High-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe and application thereof

Abstract

The invention belongs to the technical field of small-molecule fluorescent probes, and discloses a high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe and application thereof, the structural formula of the fluorescent probe is as shown in formula I, ; in the formula, X1 is a carbon atom or an oxygen atom; x2 is a halide ion or a tetrafluoroboric acid ion; n is 0-5; m is 0-2; r1 and R2 are hydrogen, methyl or ethyl; r3 is an electron donor substituent; r < 4 > is 4-dimethylamino phenyl or 6-dimethylamino naphthyl. The fluorescent probe provided by the invention can accurately identify glioma cells carrying mutant isocitrate dehydrogenase 1 and indicate tumor boundaries, can be used as a targeted fluorescent imaging agent, and has a huge potential of being applied to glioma fluorescence-guided resection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of small molecule fluorescent probe, and specifically discloses a high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe and application thereof. BACKGROUND

[0002] The statements herein are provided only to enhance understanding of the present application and are not necessarily intended to constitute the prior art.

[0003] Glioma is a central nervous system tumor originating from glial cells, and is the main cause of primary brain tumor death. Surgical resection is the preferred treatment, but due to its infiltrative growth, the boundary is unclear, it is difficult to completely resect, and it is prone to recurrence and reduces the survival rate.

[0004] Glioma is divided into four grades: grade I-II is low grade, and grade III-IV is high grade. The higher the grade, the higher the malignancy. Most of grade II-III glioma and secondary glioblastoma (grade IV) have IDH1 mutations. IDH1 is a key enzyme in the tricarboxylic acid cycle, which normally catalyzes isocitrate to α-ketoglutarate; after mutation, the function changes, catalyzing α-ketoglutarate to generate carcinogenic D-2-hydroxyglutarate. mIDH1 is an important marker for glioma grading and treatment, and IDH1 inhibitors (such as Ivosidenib) have been used in clinical research, and NCCN guidelines recommend its use for IDH mutant astrocytoma treatment.

[0005] At present, fluorescence-guided surgery using tumor-targeting imaging agents has become an effective method for intraoperative detection and resection of tumors, among which the commonly used fluorescent imaging agents include 5-aminolevulinic acid (5-ALA), fluorescein sodium, indocyanine green (ICG), and tozuleristide (BLZ-100). 5-ALA accumulates less in low-grade glioma, and the fluorescence intensity is low, which is not easy to distinguish from normal brain tissue. The specificity of fluorescein sodium and ICG is low, which limits its large-scale application. Tozuleristide is a new type of peptide fluorescent imaging agent developed in recent years, and the research on fluorescence-guided surgery for glioma is currently in the clinical trial stage. Finding a fluorescent imaging agent targeting glioma for precise positioning of glioma is crucial for the diagnosis and treatment of glioma, and is also a difficult problem that needs to be solved in the field of glioma research. SUMMARY

[0006] In view of the deficiencies in the prior art, the purpose of the present application is to provide a high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe and application thereof.

[0007] In order to achieve the above object, the present application is realized by the following technical scheme: In the first aspect, the present application provides a high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe, whose structural formula is shown as formula I:

[0008] In the formula, X1 is a carbon atom or an oxygen atom; X2 is a halogen ion or a tetrafluoroborate ion; n is 0-5; m is 0-2; R1 and R2 are hydrogen, methyl or ethyl; R3 is an electron-donating substituent; R4 is a 4-dimethylaminophenyl or a 6-dimethylaminonaphthyl.

[0009] In the second aspect, the present application provides the application of the high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe in any of the following: (1) for the mechanism and pharmaceutical research of mutant isocitrate dehydrogenase 1; (2) for screening of mutant isocitrate dehydrogenase 1 inhibitors; (3) for preparing diagnostic reagents for mutant isocitrate dehydrogenase 1 related diseases; (4) for preparing fluorescent imaging agents of mutant isocitrate dehydrogenase 1.

[0010] The beneficial effects achieved by one or more embodiments of the present application are as follows: (1) The fluorescent probe of the present application is a small molecule inhibitor of mIDH1 and a fluorescent group with blood-brain barrier permeability, which is covalently coupled to construct a small molecule fluorescent probe. The fluorescent probe has superior environmental sensitivity. With the decrease of the external environment polarity, its fluorescence significantly enhances. After binding with mutant isocitrate dehydrogenase 1, it shows significant fluorescence enhancement, which improves the signal-to-noise ratio and enables visual research of mutant isocitrate dehydrogenase 1 without washing.

[0011] (2) The fluorescent probe of the present application has a large stokes shift and a near-infrared fluorescence emission wavelength, which can reduce the interference of excitation light and biological body fluorescence during fluorescence imaging research, and improve the sensitivity of the probe.

[0012] (3) The fluorescent probe of the present application has extremely high affinity and superior selectivity for mutant isocitrate dehydrogenase 1, which realizes specific labeling of mutant isocitrate dehydrogenase 1 in proteins, cells and animals.

[0013] ​(4) The fluorescent probe of the present application can accurately identify glioma cells carrying mutant isocitrate dehydrogenase 1, indicate tumor boundaries, can be used as a targeted fluorescent imaging agent, and has great potential for application in fluorescent guided surgical resection of glioma. BRIEF DESCRIPTION OF DRAWINGS

[0014] The accompanying drawings, which form a part of this specification, are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification. The embodiments of the present application, and their

[0015] Figure 1 In FIG. 1, A is the ultraviolet-visible absorption spectrum of the fluorescent probe F01~F05 in water; B is the fluorescence emission spectrum of the fluorescent probe F01~F05 in water; C is the fluorescence emission spectrum of the fluorescent probe F01 in different solvents; D is the fluorescence emission spectrum of the fluorescent probe F02 in different solvents; E is the fluorescence emission spectrum of the fluorescent probe F03 in different solvents; F is the fluorescence emission spectrum of the fluorescent probe F04 in different solvents; G is the fluorescence emission spectrum of the fluorescent probe F05 in different solvents; the data is shown as mean ± SEM, n = 3.

[0016] Figure 2 In FIG. 2, A is the fluorescence response comparison chart of the fluorescent probes F01~F05 to mIDH1 and IDH1 protein; B is the fluorescence change chart of the fluorescent probes F01~F05 after incubation with glioma cells LN229 or LN229 cells stably transfected with mIDH1. Increased FI represents increased fluorescence intensity; the data is shown as mean ± SEM, n = 3; Figure 3 In FIG. 3, the fluorescence imaging chart of the fluorescent probes F01~F05 (0.25 μM) in LN229 cells stably transfected with mIDH1, wherein A is F01 and F03; B is F02 and antagonist Safusidenib; C is F04 and antagonist Safusidenib; D is F05 and antagonist Safusidenib; the concentration of antagonist Safusidenib is 10 μM, and the scale is 20 μm; Figure 4 In FIG. 4, the imaging chart of the fluorescent probe F04 in a nude mouse glioma orthotopic model, wherein A is the bioluminescence imaging chart; B is the fluorescence imaging chart at different administration time points after intravenous injection of probe F04 solution, and blank represents the fluorescence imaging chart before administration; C is the fluorescence imaging chart of brain tissue 1 hour after injection of probe F04. DETAILED DESCRIPTION

[0017] It should be noted that the following detailed description is illustrative only and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0018] In a first aspect, the present application provides a high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe, whose structural formula is shown as Formula I:

[0019] ; In the formula, X1 is a carbon atom or an oxygen atom; X2 is a halogen ion or a tetrafluoroborate ion; n is 0-5; m is 0-2; R1 and R2 are hydrogen, methyl or ethyl; R3 is an electron-donating substituent; R4 is a 4-dimethylaminophenyl or a 6-dimethylaminonaphthyl group.

[0020] In some embodiments, R3 is N, N-dimethylamino or N, N-diethylamino.

[0021] Preferably, R3 is N, N-dimethylamino.

[0022] In some embodiments, R1 and R2 are methyl.

[0023] In some embodiments, X2 is a tetrafluoroborate ion.

[0024] In some embodiments, n is 0-2; m is 1.

[0025] In some embodiments, the high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe is selected from any one of the following compounds: ; ; .

[0026] Preferably, the high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe is F04, whose structural formula is: .

[0027] Preferably, the high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe is F05, whose structural formula is: .

[0028] In a second aspect, the present application provides the use of the high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe in any one of the following: (1) Mechanism and pharmaceutical research for mutant isocitrate dehydrogenase 1; (2) Screening for inhibitors of mutant isocitrate dehydrogenase 1; (3) Preparation of diagnostic reagents for mutant isocitrate dehydrogenase 1 related diseases; (4) Preparation of fluorescent imaging agents for tumor cells carrying mutant isocitrate dehydrogenase 1.

[0029] In some embodiments, the mutant isocitrate dehydrogenase 1 related disease is brain glioma, chondrosarcoma, and acute leukemia.

[0030] In some embodiments, the tumor is brain glioma or chondrosarcoma.

[0031] The following examples can make the professional technical personnel more fully understand the present application, but do not limit the present application in any way.

[0032] Example 1 Preparation of fluorescent probes F01~F05:

[0033] Synthesis of intermediate L01: 2,4,6-trichlorobenzaldehyde (2095 mg, 10 mmol) and hydroxylamine hydrochloride (764 mg, 11 mmol) were dissolved in 50 mL of ethanol, pyridine (791 mg, 10 mmol) was added, stirred at room temperature for 45 minutes, pH was adjusted to 2 with 1M hydrochloric acid solution, 200 mL of dichloromethane was added, washed with saturated sodium chloride solution, water, dried over anhydrous sodium sulfate, filtered, concentrated to obtain solid product 2.11 g, yield 94.0 %. 1 H NMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 8.12 (s, 1H), 7.40(s, 2H);ESI-MS: m / z [M + 2] + calcd for C7H4Cl3NO + 224.93, found 225.04.

[0034] Synthesis of intermediate L02: Intermediate L01 (2245 mg, 10 mmol) and N-chlorosuccinimide (1375 mg, 10.3 mmol) were dissolved in 13 mL of DMF, stirred at room temperature for 1.5 h, then water and methyl tert-butyl ether were added, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, concentrated to obtain solid product 2.51 g, yield 96.9 %. 1H NMR (400 MHz, CDCl3) δ 8.32(s, 1H), 8.12 (s, 1H), 7.40 (s, 2H);ESI-MS: m / z [M +Na+ H] + calcd for C7H7Cl4N2O + 274.93, found 274.37.

[0035] Synthesis of intermediate L03: 2-Fluoroisobutyric acid (5305 mg, 50 mmol) was dissolved in tetrahydrofuran, cooled to 15-20 °C, and CDI (8513 mg, 52.5 mmol) was added portionwise. After stirring at room temperature for 30 min, it was added to a suspension of potassium monomethyl malonate (18.74 g, 120 mmol) and MgCl2(5713 mg, 60 mmol) in tetrahydrofuran, and the mixture was stirred at 50 °C until the starting material disappeared. The pH was adjusted to 4 with 2M hydrochloric acid solution, and the mixture was extracted with ethyl acetate, washed with saturated sodium bicarbonate and sodium chloride solutions, dried over anhydrous sodium sulfate, filtered, and concentrated to give the product as an oil, 6526 mg, in 80.5 % yield. 1 H NMR (400 MHz, CDCl3) δ 8.32(s, 1H), 8.12 (s, 1H), 7.40 (s, 2H);ESI-MS: m / z [M +Na+ H] + calcd for C7H 12 FNaO3 + 186.07, found 186.26.

[0036] Synthesis of intermediate L04: Intermediate L03 (973 mg, 6 mmol) was dissolved in 40 mL of tetrahydrofuran, cooled to 0-10 °C, and potassium tert-butoxide (673 mg, 6 mmol) was added. After stirring for 40 min, a solution of intermediate L02 (1295 mg, 5 mmol) in tetrahydrofuran was added dropwise, and the mixture was stirred overnight at room temperature. The solvent was evaporated, ethyl acetate and water were added, and the organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. The product was purified by column chromatography (petroleum ether / ethyl acetate = 100:1) to give 1100 mg of the product in 60.0 % yield. 1 H NMR (400 MHz, CDCl3) δ 7.45 (s, 2H), 3.70 (s, 3H), 1.97 (s, 3H), 1.92 (s,3H);ESI-MS: m / z [M +Na] + calcd for C14 H 11 Cl3FNNaO3 + 387.97, found 387.90.

[0037] Synthesis of intermediate L05: Intermediate L04 (1100 mg, 3 mmol) was dissolved in 30 mL of tetrahydrofuran, and a solution of NaOH (240 mg, 6 mmol) in water (18 mL) was added dropwise. The mixture was stirred at room temperature overnight, and the pH was adjusted to 2 with 2M hydrochloric acid solution. The mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated by suction filtration. The product was purified by column chromatography (dichloromethane / methanol = 20:1) to obtain a white solid with a yield of 858 mg (81.1 %). 1 H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 2H), 1.93 (s, 3H),1.88 (s, 3H);ESI-MS: m / z [M-H] - calcd for C 13 H8Cl3FNO3 - 349.96, found 350.14 m / z;[2M-H+2] - calcd for C 26 H 17 Cl6F2N2O6 - 702.92, found 702.81.

[0038] Synthesis of intermediate L06: To a reaction flask was added 3-methyl-4-bromo-1H-indole (1050 mg, 5 mmol), tert-butyl acrylate (1282 mg, 10 mmol), palladium acetate (17 mg, 0.075 mmol), 2-diphenylphosphino-2'-methylbiphenyl (53 mg, 0.15 mmol), triethylamine (1012 mg, 10 mmol), and 15 mL of DMF. The mixture was heated to 135°C under anhydrous and anaerobic conditions, and the reaction was allowed to proceed for 1-2 h. The mixture was cooled to room temperature, and 150 mL of ethyl acetate was added. The mixture was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and concentrated. The product was purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to obtain a yellow solid with a yield of 1270 mg (98.7 %). 1 H NMR (400 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.40 (d, J =15.7 Hz, 1H), 7.48-7.37 (m, 2H), 7.24 (dd, J= 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, + calcd for C 16 H 20 NO2 + 258.15, found257.94。

[0039] Synthesis of intermediate L07: Intermediate L05 (300 mg, 0.85 mmol) was dissolved in 10 mL of tetrahydrofuran, and sulfurous dichloride (1517 mg, 12.75 mmol) and DMF (40 μL) were added, and refluxed for 2 h, and the solvent and residual sulfurous dichloride were evaporated, and dissolved in 10 mL of tetrahydrofuran, and potassium tert-butoxide (202 mg, 1.8 mmol) was added, and stirred at room temperature for 2 h, and the above acyl chloride solution was added dropwise, and stirred at room temperature for 2-3 h, and the solvent was evaporated, and ethyl acetate and water were added, and the organic phase was washed with saturated sodium bicarbonate, saturated sodium chloride solution, and dried over anhydrous sodium sulfate, and suction filtered, and concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 60: 1) to obtain 240 mg of white solid product, with a yield of 67.6 %. 1 H NMR (400 MHz, CDCl3) δ8.42 (dd, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, J = 7.7 Hz, 1H), 6.43 (d, + calcd for C 29 H 28 Cl3FKN2O5+ 647.07, found 646.97.

[0040] Synthesis of intermediate L08: Intermediate L07 (231 mg, 0.39 mmol) was dissolved in 10 mL of dichloromethane, trifluoroacetic acid (1112 mg, 9.75 mmol) was added, stirred at room temperature overnight, evaporated the solvent and residual trifluoroacetic acid to get the white solid product 200 mg, yield 95.7 %. 1 H NMR (400 MHz, CDCl3) δ 8.54-8.43 (m, 2H), 7.58 (d, J =7.7 Hz, 1H),7.36 (s, 2H), 7.35 (d, J =16.1 Hz, 1H), 7.02 (s, 1H), 6.45 (d, J =15.7 Hz, 1H),2.42 (s, 3H), 1.91 (s, 3H), 1.86 (s, 3H);ESI-MS: m / z [M+2+H] + calcd for C 25 H 19 Cl3FN2O4 + 537.04, found 537.01.

[0041] Synthesis of intermediate L09a: Intermediate L08 (107 mg, 0.2 mmol) was dissolved in 4 mL of DMF, HATU (80 mg, 0.21 mmol) was added, stirred at room temperature for 1 h, then N-tert-butoxycarbonyl-1,2-ethanediamine (35 mg, 0.22 mmol) and DIPEA (52 mg, 0.4 mmol) were added, continued to stir for 1 h, added water and ethyl acetate, separated the organic layer, washed with saturated sodium bicarbonate solution, 1M hydrochloric acid solution and saturated sodium chloride solution in turn, dried with anhydrous sodium sulfate, concentrated, washed with petroleum ether, evaporated to dryness to get the white solid product 98 mg, yield 72.1 %. 1 H NMR (400 MHz, CDCl3) δ 8.41 (d, J =8.2 Hz, 1H), 8.31 (d, J =15.3 Hz, 1H), 7.47 (d, J= 7.7 Hz, 1H), 7.35 (s, 2H), 7.39- 7.27 (m, 1H), 6.97(s, 1H), 6.47 (s, 1H), 6.36 (d, J = 15.3 Hz, 1H), 4.95 (s, 1H), 3.51 (t, J =5.6Hz, 2H), 3.36 (d, J =6.0 Hz, 2H), 2.41 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H),1.44 (s, 9H);ESI-MS: m / z [M] + calcd for C 32 H 32 Cl3FN4O5 + 676.14, found 676.68.

[0042] Synthesis of intermediate L09b: Intermediate L09b was prepared according to the procedure for the synthesis of L09a using N-BOC-1,5-diaminopentane as starting material to give the off-white solid product 136 mg in 94.4 % yield. 1 H NMR (400 MHz, CDCl3) δ 8.40 (d, J =8.2 Hz,1H), 8.33 (d, J =15.4 Hz, 1H), 7.48 (d, J =7.6 Hz, 1H), 7.38-7.27 (m, 3H), 6.97(s, 1H), 6.39 (d, J =15.2 Hz, 1H), 5.90 (s, 1H), 4.59 (s, 1H), 3.40 (q, J =6.6Hz, 2H), 3.19-3.06 (m, 2H), 2.41 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H), 1.56-1.33 (m, 15H);ESI-MS: m / z [M +2] + calcd for C 35 H 38 Cl3FN4O5 + 720.19, found 720.94.

[0043] Synthesis of intermediate L09c: Intermediate L09c was prepared according to the synthetic procedure of L09a using N-BOC-1,8-octanediamine as starting material to give the white solid product 150 mg in 98.7% yield. 1 H NMR (400 MHz, CDC13) δ 8.40 (d, J = 8.2 Hz, 1H), 8.33 (d, J = 15.2 Hz, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.35 (s, 2H), 7.30 (t, J = 8.7 Hz, 1H), 6.97 (s, 1H), 6.35 (d, J = 15.2 Hz, 1H), 5.66 (s, 1H), 4.52 (s, 1H), 3.39 (q, J = 6.7 Hz, 2H), 3.10 (d, J = 6.8 Hz, 2H), 2.41 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H), 1.44 (s, 10H), 1.36 - 1.23 (m, 11H); ESI-MS: m / z [M+2] + C 38 H 44 Cl3FN4O5 + 762.23, found 762.85.

[0044] Synthesis of intermediate L09d: Intermediate L09d was prepared according to the synthetic procedure of L09a using N-BOC-2-(2-aminoethoxy)ethanamine as starting material to give the off-white solid product 135 mg in 93.8% yield. 1 H NMR (400 MHz, CDC13) δ 8.40 (d, J = 8.2 Hz, 1H), 8.33 (d, J = 15.3 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.35 (s, 2H), 7.30 (t, J = 7.9 Hz, 1H), 6.97 (s, 1H), 6.47 (d, J= 15.3 Hz, 1H), 6.35 (s, 1H), 4.85 (s, 1H), 3.63-3.58 (m, 4H), 3.55 (t, J = 5.1 Hz, 2H), 3.34 (d, J = 5.6 Hz, 2H),2.42 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H), 1.45 (s, 9H);ESI-MS: m / z [M+2] + calcd for C 34 H 36 Cl3FN4O6 + 722.17, found 722.84.

[0045] Synthesis of intermediate L09e: Intermediate L09e was prepared according to the procedure for the synthesis of L09a, using N-BOC-2-[2-(2-amino-ethoxy)ethoxy]-ethylamine as starting material to give the off-white solid product 150 mg in 98.0 % yield. 1 H NMR (400 MHz, CDCl3) δ8.40 (d, J = 8.0 Hz, 1H), 8.34 (d, J = 15.2 Hz, 1H), 7.49 (d, J = 7.7 Hz, 1H), 7.35 (s, 2H), 7.31 (s, 1H), 6.97 (s, 1H), 6.41 (d, J = 15.2 Hz, 1H), 6.28 (s, 1H),5.00 (s, 1H), 3.64 (s, 9H), 3.58 (s, 1H), 3.34 (s, 2H), 2.41 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H), 1.44 (s, 9H);ESI-MS: m / z [M+2] + calcd for C 36 H 40 Cl3FN4O7 + 766.19, found 766.85.

[0046] Synthesis of intermediate L10a: Intermediate L09a (88 mg, 0.13 mmol) was dissolved in 3 mL of methanol solution of hydrogen chloride (4 M), stirred at room temperature until the starting material disappeared, the solvent was evaporated, dissolved in dichloromethane, evaporated to dryness, washed with petroleum ether, and dried to give 75 mg of off-white solid product, with a yield of 88.2%. 1 H NMR (400 MHz, CD3OD) δ 8.31 (dd, J =11.8, 3.6 Hz, 2H), 7.58 (d, J =7.8 Hz, 1H), 7.55 (s, 2H), 7.33 (t, J = 8.0 Hz, 1H), 7.18 (s, 1H), 6.62 (d, J =15.5 Hz, 1H), 3.59 (t, J =6.0 Hz, 2H), 3.13 (t, J = 5.9 Hz, 2H), 2.42-2.37 (m,3H), 1.90 (s, 3H), 1.85 (s, 3H); ESI-MS: m / z [M+2+H] + calcd for C 27 H 25 Cl3FN4O3 + 579.09, found 579.29.

[0047] Synthesis of intermediate L10b: Using the synthetic method of L10a, intermediate L10b was prepared. Using intermediate L09b as a raw material, 115 mg of off-white solid product was obtained, with a yield of 97.5%. 1 H NMR (400 MHz, CDCl3) δ 8.42 – 8.03 (m, 4H), 7.45 (s,1H), 7.27 (s, 3H), 6.94 (s, 1H), 6.62 (s, 1H), 3.30 (s, 2H), 3.03 (s, 2H),2.34 (s, 3H), 2.13 (s, 6H), 1.86 (s, 3H), 1.81 (s, 3H); ESI-MS: m / z [M+2+H] + calcd for C 30 H 31 Cl3FN4O3 + 621.14, found 621.26.

[0048] Synthesis of intermediate L10c: Intermediate L10c was prepared by the synthetic method of L10a, using intermediate L09c as the starting material, to give a white solid product 131 mg in 93.6% yield. 1 H NMR (400 MHz, CD3OD) δ 8.33-8.22 (m, 2H), 7.55 (d, J = 4.5 Hz, 3H), 7.32 (t, J = 8.0 Hz, 1H), 7.17 (s, 1H), 6.58 (d, J = 15.5 Hz, 1H), 3.31 (s, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.39 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H), 1.64 (d, J = 8.1 Hz, 2H), 1.58 (s, 2H), 1.40 (s, 8H); ESI-MS: m / z [M+2] + calcd for C 33 H 36 Cl3FN4O3 + 662.18, found 662.35.

[0049] Synthesis of intermediate L10d: Intermediate L10d was prepared by the synthetic method of L10a, using intermediate L09d as the starting material, to give a white solid product 123 mg in 98.4% yield. 1 H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 8.31 (s, 3H), 7.48 (s, 2H), 7.33 (s, 2H), 6.96 (s, 1H), 6.79 (s, 1H), 3.54 (s, 6H), 3.06 (s, 3H), 2.39 (s, 3H), 1.89 (s, 3H), 1.84 (s, 3H); ESI-MS: m / z [M+2] + calcd for C 29 H 28 Cl3FN4O4 + 622.11, found 622.28.

[0050] Synthesis of intermediate L10e: Intermediate L10e was prepared by using the synthetic method of L10a, using intermediate L09e as the raw material to obtain a white solid product 110 mg in a yield of 89.4 %. 1 H NMR (400 MHz, CDCl3) δ 8.43 – 8.23 (m, 4H), 7.54 (s,1H), 7.33 (s, 2H), 6.96 (s, 1H), 6.78 (s, 1H), 3.63 (s, 10H), 3.19 (s, 2H),2.40 (s, 3H), 1.89 (s, 2H), 1.84 (s, 4H);ESI-MS: m / z [M + 2] + calcd forC 31 H 32 Cl3FN4O5 + 666.14, found 666.36。

[0051] Synthesis of target compound F01 (4-((1E,3E)-4-(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-(2-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-1H-indol-4-yl)acrylamido)ethyl)-2,6-dimethylpyridin-1-ium tetrafluoroborate): Intermediate L10a (46 mg, 0.07 mmol) and Py-5 (36 mg, 0.098 mmol) were dissolved in 4 mL of dichloromethane, DIPEA (22 mg, 0.168 mmol) was added, stirred at room temperature until the raw material disappeared, the solvent was evaporated, and column chromatography was purified (neutral alumina, dichloromethane / methanol=100:1) to obtain a dark red solid product 51 mg in a yield of 78.5 %. 1 H NMR (400MHz, CDCl3) δ 8.37 (d, J =8.2 Hz, 1H), 8.31 (d, J =7.6 Hz, 1H), 8.21 (d, J =15.4Hz, 1H), 7.56 (d, J =7.6 Hz, 1H), 7.47- 7.35 (m, 4H), 7.35-7.26 (m, 3H), 6.96(d, J =17.4 Hz, 2H), 6.82 (d, J=11.8 Hz, 1H), 6.71-6.56 (m, 3H), 6.36 (d, J =15.3Hz, 1H), 4.59 (d, J =8.6 Hz, 2H), 3.81 (d, J =10.2 Hz, 2H), 3.07- 2.93 (m, 12H), 2.37 (s, 3H), 1.89 (s, 3H), 1.84 (s, 3H); ESI-HRMS: m / z [M+2] + calcd forC 46 H 44 Cl3FN5O3 + 840.2459, found 840.2464; m / z [M] + calcd for C 46 H 44 Cl3FN5O3 + 838.2489, found 838.2488; HPLC purity: 96.81%.

[0052] Synthesis of target compound F02 (4-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-(5-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazo-4-carbonyl)-3-methyl-1H-indol-4-yl)acrylamido)pentyl)-2,6-dimethylpyridin-1-onium tetrafluoroborate): The synthesis method of F02 is similar to that of F01. Using intermediate L10b as raw material, 25 mg of dark red solid product was obtained with a yield of 43.1%. 1 H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 8.33 (d, J =8.3 Hz, 1H), 8.26 (d, J =15.4 Hz, 1H), 7.64 (d, J =7.8 Hz, 1H), 7.47-7.32 (m, 6H), 7.24(d, J =7.5 Hz, 1H), 7.00-6.90 (m, 3H), 6.78 (dd, J =15.6, 10.3 Hz, 1H), 6.68 (d, J =8.5 Hz, 2H), 6.33 (d,J = 15.2 Hz, 1H), 4.59 (s, 2H), 3.46 (d, J = 5.6 Hz, 2H), 3.05 (s, 6H), 2.98-2.90 (m, 1H), 2.89 (s, 5H), 2.41 (s, 3H), 1.90 (s, 3H), 1.84 (s, 3H), 1.26 (s, 6H); ESI-HRMS: m / z [M] + calcd for C 49 H 50 Cl3FN5O3 + 880.2958, found 880.2954; [M+2] + calcd for C 49 H 50 Cl3FN5O3 + 882.2929, found 882.2930; HPLC purity: 99.74%.

[0053] Synthesis of target compound F03 (4-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-(8-((E)-3-(l-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-lH-indol-4-yl)acrylamido)octyl)-2,6-dimethylpyridin-l-ium tetrafluoroborate): The synthesis method of F03 is similar to that of F01, and intermediate L10c is used as raw material to obtain dark red solid product 60 mg in a yield of 59.4%. 1 H NMR (400 MHz, CDCl3) δ 8.32 (d, J = 8.2 Hz, 1H), 8.27 (d, J = 15.3 Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.45-7.30 (m, 5H), 7.30-7.18 (m, 2H), 6.96-6.86 (m, 2H), 6.78- 6.64 (m, 3H), 6.66-6.58 (m, 2H), 6.29 (d, J = 15.2 Hz, 1H), 4.19 (t, J = 8.3 Hz, 2H), 3.36 (q, J= 6.5 Hz, 2H), 3.00 (s, 6H), 2.78-2.68 (m, 1H), 2.66 (s, 5H), 2.39 (s, 3H), 1.89 (s, 3H), 1.84 (s, 3H), 1.66-1.51 (m, 4H), 1.34 (s, 8H); ESI-HRMS: m / z [M+2] + calcd for C 52 H 56 Cl3FN5O3 + 924.3398, found 924.3403; m / z [M] + calcd for C 52 H 56 Cl3FN5O3 + 922.3428, found 922.3426; HPLC purity: 98.35%.

[0054] Synthesis of target compound F04 (4-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-(2-(2-((E)-3-(l-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-lH-indol-4-yl)acrylamido)ethoxy)ethyl)-2,6-dimethylpyridin-l-ium tetrafluoroborate): The synthesis method of F04 is similar to that of F01, and intermediate L10d is used as raw material to obtain dark red solid product 80 mg in a yield of 82.5%. 1 H NMR (400 MHz, CDCl3) δ 8.29 (d, J = 8.2 Hz, 1H), 8.22 (d, J = 15.3 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 7.40-7.26 (m, 7H), 7.23-7.11 (m, 2H), 7.03-6.98 (m, 1H), 6.95 (d, J = 9.2 Hz, 1H), 6.84 (d, J = 15.2 Hz, 1H), 6.60 (d, J = 14.7 Hz, 2H), 6.14 (d, J= 14.8 Hz, 1H), 4.61 (s, 2H), 3.91 (s, 2H), 3.59 (s, 2H), 3.47 (s, 2H), 3.07 (s, 6H), 2.77 (s, 6H), 2.40 (s, 3H), 1.90 (s, 3H), 1.85 (s, 3H); ESI-HRMS: m / z [M+2] + calcd for C 48 H 48 Cl3FN5O4 + 884.2721, found 884.2727; m / z [M] + calcd for C 48 H 48 Cl3FN5O4 + 882.2751, found 882.2752; HPLC purity: 98.65%.

[0055] Synthesis of target compound F05 (4-((lE,3E)-4-(4-(dimethylamino)phenyl)buta-l,3-dien-l-yl)-l-(2-(2-(2-((E)-3-(l-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-lH-indol-4-yl)acrylamido)ethoxy)ethoxy)ethyl)-2,6-dimethylpyridin-l-ium tetrafluoroborate): The synthesis method of F05 is similar to that of F01, and intermediate L10e is used as raw material to obtain dark red solid product 72 mg in a yield of 70.6%. 1 H NMR (400 MHz, CDCl3) δ 8.36-8.22 (m, 2H), 7.57 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 10.7 Hz, 3H), 7.36 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 5.6 Hz, 2H), 7.23 (t, J = 8.0 Hz, 1H), 7.06-6.98 (m, 1H), 6.95 (s, 2H), 6.92 (s, 1H), 6.81-6.61 (m, 2H), 6.36 (d, J = 15.2 Hz, 1H), 4.63 (s, 2H), 3.95 (t, J=4.6Hz, 2H), 3.57- 3.46 (m, 8H), 3.05 (s, 6H), 2.92-2.80 (m, 1H), 2.78 (s, 5H), 2.39 (s, 3H), 1.89 (s, 3H), 1.84 (s, 3H); ESI-HRMS: m / z [M+2] + calcd forC 50 H 52 Cl3FN5O5 + 928.2984, found 928.2986; m / z [M] + calcd for C 50 H 52 Cl3FN5O5 + 926.3013, found 926.3009; HPLC purity: 98.29%.

[0056] Example 2 Preparation of fluorescent probes P01~P02: ; Synthesis of intermediate L11: 4-Dimethylaminocinnamaldehyde (578 mg, 3.3 mmol), (1,3-dioxolane-2-yl)methyltriphenyl bromide (2833 mg, 6.6 mmol), and 18-crown ether-6 (132 mg, 0.5 mmol) were dissolved in 40 mL of acetonitrile. The solution was cooled to 0 °C, and sodium hydride (60%, 660 mg, 16.5 mmol) was added in portions. The mixture was stirred in an ice bath for 3 h. 2M aqueous hydrogen chloride solution was added to adjust the pH to 2-3, and the mixture was stirred for 30 min. The solution was then adjusted to neutral with saturated sodium bicarbonate solution, extracted with ethyl acetate, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 15:1) to give 490 mg of orange solid product, with a yield of 73.8%. 1 H NMR (400 MHz, CDCl3) δ 9.58 (d, J = 8.0 Hz, 1H), 7.45 (d, J =8.3 Hz, 2H), 7.27 (s, 2H), 6.96 (d, J = 15.3 Hz, 1H), 6.91 (d, J = 6.7 Hz, 1H), 6.85 (d, J = 15.0 Hz, 1H), 6.21 (dd, J= 15.1, 8.1 Hz, 1H), 3.06 (s, 6H);ESI-MS: m / z [M +H] + calcd for C 13 H 16 NO + 202.12, found 202.05.

[0057] Synthesis of intermediate L12: Intermediate L11 (201 mg, 1 mmol), methyl cyanoacetate (119 mg, 1.2 mmol) and piperidine (21 mg, 0.25 mmol) were dissolved in 8 mL of dichloromethane, stirred at 15-20 °C for 4 h, the solvent was evaporated, the residue was purified with dichloromethane and petroleum ether, and the solid product was obtained after drying, 251 mg, yield 89.0 %. 1 H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 12.0 Hz, 1H), 7.43 (d, J = 8.5 Hz, 2H), 7.10 (dd, J = 14.3, 10.7 Hz, 1H), 6.93(d, J = 15.2 Hz, 1H), 6.90 – 6.83 (m, 1H), 6.83 – 6.71 (m, 3H), 3.88 (s, 3H),3.07 (s, 6H);ESI-MS: m / z [M + H] + calcd for C 17 H 19 N2O2 + 283.14, found 283.18;m / z [2M ] + calcd for C 34 H 36 N4O4 + 564.27, found 564.41.

[0058] Synthesis of intermediate L13: Intermediate L12 (226 mg, 0.8 mmol) was dissolved in 15 mL of tetrahydrofuran and 9 mL of methanol, 0.8 mL of 1.5 M aqueous NaOH was added, stirred at 15-20 °C overnight, 2 M aqueous hydrogen chloride solution was used to adjust the pH to 2-3, 100 mL of ethyl acetate was added, the organic phase was washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, the residue was washed with petroleum ether, filtered and dried to give a solid product 190 mg, the yield was 88.4 %. 1 H NMR (400 MHz, DMSO-d6) δ 13.40 (s, 1H),7.97 (d, J =12.1 Hz, 1H), 7.61-7.45 (m, 2H), 7.39 (t, J =12.7 Hz, 1H), 7.10 (t, J =13.4 Hz, 1H), 6.99 (d, J =15.5 Hz, 1H), 6.76-6.60 (m, 3H), 2.99 (s, 6H);ESI-MS:m / z [M-H] - calcd for C 16 H 15 N2O2 - 267.11, found 266.98;m / z [2M-H] - calcd forC 32 H 31 N4O4 - 535.23, found 534.66。

[0059] Synthesis of the target compound P01 ((2E,4E,6E)-2-cyano-7-(4- (dimethylamino)phenyl)-N-(2-(2-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6- trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-1H-indol-4-yl)acrylamido)ethoxy) ethyl)hepta-2,4,6-trienamide): Intermediate L13 (59 mg, 0.22 mmol) was dissolved in 3 mL of DMF, and HATU (87 mg, 0.23 mmol) was added. The mixture was stirred at 10–20 °C for 1.5 h. Intermediate L10d (125 mg, 0.18 mmol) and DIPEA (116 mg, 0.9 mmol) were then added, and the mixture was stirred for another 1 h. Water and ethyl acetate were added, and the organic layer was separated. The organic layer was washed successively with 1 M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution. The mixture was dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (neutral alumina, dichloromethane / methanol = 100:1) to obtain 126 mg of dark red solid product, with a yield of 80.3%. 1 H NMR (400 MHz, CDCl3) δ 8.47-8.32(m, 2H), 8.02-7.89 (m, 1H), 7.56 (t, J =9.2 Hz, 1H), 7.37 (dp, J =16.9, 8.9 Hz,4H), 7.01-6.90 (m, 1H), 6.87-6.77 (m, 2H), 6.76-6.64 (m, 3H), 6.64-6.50 (m,2H), 6.47-6.21 (m, 1H), 3.71-3.62 (m, 8H), 3.05 (d, J =8.0 Hz, 6H), 2.44 (d, J =5.4 Hz, 3H), 1.94-1.89 (m, 3H), 1.88-1.85 (m, 3H); ESI-HRMS: m / z [M+2] + calcdfor C 45 H 43 Cl3FN6O5 + 873.2310, found 873.2311; m / z [M] + calcd for C 45 H 43 Cl3FN6O5 + 871.2340, found 871.2336; HPLC purity: 99.81%.

[0060] Synthesis of target compound P02 ((2E,4E,6E)-2-cyano-7-(4- (dimethylamino)phenyl)-N-(2-(2-(2-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6- trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-1H-indol-4-yl)propenoylamido)ethoxy) ethoxy)ethyl)hepta-2,4,6-trienamide): The synthesis method of P02 is similar to that of P01, and the dark red solid product 132 mg is obtained with a yield of 82.5% by taking intermediate L10e as raw material. 1 H NMR (400 MHz, CDCl3) δ 8.36-8.22 (m, 2H), 7.88 (d, J =12.1 Hz, 1H), 7.46 (d, J =7.9 Hz, 1H), 7.31 (d, J =8.3 Hz, 2H), 7.21 (d, J =14.9Hz, 1H), 6.95 (dd, J =14.3, 10.4 Hz, 1H), 6.89 (s, 1H), 6.83-6.66 (m, 2H), 6.70(s, 3H), 6.66 -6.51 (m, 2H), 6.46 (d, J =15.3 Hz, 1H), 3.56 (dd, J =11.4, 6.8 Hz,12H), 2.97 (s, 6H), 2.35 (s, 3H), 1.82 (s, 3H), 1.77 (s, 3H);ESI-HRMS: m / z[M] + calcd for C 47 H 47 Cl3FN6O6 + 915.2602, found 915.2598; m / z [M+2] + calcd forC 47 H 47 Cl3FN6O6 + 917.2572, found 917.2570;HPLC purity: 99.59%.

[0061] Preparation of fluorescent probes N01~N02:

[0062] Synthesis of intermediate L14: 6-(dimethylamino)-2-naphthaldehyde (205 mg, 1.03 mmol), (1,3-dioxolan-2-yl)methyltriphenylphosphonium bromide (884 mg, 2.06 mmol) and 18-crown-6 (41 mg, 0.155 mmol) were dissolved in 16 mL of acetonitrile, cooled to 0 °C, sodium hydride (60%, 206 mg, 5.15 mmol) was added in portions, and the temperature was allowed to rise to room temperature naturally, stirred overnight, 2M aqueous hydrogen chloride solution was added to pH 2-3, stirred for 30 minutes, and then the pH was adjusted to 7-8 with saturated NaHC03solution, extracted with ethyl acetate, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 13:1) to give 174 mg of yellow solid product with a yield of 75.0 %. 1 H NMR (400 MHz, CDCl3) δ 9.72 (d, J =7.8 Hz, 1H),7.88 (s, 1H), 7.82 (d, J=8.7 Hz, 1H), 7.73 (d, J= 8.9 Hz, 1H), 7.66-7.55 (m,2H), 7.29 (d, J=15.9 Hz, 2H), 6.77 (dd, J=15.7, 7.7 Hz, 1H), 3.15 (s, 6H);ESI-MS: m / z [M+ H] + calcd for C 15 H 16 NO + 226.21, found 226.11。

[0063] Synthesis of intermediate L15: Intermediate L14 (261 mg, 1.16 mmol), (1,3-dioxolan-2-yl)methyltriphenylphosphonium bromide (996 mg, 2.32 mmol) and 18-crown-6 (61 mg, 0.232 mmol) were dissolved in 25 mL of acetonitrile, cooled to 0 °C, sodium hydride (60%, 232 mg, 5.8 mmol) was added in portions, and the temperature was allowed to rise to room temperature naturally, stirred overnight, 2M aqueous hydrogen chloride solution was added to pH 2-3, stirred for 30 minutes, and then the pH was adjusted to 7-8 with saturated NaHC03solution, extracted with ethyl acetate, washed with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 10:1) to give 225 mg of solid product with a yield of 77.1 %. 1H NMR (400 MHz, CDCl3) δ 9.54 (d, J=8.1 Hz, 1H), 7.65 (d, J=12.8Hz, 2H), 7.60-7.43 (m, 2H), 7.30-7.17 (m, 2H), 7.06 (d, J=15.4 Hz, 1H), 6.96(dd, J=15.4, 10.6 Hz, 1H), 6.84 (s, 1H), 6.19 (dd, J=15.1, 8.0 Hz, 1H), 3.03(s, 6H);ESI-MS: m / z [M+H] + calcd for C 17 H 18 NO + 252.14, found 252.21.

[0064] Synthesis of intermediate L16: Intermediate L15 (183 mg, 0.73 mmol) and methyl cyanoacetate (87 mg, 0.876 mmol) were dissolved in 15 mL of dichloromethane, piperidine (31 mg, 0.365 mmol) was added, stirred at room temperature overnight, evaporated the solvent, washed with petroleum ether, dried to obtain the product 220 mg, yield 90.9 %. 1 H NMR (400 MHz, CDCl3) δ 7.96 (d, J=12.1 Hz, 1H),7.81 (d, J=10.8 Hz, 2H), 7.73 (d, J=8.7 Hz, 1H), 7.64 (d, J=8.7 Hz, 1H), 7.36(d, J=15.3 Hz, 2H), 7.21-7.06 (m, 3H), 6.87 (t, J=12.8 Hz, 1H), 3.90 (s, 3H),3.17 (s, 6H);ESI-MS: m / z [M+H] + calcd for C 21 H 21 N2O2 + 333.16, found 333.21.

[0065] Synthesis of intermediate L17: Intermediate L16 (206 mg, 0.62 mmol) was dissolved in 12 mL of tetrahydrofuran and 3 mL of methanol, and an aqueous NaOH solution (37 mg dissolved in 0.6 mL of water) was added. The mixture was stirred until the starting material disappeared. The pH was adjusted to 1-2 with 2M aqueous hydrogen chloride solution. 60 mL of ethyl acetate, water, and saturated sodium chloride solution were added to wash the organic phase. The organic phase was dried over anhydrous sodium sulfate, concentrated, and then slurried in a mixture of petroleum ether and ethyl acetate. The product was obtained as a black solid in a yield of 138 mg (70.1%). 1 H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 12.1 Hz, 1H), 7.88 (s, 1H), 7.75 (d, J = 9.1 Hz, 1H), 7.71-7.63 (m, 2H), 7.47 (dd, J = 14.1, 11.1 Hz, 1H), 7.35 (dd, J = 15.1, 11.1 Hz, 1H), 7.23 (dd, J = 9.0, 2.7 Hz, 1H), 7.17 (d, J = 15.1 Hz, 1H), 6.94 (d, J = 2.7 Hz, 1H), 6.84-6.73 (m, 1H), 3.05 (s, 6H); ESI-MS: m / z [M + H] + calcd for C 20 H 19 N2O2 + 319.14, found 319.23.

[0066] The synthesis method of N01 ((2E,4E,6E)-2-cyano-7-(6-(dimethylamino)naphthalen-2-yl)-N-(2-(2-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazole-4-carbonyl)-3-methyl-1H-indol-4-yl)propenoylamido)ethoxy)ethyl)hepta-2,4,6-trienamide) is as follows: Intermediate L17 (66 mg, 0.208 mmol) was dissolved in 3 mL of DMF, and HATU (83 mg, 0.22 mmol) was added. The mixture was stirred at 10-20°C for 1.5 h, and then intermediate L10d (111 mg, 0.16 mmol) and DIPEA (103 mg, 0.8 mmol) were added. The mixture was stirred for another 3 h. Water and ethyl acetate were added, and the organic layer was separated. The organic layer was washed with 1M hydrochloric acid solution, saturated sodium bicarbonate solution, and saturated sodium chloride solution, respectively. The organic layer was dried over anhydrous sodium sulfate, concentrated, and then purified by column chromatography (neutral alumina, dichloromethane / methanol = 300:1) to obtain the product as a black solid in a yield of 113 mg (76.4%).1 H NMR (400 MHz, CDCl3) δ 8.44 (d, J=8.2 Hz, 1H), 8.34 (d, J=15.2 Hz, 1H), 7.92 (d, J=12.0 Hz, 1H), 7.75-7.66 (m,2H), 7.66 -7.58 (m, 1H), 7.58-7.48 (m, 2H), 7.36-7.31 (m, 1H), 7.30 (s, 1H), 7.13 (dd, J = 9.2, 2.5 Hz, 1H), 6.99-6.92 (m, 3H), 6.92-6.84 (m, 1H), 6.77-6.65 (m, 1H), 6.61-6.50 (m, 2H), 6.41 (d, J=5.6 Hz, 1H), 3.70-3.57 (m, 8H), 3.10 (d, J=11.3 Hz, 1H), 3.09 (s, 5H), 2.42 (d, J=8.0 Hz, 3H), 1.88 (d, J=6.4Hz, 3H), 1.83 (d, J=6.4 Hz, 3H); ESI-HRMS: m / z [M+2] + calcd for C 49 H 45 Cl3FN6O5 + 923.2467, found 923.2472; m / z [M] + calcd for C 49 H 45 Cl3FN6O5 + 921.2496, found921.2497; HPLC purity: 99.19%.

[0067] Synthetic method of NO2 ((2E,4E,6E)-2-cyano-7-(6-(dimethylamino)naphth-2-yl)-N-(2-(2-(2-((E)-3-(1-(5-(2-fluoroprop-2-yl)-3-(2,4,6-trichlorophenyl)isoxazo-4-carbonyl)-3-methyl-1H-indol-4-yl)acrylamido)ethoxy)ethoxy)ethyl)hept-2,4,6-trienamide): The synthesis method of NO2 is similar to that of NO1. Using intermediate L10e as raw material, 107 mg of black solid product was obtained with a yield of 69.0%. 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J=8.2 Hz, 1H), 8.33 (dd, J=15.4, 3.9 Hz, 1H), 7.97 (d, J=12.1 Hz, 1H), 7.79-7.65 (m, 2H), 7.64-7.47 (m, 3H), 7.37-7.24 (m, 2H), 7.18 -7.08 (m, 1H), 7.08-6.91 (m, 3H), 6.86(d, J=2.6 Hz, 1H), 6.80-6.66 (m, 2H), 6.60-6.44 (m, 2H), 3.64 (dd, J=10.1,5.9 Hz, 11H), 3.60 (d, J=4.8 Hz, 1H), 3.08 (s, 6H), 2.41 (d, J=4.0 Hz, 3H), 1.89 (s, 3H), 1.84 (s, 3H); ESI-HRMS: m / z [M] + calcd for C 51 H 49 Cl3FN6O6 + 965.2758,found 965.2755; m / z [M+2] + calcd for C 51 H 49 Cl3FN6O6 + 967.2729, found 967.2731; HPLC purity: 99.39%.

[0068] Example 3 Affinity test of fluorescent probe: The affinity of the fluorescent probes for mIDH1 was tested using the fluorescence polarization method. The results are shown in Table 1. All probe molecules showed high affinity for the target protein mIDH1, all at the nanomolar level.

[0069] Table 1. Affinity of fluorescent probes to mutant isocitrate 1 (mIDH1)

[0070] Example 4 Optical property testing of fluorescent probes F01~F05: The absorption spectrum and fluorescence emission spectrum of the fluorescent probe were first tested. The maximum absorption wavelength of the probe was about 480 nm, and the maximum emission wavelength was about 690 nm, with a large Stokes shift. In the subsequent imaging studies, the interference of the excitation light on the emission light could be reduced, and the signal-to-noise ratio could be improved. Figure 1 The environmental sensitivity of the fluorescent probe was further tested. The fluorescence emission spectrum of the probe in different solvents showed that the fluorescence intensity increased significantly as the polarity of the solvent decreased. Figure 1

[0071] Example 5 Evaluation of the selectivity of fluorescent probes F01-F05 for mIDH1: The fluorescence response of the fluorescent probe to mIDH1 and IDH1 protein was first investigated. After incubation of the probe with the protein, all molecules showed fluorescence enhancement. In particular, F04 and F05 had significant fluorescence enhancement, and the fluorescence intensity of F04 combined with mIDH1 was about 10 times that of IDH1, indicating that F04 had superior selectivity for mIDH1. Figure 2 A).

[0072] Next, the response of the fluorescent probe to mIDH1 in living cells was evaluated. For this purpose, the probe was incubated with glioma cells LN229 and LN229 stably transfected with mIDH1 (LN229-mIDH1) cells, respectively, and the change in fluorescence intensity was measured. The results showed that only probes F04 and F05 exhibited significant fluorescence enhancement, and the fluorescence intensity of LN229-mIDH1 cells was significantly higher than that of LN229 cells, confirming the selectivity of F04 and F05 for mIDH1. Figure 2 B).

[0073] Example 6 Investigation of the cell imaging ability of fluorescent probes F01-F05: After incubation of the fluorescent probe with LN229 cells stably transfected with mIDH1, no washing was performed, and an inverted fluorescence microscope (Axio Observer A1) was used for imaging. The results showed that F01 and F03 only had weak fluorescence enhancement, while F02, F04 and F05 all exhibited significant fluorescence after interaction with LN229-mIDH1 cells. However, when incubated with the antagonist Safusidenib, only the fluorescence of F04 and F05 was essentially eliminated, and F02 still maintained strong fluorescence. This indicated that the fluorescent probes F04 and F05 could specifically bind to mIDH1 in living cells, thereby visualizing LN229-mIDH1 glioma cells. Figure 3 ).

[0074] Example 7 ​Investigation of the fluorescence imaging ability of fluorescent probe F04 in vivo: Since the fluorescent probe F04 has high affinity, selectivity and excellent in vitro labeling ability, the fluorescence imaging ability of F04 in animals was further investigated. For this purpose, LN229 cells stably transfected with mIDH1 and Fluc were used to construct an orthotopic model of glioma, and bioluminescence imaging was used to determine the stable formation of the tumor (Fig. 4A). Figure 4 A), and fluorescence imaging was performed under a small animal live optical imaging system (IVIS Spectrum 2) after tail vein injection of F04 probe solution. The results showed that the fluorescence of the mouse's head was enhanced by about 10 times after injection of the fluorescent probe F04, indicating that F04 has the ability to cross the blood-brain barrier and bind to brain tissue, causing fluorescence enhancement (Fig. 4B). Figure 4 B).

[0075] In addition, fluorescence imaging of the brain showed that the probe only exhibited fluorescence enhancement in specific locations, overlapping with the tumor site, confirming that the fluorescent probe F04 can specifically recognize glioma cells carrying mIDH1 and does not bind to normal brain cells, thereby accurately locating glioma tissue (Fig. 4C). Figure 4 C).

[0076] The above results show that the fluorescent probe F04 can be used as a targeted fluorescence imaging agent and has great potential for fluorescence-guided surgical resection of glioma.

[0077] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A high-affinity, selective mutant isocitrate dehydrogenase 1 fluorescent probe, characterized by: The structural formula is shown as formula I: ； In the formula, X1 is carbon atom or oxygen atom; X2 is halogen ion or tetrafluoroboric acid ion; n is 0-5; m is 0-2; R1 and R2 are hydrogen, methyl or ethyl; R3 is electron donating substituent; R4 is 4-dimethylaminophenyl or 6-dimethylaminonaphthyl.

2. The high-affinity, selectively mutant isocitrate dehydrogenase 1 fluorescent probe of claim 1, wherein: R3 is N, N-dimethylamino or N, N-diethylamino.

3. The high affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe of claim 2, wherein: R3 is N, N-dimethylamino.

4. The high affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe of claim 1, wherein: R1 and R2 are methyl.

5. The high affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe of claim 1, wherein: X2 is tetrafluoroboric acid ion.

6. The high affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe of claim 1, wherein: n is 0-2; m is 1.

7. The high affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe of claim 1, wherein: The high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe is selected from any one of the following compounds: ； ； 。 8. The high-affinity selective mutant isocitrate dehydrogenase 1 fluorescent probe according to any one of claims 1-7 is applied to any one of the following: (1) mechanism and pharmaceutical research of mutant isocitrate dehydrogenase 1; (2) screening of mutant isocitrate dehydrogenase 1 inhibitor; (3) preparation of diagnostic reagent for mutant isocitrate dehydrogenase 1 related disease; (4) preparation of fluorescent imaging agent for tumor cell carrying mutant isocitrate dehydrogenase 1.

9. Use according to claim 8, characterized in that: The mutant isocitrate dehydrogenase 1 related disease is brain glioma, chondrosarcoma and acute leukemia.

10. Use according to claim 8, characterized in that: The tumor is brain glioma or chondrosarcoma.

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