Preparation method and application of near-infrared fluorescent probe for specific detection of SHP2

Abstract

The application belongs to the technical field of tumor cell optical imaging, and particularly relates to a preparation method and application of a near-infrared fluorescent probe for specifically detecting SHP2. H The near-infrared fluorescent probe has a structure as shown in I, wherein R is a dicyanoisophorone fluorescent group or a dicyano methylene-4 The probe has a novel structure, strong binding affinity to SHP2 protein, good fluorescence characteristics, fast reaction time, strong specificity, and can specifically recognize SHP2 protein. The near-infrared fluorescent probe can be used for labeling tumor cells with high expression of SHP2 protein. The probe raw material is cheap and easy to obtain, the reaction steps are simple, and the post-treatment process is simple.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of optical imaging of tumor cells, and particularly relates to a preparation method and application of a near-infrared fluorescent probe for specifically detecting SHP2. BACKGROUND

[0002] Protein tyrosine phosphatase, in English, is abbreviated as PTP, Src homology 2-containing protein tyrosine phosphatase 2, in English, is abbreviated as SHP2, which is encoded by the proto-oncogene PTPN11, is a carcinogenic non-receptor protein tyrosine phosphatase first discovered in the cytoplasm. SHP2 drives cell signaling under the recruitment of receptor tyrosine kinases and participates in various oncogenic signaling cascades, positively regulates cell signaling pathways activated by growth factors, cytokines and hormones, promotes cell growth, movement, differentiation and survival, and regulates the signal transduction of various signaling pathways in the body together with other protein tyrosine kinases that can catalyze tyrosine phosphorylation.

[0003] SHP2 can regulate various cancer cell signaling pathways such as Ras-Raf-MAPK, PI3K-Akt and JAK-STAT, participate in cell proliferation, differentiation, survival and apoptosis, and is closely related to tumor growth, metastasis and drug resistance. Studies have found that several cancers such as breast cancer, gastric cancer, lung cancer, liver cancer, prostate cancer and cervical cancer are related to overexpression of SHP2. SHP2 can induce the initiation, progression and metastasis of cancer, indicating that it has the role of a cancer protein. Overexpression of SHP2 has been regarded as a prognostic and predictive biomarker. By analyzing the expression level of SHP2 during tumor formation, it is helpful for early clinical diagnosis of cancer. Therefore, developing an effective tool for specifically detecting SHP2 and establishing a detection system targeting SHP2 play a very important role in the diagnosis and treatment of cancer.

[0004] Fluorescence imaging technology can realize high sensitivity and high spatiotemporal resolution noninvasive real-time imaging, and plays an indispensable role in preclinical and basic research, and is a powerful tool for early diagnosis and subsequent treatment of diseases. In order to improve the accuracy and sensitivity of fluorescence imaging and expand the application range of optical imaging, researchers are constantly seeking to develop new chemical tools to detect more and more disease-related bioactive molecules and monitor key physiological processes of diseases.

[0005] Small molecule fluorescent probes are important tools for visualizing cellular and in vivo pathological processes. They can directly detect and image abnormal levels of enzymes in living cells and have been widely used in the biological and pharmacological detection of important biomolecules such as proteins and nucleic acids. They are of great significance for the development of disease mechanism research, clinical diagnosis, and drug screening. Near-infrared fluorescent probes, in particular, have advantages such as deep penetration, low background, and high resolution. They can penetrate tissues relatively deeply with minimal damage to biological samples, enabling in vivo imaging and have become important tools for basic research and clinical diagnosis. Although fluorescent probes for detecting phosphatases have been developed, the development of small molecule near-infrared probes for selectively detecting specific PTPs remains a challenge due to the structural similarity of protein tyrosine phosphatases and their inherent substrate specificity to phosphotyrosine. Currently, no near-infrared fluorescent probe has been developed for the specific detection of SHP2. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for preparing and applying a near-infrared fluorescent probe for the specific detection of SHP2.

[0007] This invention is achieved through the following technical solution:

[0008] In a first aspect, the present invention provides a near-infrared fluorescent probe for the specific detection of SHP2, having a chemical structural formula as shown in Formula I:

[0009] ;

[0010] Wherein, R is a dicyanoisophorone fluorophore or dicyanomethylene-4 H -Pyran fluorophores.

[0011] Furthermore, the dicyanoisophorone fluorophore is ( E) -2-[3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl]malonitrile, wherein the dicyanomethylene-4 H -Pyran fluorophores are 2-[2-[( E )-2-(4-aminophenyl)vinyl]-4 H [-1-Benzopyran-4-yl]malononitrile.

[0012] Furthermore, when the dicyanoisophorone fluorophore is ( E When 2-[3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl]malonitrile is used, the chemical structure of the near-infrared fluorescent probe is shown in Formula 11: ;

[0013] When the dicyanomethylene-4 H-Pyran fluorophores are 2-[2-[( E )-2-(4-aminophenyl)vinyl]-4 H When [-1-benzopyran-4-yl]malonitrile is used, the chemical structural formula of the near-infrared fluorescent probe is shown in Formula 12: .

[0014] SHP2 possesses a highly conserved structure, comprising two SH2 domains located at the N- and C-termini, a catalytically active PTP domain, and a C-terminal tail rich in two tyrosine phosphorylation sites (Y542 and Y580) and a proline motif. SHP2 exhibits multiple potential allosteric binding sites, including tunnel, latch, and groove binding sites formed between the SH2 and PTP domain interfaces. Compound SHP099 specifically binds to the tunnel allosteric pocket of SHP2, maintaining SHP2 in a self-inhibited state and thus inhibiting its phosphatase activity. Studies have shown that this tunnel allosteric site is unique to SHP2; therefore, SHP099 exhibits high selectivity for SHP2 but no activity against its homolog, SHP1. Furthermore, it was found that the amino group linked to piperidine in SHP099 is exposed to the solvent. To ensure the activity of the target compound, other groups in SHP099 were retained, and the amino group underwent structural modification.

[0015] This invention is based on the structural model of the SHP2 complex with the high-affinity ligand SHP099, and cleverly selects the non-binding region of the ligand and target protein as the design entry point. By introducing carefully designed linker arms and fluorescent groups, a series of near-infrared fluorescent probes specifically targeting SHP2 were successfully designed and synthesized. Experimental results reveal a key phenomenon: different combinations of linker arms and fluorescent groups significantly affect the performance of the probes. Specifically, when the amino group attached to piperidine in SHP099 is linked to the carboxyl group of the linker arm through an amidation reaction to form an amide group, its binding stability with SHP2 is greatly reduced, and it cannot effectively maintain the self-inhibitory state of SHP2. Conversely, when the amino group is cleverly modified into a secondary amine group, the probe can achieve stable binding with SHP2.

[0016] In our initial experiments, we attempted to link compound SHP099 to a fluorescent group via an amide bond, synthesizing compounds A, B, and C. However, activity tests showed that at a concentration of 10 μM, these compounds exhibited inhibition rates of only 26.4%, 11.4%, and 24.7% against SHP2, respectively. This clearly indicates that the inhibitory activity of these compounds is relatively weak and they cannot effectively bind to SHP2. This further confirms the crucial importance of the choice of linker and fluorescent group for probe performance. . . .

[0017] In this invention, the inhibitory activity experiments of two near-infrared fluorescent probes against SHP2 showed that both probes can bind to SHP2. Cellular experiments showed that these two probes could achieve cellular fluorescence imaging, but their effect on inhibiting tumor cell proliferation was limited. This may be because the cellular environment is complex; even if the probes can bind to the target protein, tumor cells may maintain survival through compensatory pathways, or tumor cells may depend on multiple signaling pathways, requiring multi-target intervention to achieve a significant inhibitory effect.

[0018] Secondly, the present invention provides a method for preparing a near-infrared fluorescent probe for specific detection of SHP2, comprising the following steps:

[0019] (1) Starting from compounds 1 and 2, compound 3 was prepared by the Suzuki reaction; a tert-butoxycarbonyl group was introduced into compound 3 to protect the amino group, forming compound 4; compound 4 underwent an aromatic nucleophilic substitution reaction to obtain compound 5; the tert-butoxycarbonyl group on compound 5 was removed to obtain compound 6; compound 6 underwent a nucleophilic substitution reaction to obtain compound 7; the synthetic reaction formula is shown below:

[0020] ;

[0021] (2) 4-( N -methyl- N (-hydroxyethyl)aminobenzaldehyde undergoes an azidation reaction to generate compound 8. Compound 8 then reacts with (3,5,5-trimethylcyclohexyl-2-enyl)malonitrile via a condensation reaction to generate compound 9. Compound 9 is formed by the reaction of 8 with 2-(2-methyl-4-yl)aminobenzaldehyde. H benzopyran-4-yl)malononitrile is synthesized into compound 10 via a condensation reaction; the synthetic reaction formula is shown below:

[0022] ;

[0023] (3) Compound 7 reacts with Compound 9 and Compound 10 via click chemistry to generate Compound 11 and Compound 12, which are the near-infrared fluorescent probes; the synthesis reaction formulas are shown below:

[0024] .

[0025] Thirdly, the present invention provides the application of the near-infrared fluorescent probe in the preparation of SHP2 expression detection products.

[0026] Fourthly, the present invention provides the application of the near-infrared fluorescent probe in screening SHP2 inhibitors.

[0027] Fifthly, the present invention provides the application of the near-infrared fluorescent probe in the preparation of tumor detection products, wherein the near-infrared fluorescent probe is used to label tumor cells with high expression of SHP2 protein.

[0028] Furthermore, the tumor is breast cancer, refractory liver cancer, cervical cancer, prostate cancer, ovarian cancer, gastric cancer, non-small cell lung cancer, or melanoma.

[0029] The beneficial effects of this invention are:

[0030] 1. The probe structure of this invention is novel, has a strong affinity for binding to SHP2 protein, and is relatively sensitive to changes in the surrounding environment, indicating that the probe has good fluorescence properties, fast reaction time, and strong specificity.

[0031] 2. The probe of this invention can specifically recognize the SHP2 protein. The probe of this invention enables high-throughput screening of SHP2 protein inhibitors and its application in the preparation of reagents for detecting SHP2 protein inhibition. The fluorescent probe of this invention can be used for labeling tumor cells with high SHP2 protein expression. The application of the SHP2 near-infrared fluorescent probe of this invention in the preparation of pharmaceutical formulations for screening SHP2 protein-related diseases, wherein the related diseases are malignant tumors; preferably, the SHP2 protein overexpression-related malignant tumors are breast cancer, refractory liver cancer, cervical cancer, prostate cancer, ovarian cancer, gastric cancer, non-small cell lung cancer, and melanoma.

[0032] 3. The probe material of this invention is inexpensive and readily available, and the post-processing is simple. Attached Figure Description

[0033] Figure 1 This is a synthetic route diagram for compounds 1 through 7.

[0034] Figure 2 The synthetic routes for compounds 9 and 10 are shown.

[0035] Figure 3 The synthetic routes for compounds 11 and 12 are shown.

[0036] Figure 4 For the fluorescence properties of compound 11, A: Fluorescence excitation spectrum of compound 11 in PBS buffer solution at different concentrations, B: Fluorescence emission spectrum of compound 11 in PBS buffer solution at different concentrations, C: Fluorescence excitation spectrum of compound 11 in different solvents, D: Fluorescence emission spectrum of compound 11 in different solvents.

[0037] Figure 5For the fluorescence properties of compound 12, A: fluorescence excitation spectrum of compound 12 in PBS buffer solution at different concentrations, B: fluorescence emission spectrum of compound 12 in PBS buffer solution at different concentrations, C: fluorescence excitation spectrum of compound 12 in different solvents, D: fluorescence emission spectrum of compound 12 in different solvents.

[0038] Figure 6 The diagram shows the cytotoxicity of compound 11 in MDA-MB-231 cells, MCF-7 cells, and HUVEC cells. A: MDA-MB-231 cells, B: MCF-7 cells, C: HUVEC cells.

[0039] Figure 7 The diagram shows the cytotoxicity of compound 12 in MDA-MB-231 cells, MCF-7 cells, and HUVEC cells. A: MDA-MB-231 cells, B: MCF-7 cells, C: HUVEC cells.

[0040] Figure 8 Imaging of 10 µM compound 11 in MDA-MB-231 cells, MCF-7 cells, and HUVEC cells. A: Bright field channel of MDA-MB-231 cells, B: Bright field channel of MCF-7 cells, C: Bright field channel of HUVEC cells, D: Fluorescence channel of MDA-MB-231 cells, E: Fluorescence channel of MCF-7 cells, F: Fluorescence channel of HUVEC cells; Scale bar is 50 μm.

[0041] Figure 9 Imaging of 10 µM compound 12 in MDA-MB-231 cells, MCF-7 cells, and HUVEC cells. A: Bright field channel of MDA-MB-231 cells, B: Bright field channel of MCF-7 cells, C: Bright field channel of HUVEC cells, D: Fluorescence channel of MDA-MB-231 cells, E: Fluorescence channel of MCF-7 cells, F: Fluorescence channel of HUVEC cells; Scale bar is 50 μm. Detailed Implementation

[0042] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0043] The following examples include some compound names and their corresponding CAS numbers:

[0044] Compound 1: 2,3-dichlorophenylboronic acid, CAS number 151169-74-3.

[0045] Compound 2: 3-bromo-6-chloropyrazine-2-amine, CAS number 212779-21-0.

[0046] PdCl2: Palladium dichloride, CAS number 72287-26-4.

[0047] (Boc)2O: Di-tert-butyl dicarbonate, CAS number 24424-99-5.

[0048] (4-Methylpiperidin-4-yl)tert-butyl carbamate: CAS number 163271-08-7.

[0049] 4-[ N -(2-hydroxyethyl)- N [-Methyl]aminobenzaldehyde: CAS number is 27913-86-6.

[0050] 2-(3,5,5-trimethylcyclohexyl-2-enyl)malononitrile: CAS number 23051-44-7.

[0051] 2-(2-methyl-4-) H Benzopyran-4-yl)malononitrile: CAS number is 15058-15-8.

[0052] Example 1: Preparation of a near-infrared fluorescent probe for specific detection of SHP2.

[0053] Reference Figure 1 - Figure 3 The preparation steps are as follows:

[0054] Preparation of compound 3: 1 g of compound 1, 1.09 g of compound 2, 383 mg of PdCl2 and 20 mL of 50% (v / v) 1,4-dioxane aqueous solution were mixed and stirred at 110 °C under nitrogen protection for 12 h. After cooling to room temperature, the mixture was filtered with Celite diatomaceous earth and then washed with ethyl acetate. The solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography with PE / EA = 100:1-20:1. After purification, 807 mg of yellow solid was obtained, with a yield of 56%. 1 H NMR (400MHz, CDCl3) δ 8.01 (s, 1H), 7.59 (dd, J =7.8, 1.9 Hz, 1H), 7.36(t, J =7.7Hz, 1H), 7.31(dd, J =7.6,1.9Hz,1H),4.71(s,2H).

[0055] Preparation of compound 4: 200 mg of compound 3, 398 mg of (Boc)2O and 10 mg of 4-dimethylaminopyridine were dissolved in 10 mL of dichloromethane and stirred at room temperature for 8 h; the reaction mixture was diluted with 1 M HCl, washed with brine and dried with MgSO4; the crude product was purified by silica gel chromatography, PE / EA = 100:1-30:1, and 175 mg of yellow oily product was obtained after purification, with a yield of 50%. 1 H NMR (400MHz, CDCl3) δ 8.67 (s, 1H), 7.56 (dd, J =7.8, 1.7 Hz, 1H), 7.34 (dd, J =7.7, 1.7 Hz, 1H), 7.28(t, J =7.8Hz, 1H), 1.37(s, 6H).

[0056] Preparation of compound 5: 753 mg of compound 4, 410 mg of tert-butyl (4-methylpiperidin-4-yl)carbamate, and 0.4 mL of... N , N - Diisopropylethylamine was added to 8 mL of dimethylformamide and stirred at 80 °C for 8 h. The reaction mixture was diluted with ethyl acetate and washed with water and brine. The organic layer was dried with MgSO4, filtered, and concentrated to obtain the crude compound. The crude product was purified by rapid chromatography with PE / EA = 100:1-5:1. After purification, 689 mg of a yellow oil was obtained, with a yield of 68%. 1 H NMR (400MHz, DMSO-) d 6 ) δ 8.41(s,1H),7.67(dd, J =8.1, 1.5 Hz, 1H), 7.42(t, J =7.9Hz, 1H), 7.28(dd, J =7.8,1.5Hz,1H),6.67(s,1H),3.90(dd, J =13.7, 4.7 Hz, 2H), 3.29 (d, J =11.5Hz,2H),2.14(d, J =13.4Hz,2H),1.47(d, J =10.1Hz,2H),1.39(s,10H),1.27(s,20H).

[0057] Preparation of compound 6: 689 mg of compound 5 was stirred in 24 mL of 4 M HCl / EtOAc at room temperature for 8 h. After filtration, 382 mg of yellow solid was obtained, with a yield of 92%. 1H NMR (400MHz, DMSO-) d 6 ) δ 8.35(s,3H),7.71(dd, J =7.9,1.7Hz,1H),7.59(s,1H),7.45(t, J =7.8Hz, 1H), 7.39(dd, J =7.7, 1.7 Hz, 1H), 4.01 (d, J =5.0Hz,2H),3.37(ddd, J =13.4,9.4,3.6Hz,2H),1.87–1.68(m,4H),1.39(s,3H).

[0058] Preparation of compound 7: 500 mg of compound 6 was dissolved in acetonitrile. Under stirring, 169 mg of bromopropyne, 535 mg of potassium carbonate, and 21 mg of potassium iodide were added, and the mixture was heated to reflux at 80 °C. After the reaction was complete, the reaction solution was evaporated to dryness, extracted with ethyl acetate after adding water, and the organic phases were combined. The mixture was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 97%:3%), yielding 210 mg of a yellow solid (42% yield). 1 H NMR (400MHz, DMSO-) d 6) δ 7.67–7.59(m,1H),7.50(s,1H),7.40(t, J =7.8Hz,1H),7.36–7.29(m,1H),5.63(s,2H),3.72–3.56(m, 2H),3.57–3.45(m,2H),3.02(s,1H),1.68–1.52(m,2H),1.50–1.36(m,2H),1.10(s,3H).

[0059] Preparation of compound 8: 1.79 g of 4-[ N -(2-hydroxyethyl)- N [-methyl]aminobenzaldehyde and 4.13 g of diphenyl azidophosphate were dissolved in 25 mL of tetrahydrofuran. 2.28 mg of 1,8-diazabicyclo[5.4.0]undec-7-ene was added dropwise at 0 °C. The mixture was stirred at 0 °C for 1 h, then slowly heated to 70 °C and stirred for 12 h. After removing the solvent under vacuum, the crude product was purified by silica gel column chromatography (PE / EA = 96%:4%), yielding 1.747 g of a yellow oily product (85% yield). 1 H NMR (400MHz, DMSO-) d 6) δ 9.65(s,1H),7.67(d, J =8.6Hz,2H),6.84(d, J =8.6Hz,2H),3.65(t, J =5.9Hz,2H),3.50(t, J =5.8Hz,2H),3.02(s,3H).

[0060] Preparation of Compound 9: 549 mg of Compound 8 and 500 mg of 2-(3,5,5-trimethylcyclohexyl-2-enylidene)malonitrile were dissolved in 15 mL of acetonitrile. 0.5 mL of piperidine was added, and the mixture was stirred at 40 °C for 8 h under nitrogen protection. After the reaction was complete, the reaction solution was evaporated to dryness, water was added, and the mixture was extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, and purified by silica gel column chromatography (PE / EA = 94%:6%). 859 mg of a dark solid was obtained after purification, with a yield of 86%. Melting point: 136-138 °C. 1 H NMR (400MHz, DMSO-) d 6) δ 7.50(d, J =8.5Hz,2H),7.18(d, J =16.0Hz, 1H), 7.07(d, J =15.9Hz, 1H), 6.72(d, J =8.5Hz,2H), 6.68(s,1H), 3.58(t, J =5.9Hz,2H),3.45(t, J =5.8Hz,2H),2.95(s,3H),2.46(s,4H),0.94(s,6H).

[0061] Preparation of compound 10: 490 mg of compound 8 and 500 mg of 2-(2-methyl-4-ethylhexylene) were mixed. H (-Benzopyran-4-yl)malononitrile was dissolved in 15 mL of acetonitrile, and 0.5 mL of piperidine was added. The mixture was stirred at 40 °C for 8 h under nitrogen protection. After the reaction was complete, the reaction solution was evaporated to dryness, water was added, and the mixture was extracted with dichloromethane. The combined organic phases were washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed to obtain the crude compound. The crude product was purified by silica gel column chromatography (PE / EA = 93%:7%), yielding 765 mg of a dark solid (80% yield). Melting point: 138-140 °C. 1 H NMR (400MHz, DMSO-) d 6) δ 8.68(d, J =8.3Hz, 1H), 7.85(t, J=7.9Hz, 1H), 7.73(d, J =8.4Hz, 1H), 7.65(d, J =15.8Hz,1H),7.61–7.50(m,3H),7.15(d, J =15.8Hz,1H),6.86(s,1H),6.79(d, J =8.5Hz,2H),3.62(t, J =5.8Hz,2H),3.49(t, J =5.9Hz,2H),3.00(s,3H).

[0062] Preparation of Compound 11: 104 mg of Compound 7 was dissolved in an equal volume mixture of tetrahydrofuran and water. 101 mg of Compound 9, 54 mg of copper sulfate, and 160 mg of sodium ascorbate were added sequentially with stirring, and the reaction was carried out at room temperature. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 97%:3%), yielding 50 mg of a dark solid (yield 24%) with a melting point of 154-156 °C. 1 H NMR (400MHz, DMSO-) d 6) δ 7.93(s, 1H), 7.59(d, J =8.0Hz, 1H), 7.49(d, J =8.6Hz,2H),7.45(s,1H),7.36(t, J =7.8Hz, 1H), 7.27(d, J =7.6Hz, 1H), 7.20(d, J =15.9Hz, 1H), 7.11(d, J =15.9Hz,1H),6.72(s,1H),6.64(d, J =8.6Hz,2H), 5.60(s,2H), 4.50(t, J =6.4Hz,2H),3.84(t, J =6.1Hz,2H),3.69(s,2H),3.60–3.52(m,2H),3.51–3.44(m,2H),2.79(s,3H), 1.62–1.55(m,2H),1.48–1.40(m,2H),1.20(s,4H),1.11(s,3H),0.97(s,6H). 13 C NMR (101MHz, DMSO-) d6) δ 170.46,157.79,153.62,151.11,150.28,149.51,139.90,139.60,138.74,132.49,131.99,131.10,130.32,130.14,128.69,12 4.95,124.35,120.87,117.05,114.96,114.15,112.28,73.68,67.87,52.00,47.26,42.78,40.65,38.69,38.38,32.12,27.93.

[0063] Preparation of Compound 12: 104 mg of Compound 7 was dissolved in an equal volume mixture of tetrahydrofuran and water. 107 mg of Compound 9, 54 mg of copper sulfate, and 160 mg of sodium ascorbate were added sequentially with stirring, and the reaction was carried out at room temperature. After the reaction was complete, water was added to the reaction solution, and the mixture was extracted with ethyl acetate. The combined organic layers were washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography (DCM / MeOH = 96%:4%), yielding 30 mg of a dark solid (yield 14%) with a melting point of 148-150 °C. 1 H NMR (400MHz, DMSO-) d 6) δ 8.69(d, J =8.3Hz, 1H), 7.96(s, 1H), 7.86(t, J =7.8Hz, 1H), 7.75(d, J =8.4Hz, 1H), 7.66(d, J =15.7Hz,1H),7.59–7.55(m,3H),7.45(s,1H),7.35(t, J =7.6Hz, 1H), 7.26(d, J =7.6Hz, 1H), 7.17(d, J =16.0Hz,2H),6.88(s,1H),6.71(d, J =8.3Hz,2H),6.66(s,1H),5.59(s,2H),5.29(t, J =4.9Hz,2H),4.52(s,2H),3.87(s,2H),3.74–3.65(m,2H),3.60–3.52(m,2H),2.80(s,3H),1.63–1.56(m,2H),1.44–1.40(m,2H),1.12(s,3H). 13 C NMR (101MHz, DMSO-) d6) δ 159.94,153.54,153.10,152.52,151.10,150.80,140.41,139.84,135.54 ,132.48,131.96,131.07,130.78,130.12,128.68,126.39,125.02,123.2 7,119.39,118.20,117.65,117.05,116.86,114.05,112.35,105.54,58.23,52.02,47.31,38.50,35.58,31.77,29.55,29.05,27.02,25.59,22.58.

[0064] Experimental Example 1: Determination of the optical activity of probe compounds.

[0065] In Example 1, fluorescent probe compounds 11 and 12 were prepared into concentrated stock solutions with a concentration of 10 mM using dimethyl sulfoxide (DMSO). These solutions were then diluted with PBS buffer to obtain solutions of different concentrations. Finally, solutions were diluted with different organic solvents to a concentration of 5 µM. The fluorescence spectra of the probe compounds at different concentrations and in different solvents were measured using a fluorescence spectrophotometer to investigate the fluorescence characteristics of the probes. The experimental results show that the fluorescence intensity of compounds 11 and 12 increases with increasing concentration and is relatively sensitive to changes in the surrounding environment, indicating that the probes possess good fluorescence characteristics. Figure 4 and Figure 5 In addition, using cresol purple as a reference, the fluorescence quantum yields of compounds 11 and 12 were determined, as shown in Table 1.

[0066] Table 1: Optical properties of the probe compounds.

[0067]

[0068] Experimental Example 2: Determination of the bioactivity of probe compounds.

[0069] To determine the binding activity of the fluorescent probe compound SHP2 to protein, 30 µL of the probe compound, 20 µL of SHP2, and 10 µL of 2p-IRS1 peptide were added to a 96-well black ELISA plate and incubated at 4 °C for 10 min. Then, 30 µL of DiFMUP was added and the plate was incubated at 37 °C for 20 min. The fluorescence intensity at 350 nm and 450 nm was measured using an ELISA reader.

[0070] The results are shown in Table 2. The experimental results show that compounds 11 and 12 inhibited SHP2 by more than 50% at 10 μM, indicating that compounds 11 and 12 can bind to the target enzyme SHP2 and have good affinity.

[0071] Table 2: Determination of the inhibitory activity of probe compounds against SHP2 protein

[0072]

[0073] Experimental Example 3: Cytotoxicity Experiment of Probe Compounds.

[0074] Logarithmic growth phase MDA-MB-231 cells, MCF-7 cells, and HUVEC cells were seeded in clear 96-well plates, 100 μL per well, approximately 7 × 10⁶ cells / well. 3 -8×10 3 Cells were cultured at 37°C in a 5% CO2 incubator for 12 hours until adherence. Then, 100 μL of probe solutions of different concentrations were added, with three replicates for each concentration. A control group (cells only, no compound added) and a blank group (no cells or compounds added) were also included. Cells were incubated for 24 hours. After incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 hours. Absorbance values ​​were measured using a microplate reader. The cytotoxicity results of compounds 11 and 12 are shown below. Figure 6 , Figure 7 As shown in the figure. The experimental results indicate that all cells exhibited good survival rates under incubation with the above-mentioned probe compounds at different concentrations, demonstrating that the probe compounds have good biocompatibility and low cytotoxicity, and can be used for subsequent live-cell fluorescence imaging experiments, etc.

[0075] Experiment Example 4: Cell fluorescence imaging experiment of probe compound.

[0076] Cellular fluorescence imaging was performed using MDA-MB-231 cells and MCF-7 cells, with HUVEC cells used as a control. The specific steps were as follows: MDA-MB-231 cells, MCF-7 cells, and HUVEC cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Before imaging, MDA-MB-231 cells, MCF-7 cells, and HUVEC cells in logarithmic growth phase were seeded in 6-well plates. After 12 hours of adherence, the supernatant was discarded using a pipette, and the cells were washed three times with PBS buffer. 1 mL of 10 μM fluorescent probe solution was added to each of the 6-well plates containing MDA-MB-231 cells, MCF-7 cells, and HUVEC cells, respectively. The plates were incubated at room temperature in the dark for 30 minutes. The fluorescent probe solution was then discarded, and the cells were washed twice with PBS. Fluorescence imaging was performed using an inverted fluorescence microscope. The imaging results are shown below. Figure 8 , Figure 9As shown, compounds 11 and 12 exhibit fluorescence responses in MDA-MB-231 and MCF-7 cells overexpressing SHP2; however, their fluorescence response values ​​are low in normal HUVEC cells. This indicates that compounds 11 and 12 can selectively screen tumor cells overexpressing SHP2 protein, enabling fluorescence imaging of tumor cells.

[0077] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A near-infrared fluorescent probe for specifically detecting SHP2, characterized in that, The near-infrared fluorescent probe has a chemical structural formula as shown in formula I: ； wherein R is a dicyanohydroquinone or a dicyanomethylene-4 H - a pyranoid fluorophore; said dicyanohydroquinone is E -2-[3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene]prop-2-enedinitrile, said dicyanomethylene-4 H - a pyranoid fluorophore is 2-[2-[( E -2-(4-aminophenyl)vinyl]-4 H -1-benzopyran-4-ylidene]prop-2-enedinitrile; when the dicyanoisophorone-based fluorophore is E when the dicyanoisophorone-based fluorophore is ; when the dicyanomethylene-4 H - the pyran fluorophore is 2-[2-[( E )-2-(4-aminophenyl)vinyl]-4 H -1-benzopyran-4-ylidene]propanedinitrile, the chemical structure of the near-infrared fluorescent probe is shown as Formula 12: .

2. The method for preparing the near-infrared fluorescent probe for specifically detecting SHP2 according to claim 1, characterized in that, The method comprises the following steps: (1) using compound 1 and compound 2 as starting materials, compound 3 is prepared by a Suzuki reaction; a tert-butyloxycarbonyl group is introduced into compound 3 to protect the amino group, forming compound 4, compound 4 is subjected to an aromatic nucleophilic substitution reaction to obtain compound 5; the tert-butyloxycarbonyl group on compound 5 is removed to obtain compound 6; compound 6 is subjected to a nucleophilic substitution reaction to obtain compound 7; the synthesis reaction formula is shown as follows: ； (2) 4- N - methyl N - hydroxyethyl) aminobenzaldehyde to generate compound 8 by azidation reaction, compound 8 and (3, 5, 5-trimethylcyclohex-2-enylidene) malononitrile generate compound 9 by condensation reaction, compound 8 and 2-(2-methyl-4 H - benzopyran-4-ylidene) malononitrile generate compound 10 by condensation reaction; the synthesis reaction formula is shown as follows: ； (3) compound 7 is subjected to a click chemistry reaction with compound 9 and compound 10 respectively to obtain compound 11 and compound 12, i.e. the near-infrared fluorescent probe; the synthesis reaction formula is shown as follows: 。 3. The near-infrared fluorescent probe of claim 1 is used for preparing a SHP2 expression detection product.

4. The near-infrared fluorescent probe of claim 1 is used for screening a SHP2 inhibitor.

5. The near-infrared fluorescent probe of claim 1 is used for preparing a tumor detection product.

6. Use of the near-infrared fluorescent probe according to claim 5 in the manufacture of a tumor detection product, characterized by, The near-infrared fluorescent probe is used for labeling tumor cells with high expression of SHP2 protein.

7. Use of the near-infrared fluorescent probe according to claim 6 in the manufacture of a tumor detection product, characterized by, The tumor is breast cancer, refractory liver cancer, cervical cancer, prostate cancer, ovarian cancer, gastric cancer, non-small cell lung cancer or melanoma.

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