition reaction of 2,3,3-trimethyl-3H-indole and an ethane halide is performed to obtain 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide, and an amide addition reaction of cyclohexanone and N,N-dimethylformamide is performed to obtain 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde. The catalyst for the amide addition reaction is phosphorus oxychloride. 2) Reaction of 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide and 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde to obtain ; 3) Conduct and 3,5-dihydroxybenzoic acid to obtain ; 4) Conduct and (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate to obtain ; 5) Conduct and The fluorescent probe is obtained by the reaction.

3. The preparation method according to claim 2, wherein: The molar ratio of the 2,3,3-trimethyl-3H-indole to the halogenated ethane in step 1) is 1:1-2; the molar ratio of the cyclohexanone to N,N-dimethylformamide in step 1) is 1:3-4.

4. The preparation method according to claim 2, wherein: In step 2), the molar ratio of the 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium halide to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde is 1 to 3:

1.

5. The preparation method according to claim 2, wherein: Step 3) The molar ratio of 1,2-dihydroxybenzoic acid to 3,5-dihydroxybenzoic acid is 1:3-4.

6. The preparation method according to claim 2, wherein: Step 4) , (2E)-trans-cyclooctene-(4-nitrophenyl) carbonate is 1:1-2.

7. The preparation method according to claim 2, characterized in that: Step 5) 、 The molar ratio is 1:1~2.

8. The preparation method according to any one of claims 2 to 7, characterized in that: The nucleophilic addition reaction in step 1) is carried out at a temperature of 60°C to 100°C, and the reaction time is 12h to 48h; the amide addition reaction in step 1) is carried out at a temperature of 40°C to 60°C, and the reaction time is 4h to 6h; the reaction in step 2) is carried out at a temperature of 130°C, and the reaction time is 1h to 2h; the reaction in step 3) is carried out at room temperature, and the reaction time is 12h to 24h; the reaction in step 4) is carried out at room temperature, and the reaction time is 48h to 72h; the reaction in step 5) is carried out at room temperature, and the reaction time is 12h to 24h.

9. A tumor detection reagent, characterized in that: The method comprises the fluorescent probe according to claim 1.

10. A detection reagent for guiding surgical resection of cancer tissue, characterized in that: The method comprises the fluorescent probe according to claim 1.

Images