A specific fluorescent probe targeting alk and a preparation method and application thereof

Abstract

The application discloses a specific fluorescent probe targeting ALK and a preparation method and application thereof, and belongs to the technical field of molecular probes. The molecular probe has the advantages of targeting tumor cells and tissues with high expression of ALK and mutant proteins, and directionally transferring a fluorescent molecule carried by the molecular probe to an area with high expression of ALK and mutant proteins, and has good biocompatibility, stability, low toxicity and the like, and can realize specific binding and fluorescent imaging of ALK and mutant proteins in pathological sites such as tumors. The molecular probe has high targeting property, low preparation cost and high practicability; the fluorescent molecular probe has an aggregation-induced emission effect, low background interference, and is more suitable for real-time and dynamic imaging of cells with high concentration and for a long time; in addition, the molecular probe has low toxicity to cell growth, and can be used for tracing and imaging in living cells. The molecular probe has high specificity for ALK and mutant proteins, and has the characteristics of low fluorescent background.

Description

Technical Field

[0001] This invention belongs to the field of molecular probe technology, specifically relating to a specific fluorescent probe targeting ALK, its preparation method, and its application. Background Technology

[0002] In recent years, molecularly targeted drugs have become widely used, representing a major breakthrough in clinical oncology treatment. For example, crizotinib, alectinib, ceritinib, brigatinib, and lorlatinib are two targeted drugs for treating non-small cell lung cancer (NSCLC), belonging to the anaplastic lymphoma kinase inhibitor (ALK-TKI) class. Accurate detection of ALK mutation types in lung cancer is crucial for the application of molecularly targeted drugs in lung cancer treatment. Clinically, ALK gene mutation status testing is necessary before administering ALK-TKI targeted drugs to NSCLC patients.

[0003] However, with current technology, it is difficult to accurately detect ALK mutation subtypes in lung cancer patients. The main problems encountered in clinical practice include the following aspects:

[0004] 1) First, lung cancer exhibits highly complex heterogeneity, including individual, spatial, and temporal heterogeneity. Individual heterogeneity refers to the significant differences in ALK mutation types among different lung cancer patients; spatial heterogeneity refers to the variations in ALK mutation types across different lesions and at different locations within the same lesion; and temporal heterogeneity refers to the dynamic changes in ALK mutation types over different time periods. Therefore, achieving real-time, dynamic, and accurate detection of ALK mutation status at the in vivo level can address the issue of lung cancer heterogeneity.

[0005] 2) Secondly, existing ALK mutation detection methods all have certain limitations. For example, molecular pathology detection of ALK mutations has poor reproducibility and cannot overcome spatial heterogeneity. In contrast, molecular detection is more convenient and has better reproducibility, but its disadvantage is that it cannot directly obtain the mutation status of the primary lesion, and the heterogeneity problem within the primary and metastatic lesions of lung cancer cannot be solved.

[0006] Furthermore, traditional imaging methods such as CT and MRI cannot reflect ALK molecular subtyping information at the molecular level. Therefore, in order to further overcome the bottlenecks in the precise diagnosis and treatment of lung cancer, it is urgent to develop a new detection technology to accurately reveal ALK mutation status at the molecular level. Summary of the Invention

[0007] Based on targeted molecular probes, molecular imaging can be used to observe the expression processes of specific cells, genes, and molecules in real time, and to track target sites (imaging, characterization, measurement, etc.), thereby assessing disease progression at the molecular pathology level. Therefore, a highly specific ALK-targeting molecular probe can achieve qualitative and quantitative detection of ALK mutation molecular typing, with results characterized by high targeting, good accuracy, few false positives, and strong intuitiveness. This molecular probe can be developed into a tool molecule with clinical application value.

[0008] The primary objective of this invention is to provide a specific fluorescent probe targeting ALK. This molecular probe can target and locate tumor cells and tissues that highly express ALK and its mutant proteins, and directionally transfer the fluorescent molecules it carries to the regions where ALK and its mutants are highly expressed. It has good biocompatibility, stability, and low toxicity, and can achieve specific binding and fluorescence imaging of ALK and its mutants in pathological sites such as tumors.

[0009] The second objective of this invention is to provide a specific fluorescent probe for targeting ALK and a method for preparing the molecular probe. This molecular probe has high targeting, low preparation cost, and strong practicality. The fluorescent molecular probe of this invention has aggregation-induced emission effect, low background interference, and is more suitable for real-time and dynamic imaging of cells at high concentrations and for long periods of time. In addition, the molecular probe has low toxicity to cell growth and can be used for tracking and imaging in living cells.

[0010] A third objective of this invention is to provide a specific fluorescent probe targeting ALK. This molecular probe exhibits high specificity for ALK and its mutant proteins, and is characterized by low fluorescence background. The technical fields related to this molecular probe also include molecular probe design, chemical synthesis, bioactivity characterization, and cell and tissue imaging.

[0011] This invention is achieved through the following technical solution:

[0012] A specific fluorescent probe targeting ALK has the following structural formula:

[0013]

[0014] In the above structural formula, R1 can be any one of H, F, Cl, or alkynyl groups;

[0015] R2 can be any one of H, F, Cl, or alkynyl groups;

[0016] R4 can be any one of H, F, Cl, or alkynyl groups.

[0017] As a preferred embodiment, the structural formula is as follows:

[0018]

[0019] A specific fluorescent probe targeting ALK has the following structural formula:

[0020]

[0021] In the above structural formula, R1 can be any one of H, F, Cl, or alkynyl groups;

[0022] R2 can be any one of H, F, Cl, or alkynyl groups;

[0023] R4 can be any one of H, F, Cl, or alkynyl groups.

[0024] Preferably, in the structural formula, n = 2 in PEG.

[0025] The aforementioned ALK-targeting specific fluorescent probe structure comprises three components: a core reactive group targeting ALK; a linking group; and a fluorescent molecular labeling group.

[0026] The core reactive group binds to the ALK protein in tumor tissue;

[0027] Specific tracking and imaging are achieved through the fluorescent molecular labeling groups;

[0028] The fluorescent molecular labeling groups include any one or more of benzoxadiazoles, naphthimides, rhodamines, and cyanine dyes.

[0029] A method for preparing a specific fluorescent probe targeting ALK includes the following steps:

[0030] Synthesis of S1 and the first intermediate (SM2):

[0031] Add ceritinib (SM1), potassium carbonate and N,N-dimethylformamide (DMF) to a round-bottom flask and stir for 5-8 minutes at room temperature. Then add ethyl 2-bromoacetate and stir overnight at room temperature.

[0032] The resulting mixture was then diluted with water and extracted with dichloromethane (DCM). The combined organic layers were dried with sodium sulfate, filtered, concentrated under vacuum, and purified to obtain the first intermediate (SM2).

[0033] S2, Synthesis of Compound I:

[0034] The first intermediate (SM2), sodium hydroxide, and methanol were added to a round-bottom flask and stirred overnight at room temperature to obtain a mixture. The pH of the mixture was adjusted to 5 with acid, and the mixture was extracted with ethyl acetate (EA). The combined organic layers were dried with sodium sulfate, filtered, vacuum evaporated, and purified to obtain compound I (Compound 1).

[0035] S3, Synthesis of Compound II:

[0036] SM3, compound I (Compound 1), HATU, DIPEA, and N,N-dimethylformamide were added to a round-bottom flask and stirred at room temperature for 1.5-2 hours. The mixture was then diluted with water and extracted with ethyl acetate. The combined organic layers were dried with sodium sulfate, filtered, vacuum evaporated, and purified to obtain compound II.

[0037] Compound II is a specific fluorescent probe targeting ALK.

[0038] Glossary: ​​SM1 above refers to Ceritinib (LDK378).

[0039] SM3 is 7-((2-aminoethyl)amino)-N,N-dimethylbenzo[c][1,2,5]oxadiazole-4-sulfonamide.

[0040] HATU is a polypeptide condensation reagent: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0041] DIEA is N,N-diisopropylethylamine.

[0042] Preferably, in S1, the ratio of ceritinib, potassium carbonate, N,N-dimethylformamide, ethyl 2-bromoacetate, water, and dichloromethane is 300mg:148mg:5mL:107.7mg:30mL:150mL;

[0043] The first intermediate is a white solid.

[0044] Preferably, in step S2, the ratio of the first intermediate, sodium hydroxide, methanol, and ethyl acetate is 150 mg: 18.9 mg: 20 mL: 150 mL.

[0045] Compound I is a white solid.

[0046] Preferably, in S3, the ratio of SM3, compound I, HATU, DIEA, N,N-dimethylformamide, water, and ethyl acetate is 13mg:30mg:37mg:18mg:1mL:30mL:150mL;

[0047] Compound II is a yellow solid.

[0048] Application of a specific fluorescent probe targeting ALK in the preparation of a fluorescent imaging reagent for tracing and localizing tumor cells with high expression of ALK and its mutant proteins.

[0049] Application of a specific fluorescent probe targeting ALK in evaluating the drug activity of anaplastic lymphoma kinase inhibitors (ALK-TKIs).

[0050] Compared with the prior art, the present invention has at least the following technical effects:

[0051] (I) This invention provides a specific fluorescent probe targeting ALK. This specific ALK molecular probe can target and locate tumor cells and tissues that highly express ALK and its mutant proteins, and directionally transfer the fluorescent molecules it carries to the regions where ALK and its mutants are highly expressed. It has good biocompatibility, stability, and low toxicity, and can achieve specific binding and fluorescence imaging of ALK and its mutants in pathological sites such as tumors.

[0052] (ii) The molecular probe has high targeting, low preparation cost, and strong practicality; the fluorescent molecular probe of the present invention has aggregation-induced emission effect, low background interference, and is more suitable for real-time and dynamic imaging of cells with high concentration and long time; in addition, the molecular probe has low toxicity to cell growth and can be used for tracing and imaging in living cells.

[0053] (iii) This molecular probe has high specificity for ALK and its mutant proteins and has the characteristic of low fluorescence background. Attached Figure Description

[0054] Figure 1 For ALK probe HX16 1 H-NMR spectrum (DMSO solvent);

[0055] Figure 2 For ALK probe HX16 13 C-NMR spectrum (DMSO solvent);

[0056] Figure 3 To validate the fluorescence "on" reaction and molecular pharmacology of HX16 for ALK;

[0057] Figure 4 This is a schematic diagram illustrating the distribution of HX16 in cancer cells as observed using confocal microscopy.

[0058] Figure 5 To assess ALK activity in a mouse model of NSCLC tumor-bearing tumor. Detailed Implementation

[0059] The embodiments of the present invention will be described in detail below with reference to the examples. However, those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0060] One specific embodiment of the present invention:

[0061] A specific fluorescent probe targeting ALK has the following structural formula:

[0062]

[0063] In the above structural formula, R1 can be any one of H, F, Cl, or alkynyl groups;

[0064] R2 can be any one of H, F, Cl, or alkynyl groups;

[0065] R4 can be any one of H, F, Cl, or alkynyl groups.

[0066] The reactive group skeleton structure of the above molecular probe is shown in Formula I, wherein the substituents R1, R2, and R4 are not particularly limited and can be H, F, Cl, or alkynyl.

[0067] The length of the linking group is not fixed and can be adjusted according to the actual effect, with PEG (n=2) being preferred.

[0068] Example 1:

[0069] A specific fluorescent probe targeting ALK has the following structural formula:

[0070]

[0071] A method for preparing a specific fluorescent probe targeting ALK includes the following steps:

[0072]

[0073] Synthesis of S1 and the first intermediate (SM2):

[0074] To a 100 mL round-bottom flask, add SM1 (Ceritinib) (300 mg, 0.537 mmol), K2CO3 (148 mg, 1.07 mmol), and DMF (5 mL), and stir for 5 minutes at room temperature. Add ethyl 2-bromoacetate (107.7 mg, 0.64 mmol), and stir the mixture overnight at room temperature. Then dilute the resulting mixture with H2O (30 mL) and extract with DCM (3 x 50 mL). Dry the combined organic layers with Na2SO4, filter, concentrate under vacuum, and purify by chromatography (silica gel, PE:EA = 10:1, Rf = 0.5) to give SM2 (261 mg, yield: 75%, white solid).

[0075] MS calculation: 643.2; MS finding: 644.2 [M+H] +

[0076] S2, Synthesis of Compound I:

[0077] Add SM2 (150 mg, 0.236 mmol), NaOH (18.9 mg, 0.46 mmol), and MeOH (20 mL) to a 50 mL round-bottom flask, and stir the mixture overnight at room temperature. Adjust the pH of the mixture to 5 with HCl, and extract with EA (3 x 50 mL). Dry the combined organic layers with Na2SO4, filter, and evaporate under vacuum. Purify the residue by Prep-HPLC to give compound I (70 mg, yield: 48%, white solid).

[0078] MS calculation: 615.2; MS discovery: 616.2[M++1]+.

[0079] S3, Synthesis of Compound II:

[0080] To a 100 mL round-bottom flask, add SM3 (13 mg, 0.048 mmol), compound I (30 mg, 0.048 mmol), HATU (37 mg, 0.097 mmol), DIEA (18 mg, 0.146 mmol), and DMF (1 mL). Stir the mixture at room temperature for 2 hours. Then dilute the mixture with H2O (30 mL) and extract with EA (3 x 50 mL). Dry the combined organic layers with Na2SO4, filter, concentrate under vacuum, and purify by preparative HPLC to give compound II (18 mg, yield: 42%, yellow solid).

[0081] MS calculation: 882.3; MS discovery: 883.4[M++1]+.

[0082] Compound II is a specific fluorescent probe targeting ALK.

[0083] The structural confirmation data for compound II are as follows: 1 H-NMR (DMSO-d6, 400MHz): δ9.46 (s, 1H), 8.46 (d, J = 8.8Hz, 1H), 8.35 (s, 1H), 8.25 (s, 1H), 8.12-8.01 (m, 2H), 7.82(t,J=8.2Hz,2H),7.62(t,J=7.6Hz,1H),7.52(s,1H),7.35(t,J=7.4Hz,1H),6.78(s,1H),6.40(d,J=8.0 Hz,1H),4.55-4.49(m,1H),3.47-3.32(m,5H),2.94(s,2H),2.86(d,J=10.7Hz,2H),2.65(s,6H),2.62-2.54( m,1H)2.16-2.07(m,5H),1.70-1.62(m,2H),1.57-1.54(m,2H),1.24(d,J=6.0Hz,6H),1.16(d,J=6.4Hz,6H). 13 C NMR (200MHz, DMSO-d6) δ170.2,158.0,155.4,154.9,146.6,146.4,144.3,141.4,140.4,139.3,138.0,134.8,131.0,126.9,126.7,124. 5,123.9,123.7,123.6,111.7,105.4,104.3,70.9,61.8,54.8,42.9,40.0,39.9,37.4(2C),37.2,36.9,32.2,21.9(2C),18.3,14.8(2C).

[0084] Experimental verification:

[0085] like Figure 1 As shown, this is the ALK probe HX16. 1 H-NMR spectrum (DMSO solvent);

[0086] like Figure 2 As shown, this is the ALK probe HX16. 13 C-NMR spectrum (DMSO solvent).

[0087] like Figure 3The image shows the fluorescence "on" reaction and molecular pharmacological verification of HX16 for ALK. (A) shows the fluorescence spectra of HX16 (5 μM) in PBS buffer with and without ALK tyrosine kinase, and after the addition of the ALK inhibitor ceritinib (50 μM).

[0088] (B) Fluorescence spectrum of HX16 (5 μM) in the presence of ALK or BSA.

[0089] The fluorescence spectrum of (C)HX16 (5 μM) changed with increasing ALK kinase (0 to 1 μM) concentration.

[0090] (D, E) Quantitative analysis of fluorescence intensity at emission wavelength of 560 nm (A, B). Data represent mean ± SEM. Each group had n = 3 independent experiments. Statistical p-values ​​were calculated using a two-tailed Student's test. ***P < 0.001.

[0091] (F) Titration curves of HX16 (5 μM) as ALK concentration increases. Data represent mean ± SD, with n = 3 independent experiments per group.

[0092] (G) The kinase inhibitory activity of HX16 against recombinant ALK was determined by fluorescence mobility shift assay. Data represent mean ± SEM, with n = 3 independent experiments per group.

[0093] Effects of (HI) probe HX16 (0 to 10 μM) on ALK activation and downstream ERK1 / 2 phosphorylation levels in cancer cells H3122(H) and H2228(I). Data represent mean ± SEM, n = 3 independent experiments per group.

[0094] like Figure 3 Results (B and E) show that HX16 exhibits fluorescence emission in the green range of 563 nm with an excitation wavelength of 443 nm. HX16 shows no fluorescence in PBS, but exhibits significant high fluorescence upon the addition of ALK. Furthermore, HX16 shows significant low fluorescence when applied to bovine serum albumin (BSA), indicating that the probe is selective for ALK but less sensitive to other proteins.

[0095] The important thing is, Figure 3 In (C), the enhanced fluorescence was eliminated by pre-incubation with ALK-TKI, further confirming the binding specificity of HX16 to the ALK tyrosine kinase domain.

[0096] Furthermore, a concentration-dependent fluorescence enhancement of HX16 for ALK kinase was observed. These results demonstrate the specific binding of HX16 to ALK kinase. The inhibitory activity of the probe against human ALK was determined using fluorescence mobility shift assay.

[0097] Figure 3 In (G), HX16 showed concentration-dependent inhibition of ALK kinase with an IC50 of 11 nM (8–15 nM; 95% CI, n = 3), indicating that the effect of introducing environmentally sensitive fluorophores into the pharmacophore of ALK-TKIs was minimal.

[0098] The kinase inhibition results further demonstrate that HX16 exhibits a high binding affinity for the ALK kinase domain.

[0099] like Figure 4 The diagram shows the distribution of HX16 in cancer cells observed using confocal microscopy. (AB) Fluorescence imaging of H3122(A) and H2228(B) cells with HX16 (5 μM, 30 min). Uptake of HX16 in cells can be blocked by incubation with ALK-TKI (10 μM, 30 min). Scale bar: 50 μm. (C) Quantitative analysis of the mean fluorescence intensity of the probe in the cell images. Data represent mean ± SEM. Statistical p-values ​​were calculated using the two-tailed Student's test; **P < 0.01, *P < 0.05. (D) Subcellular co-localization study of HX16 with mitochondrial markers, lysosomal markers, or endoplasmic reticulum markers. Scale bar: 50 μm or 25 μm.

[0100] Figure 4 (A) The results showed that a significant increase in fluorescence intensity was observed in H2228 and H3122 cells treated with HX16 by subsequent cell imaging experiments using confocal laser scanning fluorescence microscopy in cells with ALK-EML4 fusion.

[0101] To confirm the specificity of these cell markers, Figure 4 (AC) results showed that a competition experiment was conducted using ALK-TKI. As expected, cells pretreated with ALK-TKI showed approximately 50% lower uptake of HX16 compared to untreated cells.

[0102] The fluorescence signal was weak in cells treated with DMSO or SBD fluorophores, further indicating that the fluorescence labeling specificity depends on the ALK probe HX16.

[0103] These results indicate that HX16 can serve as an imaging tool for detecting ALK activity in cancer cells.

[0104] like Figure 5The following figures illustrate the assessment of ALK activity in a mouse model of NSCLC tumor bearing. (A) HE pathological analysis and immunohistochemical analysis (ALK antibody) of tumor tissue from the H3122 tumor-bearing mouse model. (BC) Flow cytometry analysis of the labeling effect of the HX16 probe on H3122 tumor tissue, and the fluorescence intensity differences between untreated samples (veh), HX16-treated samples (1 mg / kg or 5 mg / kg), and samples pretreated with ALK-TKI (5 mg / kg). Data represent mean ± SEM, n = 3 independent experiments. (DE) Confocal fluorescence imaging of tumor tissue sections from the H3122 tumor-bearing transplant mouse model treated with veh or HX16 (1 mg / kg or 5 mg / kg), or pretreated with ALK-TKI (5 mg / kg).

[0105] Figure 5 Results showed that HX16 was administered intravenously (iv) to a mouse model of NSCLC at doses ranging from 0 to 5 mg / kg. Two hours later, the animals were sacrificed, and the tumor tissue was separated into two fractions for subsequent in vitro evaluation. One fraction of the tissue was rapidly digested into a single-cell suspension, and the HX16 labeling was then evaluated by FACS.

[0106] To accurately quantify probe labeling in tumors, such as Figure 5 As shown in (B), a high proportion of HX16(+) tumor cells were observed at 5 mg / kg, followed by 1 mg / kg, which indicates a dose-dependent increase in fluorescence in HX16-treated mice.

[0107] In addition, such as Figure 5 As shown in (C), the mean fluorescence intensity of HX16 was also significantly reduced (by about 50%) after ALK-TKI pretreatment, indicating that HX16 is a specific marker of mouse tumors in vivo.

[0108] On the other hand, such as Figure 5 As shown in (D), the remaining portion of the tumor was prepared into paraffin sections for tissue imaging using confocal microscopy. Consistent with FACS analysis, the H3122 tumor sections showed significant fluorescence accumulation at HX16 concentrations of 1 mg / kg and 5 mg / kg.

[0109] Similarly, such as Figure 5 As shown in (E), pre-injection of ALK-TKI significantly suppressed the fluorescence signal, further demonstrating the specific labeling of ALK activity by HX16 in the mouse model.

[0110] In addition, such as Figure 5As shown in (A), hematoxylin-eosin (HE) staining was performed to confirm that the H3122 tumor was histopathologically cancerous. Furthermore, ALK expression in the tumor was detected by immunohistochemical (IHC) staining, and high levels of ALK were observed in all H3122 tumors using an ALK antibody.

[0111] These results demonstrate the feasibility of HX16 selectively targeting ALK activity in mouse tumor tissues and can serve as a useful chemical tool for rapidly assessing ALK activity in translational medicine to predict ALK-TKI sensitivity.

[0112] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A specific fluorescent probe targeting ALK, characterized in that, The structural formula of the compound is as follows: Equation (II).

2. A method for preparing a specific fluorescent probe targeting ALK as described in claim 1, characterized in that, Includes the following steps: S1. Synthesis of the first intermediate: Add ceritinib, potassium carbonate and N,N-dimethylformamide to a round-bottom flask and stir for 5-8 minutes at room temperature. Then add ethyl 2-bromoacetate and stir overnight at room temperature. The resulting mixture was then diluted with water and extracted with dichloromethane. The combined organic layers were dried with sodium sulfate, filtered, concentrated under vacuum, and purified to obtain the first intermediate. The first intermediate structure is as follows: ； S2, Synthesis of Compound I: The first intermediate, sodium hydroxide, and methanol were added to a round-bottom flask and stirred overnight at room temperature to obtain a mixture. The pH of the mixture was adjusted to 5 with acid, and the mixture was extracted with ethyl acetate. The combined organic layers were dried with sodium sulfate, filtered, vacuum evaporated, and purified to obtain compound I. The structural formula of compound I is as follows: ； S3, Synthesis of Compound II: SM3, compound I, HATU, DIEA, and N,N-dimethylformamide were added to a round-bottom flask and stirred at room temperature for 1.5-2 hours. The mixture was then diluted with water and extracted with ethyl acetate. The combined organic layers were dried with sodium sulfate, filtered, vacuum evaporated, and purified to obtain compound II. The structural formula of SM3 is as follows: 。 3. The method for preparing a specific fluorescent probe targeting ALK according to claim 2, characterized in that, In S1, the ratio of ceritinib, potassium carbonate, N,N-dimethylformamide, ethyl 2-bromoacetate, water, and dichloromethane used is: 300mg: 148mg: 5mL: 107.7mg: 30mL: 150mL; The first intermediate is a white solid.

4. The method for preparing a specific fluorescent probe targeting ALK according to claim 2, characterized in that, In S2, the ratio of the first intermediate, sodium hydroxide, methanol, and ethyl acetate used is 150 mg: 18.9 mg: 20 mL: 150 mL; Compound I is a white solid.

5. The method for preparing a specific fluorescent probe targeting ALK according to claim 2, characterized in that, In S3, the ratio of SM3, compound I, HATU, DIEA, N,N-dimethylformamide, water, and ethyl acetate used is: 13mg:30mg:37mg:18mg:1 mL:30 mL:150 mL; Compound II is a yellow solid.

6. The application of a specific fluorescent probe targeting ALK as described in claim 1 in the preparation of a tracking and localization fluorescent imaging reagent for tumor cells with high expression of ALK and its mutant proteins.

7. The application of a specific fluorescent probe targeting ALK as described in claim 1 in the preparation of a reagent for evaluating the drug activity of anaplastic lymphoma kinase inhibitors.

Images