Near-infrared fluorescent probe, preparation method therefor and use thereof

Abstract

A near-infrared fluorescent probe, a preparation method therefor, and a use thereof. The preparation method comprises: mixing 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, an alkali, and a polar solvent, and adding crizotinib and a linker molecule for a reaction to obtain a product a; dropwise adding the product a to water to form a suspension, adding an organic solvent for extraction, drying an extract, then concentrating the extract, then adding trifluoroacetic acid, and after the reaction is complete, performing column chromatography to obtain an intermediate b; and adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, an alkali, a dye molecule, and a polar solvent for a reaction to obtain a mixture c, and purifying the mixture c to obtain the near-infrared fluorescent probe. The provided probe can actively target a cellular mesenchymal-epithelial transition factor, can be quickly removed in normal tissues, and can be retained for a long time at a tumor site, thereby achieving the effect of in vivo diagnosis.

Description

A near-infrared fluorescent probe and its preparation method and application Technical Field

[0001] The present invention relates to the field of organic fluorescent probe molecules, and in particular to a near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Cancer poses a serious threat to human life and health. With the deepening of the aging population and changes in people's lifestyles, the challenges posed by cancer are becoming more severe. Clinically, the main treatments for cancer include surgical resection, chemotherapy, radiotherapy, etc. Among them, surgical resection is currently the preferred option for treating solid tumors. Accurate resection of tumors can effectively improve patient prognosis and prolong patient survival. In order to achieve accurate identification of tumors and surgical navigation, technologies including computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET) and ultrasound (US) have been used for imaging and diagnosis of cancer tissues. However, due to inherent limitations such as low spatial resolution or sensitivity, their ability to accurately identify the boundaries of diseased tissues is affected. Optical molecular imaging surgical navigation technology has the characteristics of real-time, non-invasive and high resolution, providing a new imaging-assisted means for precise tumor surgery. Near-infrared fluorescence imaging diagnosis has advantages in tumor diagnosis (early, middle and late), intraoperative navigation, prognosis and recurrence monitoring diagnosis. This diagnostic method mainly uses near-infrared fluorescent probes (650-1000nm) for specific detection. It can provide high-resolution tissue and organ images, has the advantages of low biological toxicity and low autofluorescence, and is conducive to minimizing background interference.

[0003] Indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye that readily binds to plasma proteins after intravenous injection, is metabolized by the liver into bile, and can be retained in tumor tissue via the EPR effect. However, ICG has poor specificity, making it difficult to distinguish tumor tissue from peritumoral inflammatory tissue and benign nodules, which can easily lead to false-positive results. Near-infrared fluorescent probes with specific targeting and renal excretion can effectively avoid false-positive results and significantly improve the tumor-to-background ratio, allowing surgeons to more accurately identify tumor boundaries during surgery, completely remove residual tumor lesions, and maximize patient survival and improve quality of life.

[0004] Cellular mesenchymal-epithelial transition factor (c-MET) is a tyrosine kinase receptor, and hepatocyte growth factor (HGF) is the sole ligand for c-MET. It is a paracrine signaling molecule produced and secreted by mesenchymal cells during development. Under normal physiological conditions, HGF / c-MET mediates embryonic development, cell proliferation, tissue repair, and neuromuscular formation. Many targets involved in or implicated in the c-MET signaling pathway are crucial for the development of various cancers. Abnormal activation of these targets can contribute to the development of various cancers, thus highlighting the crucial role of c-MET in the pathogenesis of cancer. Numerous studies have demonstrated that c-MET targets are susceptible to overactivation, which can potentially transform normal cells into cancer cells and further promote subsequent events such as invasion, metastasis, and spread.

[0005] Crizotinib is an anti-tumor drug and tyrosine kinase inhibitor whose primary target is c-MET. It is used to treat patients with locally advanced or metastatic non-small cell lung cancer (NSCLC) that is positive for anaplastic lymphoma kinase (ALK) as determined by a CFDA-approved test. Crizotinib specifically targets tumor tissue, activating pathways that further lead to tumor cell apoptosis, thus playing a therapeutic role. In the development of tumor-specific fluorescent probes, anti-tumor drugs with tumor-specific killing effects have considerable research value and can be used in the development of specifically targeted fluorescent probes, further expanding the application value of drugs for surgical navigation and tumor resection.

[0006] Patent publication number CN116200188A discloses a near-infrared zone II indocyanine green nanoparticle-specific tumor imaging fluorescent probe, its preparation method and application. It uses hexadecyltrimethylammonium bromide as a template and uses a one-pot co-condensation method to synthesize monodisperse thiol-functionalized nanoparticles. Anti-PD-L1 antibodies are then coupled to the obtained nanoparticles through a polyethylene glycol linker. After dispersion, an antibody-polyethylene glycol functionalized nanoparticle solution is obtained. Indocyanine green (ICG) solution is added to the above solution. After dispersion, a near-infrared zone II indocyanine green nanoparticle-specific tumor imaging fluorescent probe is formed. The thiol-functionalized nanoparticles in this fluorescent probe can prevent ICG aggregation and reduce fluorescence quenching, but the problem is that false positives are easily generated.

[0007] Summary of the Invention

[0008] The purpose of the present invention is to provide a near-infrared fluorescent probe and its preparation method and application. The probe can actively target the mesenchymal-epithelial transition factor, can be quickly cleared in normal tissues, and can be retained in the tumor site for a long time, thereby achieving the effect of in vivo diagnosis.

[0009] To achieve the above object, the present invention provides a near-infrared fluorescent probe, which is a compound represented by formula (I): (Ⅰ)

[0010] In formula (I), X is a linker molecule selected from any one of: n-terminal, PEG, and polyglycine chain; and Y is a dye molecule having fluorescence excitation and emission spectra in the near-infrared range.

[0011] Preferably, the structural formula of Y is:

[0012] In the structural formula, R1 and R2 are selected from (CH2)nSO3H, n=3 or 4; Z is selected from 3-mercaptopropionic acid or 4-hydroxyphenylpropionic acid.

[0013] Preferably, the near-infrared fluorescent probe actively targets mesenchymal-epithelial transition factor.

[0014] The present invention also provides a method for preparing a near-infrared fluorescent probe, comprising the following steps:

[0015] S1: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, and a polar solvent are mixed evenly, crizotinib and a linker molecule are added, and the mixture is reacted at room temperature for 1-2 hours to obtain product a, which is then added dropwise to water to form a suspension;

[0016] S2: Ethyl acetate was added to the suspension for extraction, the extract was dried and concentrated, and trifluoroacetic acid was added to react at room temperature for 30-50 minutes. After column chromatography, intermediate b was obtained;

[0017] S3: adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, a dye molecule having a fluorescence excitation and emission spectrum in the near-infrared range, and a polar solvent to the intermediate b, and stirring at room temperature for 1 to 2 hours to obtain a mixture c;

[0018] S4: Purify the mixture c to obtain a near-infrared fluorescent probe.

[0019] Preferably, the molar ratio of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, base, crizotinib, linker molecule, and polar solvent in S1 is (1-2):(1-3):1:1.2:70.

[0020] Preferably, the volume ratio of ethyl acetate to trifluoroacetic acid in S2 is 1:(1-2).

[0021] Preferably, the molar ratio of intermediate b, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, base, dye molecule having fluorescence excitation and emission spectrum in the near-infrared range, and polar solvent in S3 is (1-2):2:(1-3):1.1:(90-95).

[0022] Preferably, the base is any one or more of triethylamine and diisopropylethylamine; and the polar solvent is any one or more of dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone.

[0023] The present invention provides an application of a near-infrared fluorescent probe in the preparation of a fluorescent contrast agent.

[0024] The present invention also provides an application of a near-infrared fluorescent probe in the preparation of tumor diagnostic drugs.

[0025] Preferably, the tumor is liver cancer or colorectal cancer.

[0026] The beneficial effects achieved by the present invention are:

[0027] 1. Crizotinib is an anti-tumor drug whose primary target is c-MET. It can specifically target tumor tissue, activate pathways that further lead to tumor cell apoptosis, and thus play a role in tumor treatment. The present invention connects crizotinib and dye molecules via a linker molecule. Under the action of the HATU condensing agent, the carboxyl and amino groups of these three compounds react to form an amide bond, allowing for coupling to create a near-infrared fluorescent probe. This further expands the drug's application value and can be used for surgical navigation to remove tumors.

[0028] 2. The near-infrared fluorescent probe prepared by the present invention can actively target mesenchymal-epithelial transition factor. It has the advantages of active tumor targeting ability, good water solubility, long tumor retention time, and no accumulation in normal tissues. It can not only be used to prepare fluorescent contrast agents or tumor diagnostic drugs, but also has application potential in the field of fluorescence-guided tumor surgical resection during clinical surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 is the absorption spectra of NY-cMet-01 to 06 prepared in Examples 1 to 6;

[0030] FIG2 is the fluorescence spectra of NY-cMet-01 to 06 prepared in Examples 1 to 6;

[0031] FIG3 shows the in vivo imaging results of NY-cMet-01-05 prepared in Examples 1-5 in HepG2 liver cancer-bearing mice;

[0032] FIG4 shows the in vivo imaging results of NY-cMet-02 and NY-cMet-04 prepared in Example 2 and Example 4 in mice bearing colorectal cancer HCT116 tumors. DETAILED DESCRIPTION

[0033] The present invention will be further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention.

[0034] The materials and sources used in the examples are shown in Table 1 below:

[0035] Table 1 Materials and sources

[0036] Example 1

[0037] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0038] (1) Crizotinib (100 mg, 1.0 eq), S0456-Der-01 (248 mg, 1.1 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 169 mg, 2.0 eq), N,N-diisopropylethylamine (86 mg, 3.0 eq), and DMSO (1 mL, 70.0 eq) were mixed and stirred, and reacted at room temperature in the dark for 1 hour. The reaction was monitored by liquid chromatography (HPLC). After the reaction was complete, a mixture was obtained.

[0039] (2) The mixture is purified by liquid phase to obtain a target fraction solution, which is lyophilized to obtain a green solid, namely the near-infrared fluorescent probe NY-cMet-01.

[0040] The synthetic route of the near-infrared fluorescent probe NY-cMet-01 is as follows:

[0041] (3) The structure of NY-cMet-01 was characterized by mass spectrometry, H NMR spectrum, and C NMR spectrum. The results of the structure determination are as follows: [M-2H] / 2-=723.4; 1H NMR (600MHz, DMSO-d6) δ8.17 (s, 1H), 7.98 (d, J=13.5Hz, 2H), 7.81–7.71 (m, 3H), 7.63–7.57 (m, 6H), 7.46 (td, J=8.7 , 5.6Hz, 1H), 7.32–7.25 (m, 4H), 7.18 (s, 1H), 7.04 (d, J=8.5Hz, 2H), 6.35 (td, J=13.2, 6.6Hz, 3H), 6.21–6.13 (m, 2H) , 4.36 (d, J = 10.4Hz, 2H), 4.11 (s, 4H), 3.86–3.76 (m, 1H), 3.08 (t, J = 11.4Hz, 1H), 2.80 (dt, J = 20.8, 6.8Hz, 2H), 2.7 2–2.51 (m, 10H), 1.99 (d, J=31.4Hz, 2H), 1.82 (dd, J=48.5, 23.8Hz, 13H), 1.66–1.50 (m, 2H), 1.23 (d, J=5.7Hz, 12H); 13 C NMR (151MHz, DMSO-d6) δ172.01, 170.14, 163.52, 158.98, 158.72, 158.53, 158.47, 156.55, 146.44, 145.43, 142.56, 14 1.45, 141.32, 140.77, 136.10, 135.66, 131.26, 130.97, 129.35, 126.59, 125.71, 125.62, 122.51, 122.13, 121.70, 120. 07, 118.66, 118.56, 118.10, 116.67, 116.59, 116.45, 114.88, 114.54, 110.78, 101.05, 73.98, 58.42, 51.10, 48.91, 44.06, 43.90, 34.56, 32.84, 32.64, 32.07, 31.85, 30.17, 27.61, 27.58, 27.52, 27.47, 26.32, 24.18, 22.75, 21.16, 18.89.

[0042] Example 2

[0043] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0044] (1) Crizotinib (100 mg, 1.0 eq), BOC-NH-PEG4 acid (98 mg, 1.2 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (169 mg, 2.0 eq), N,N-diisopropylethylamine (86 mg, 3.0 eq), and DMSO (1 mL, 70.0 eq) were reacted at room temperature for 1 hour. The reaction was monitored by thin layer chromatography (TLC). After the reaction was complete, the reaction solution was dropped into water to form a suspension.

[0045] (2) 10 mL of ethyl acetate was added to the suspension for extraction. The organic layers were combined and concentrated, and then 10 mL of trifluoroacetic acid was added to react at room temperature for 30 min. The reaction was monitored by thin layer chromatography (TLC). After the reaction was complete, the mixture was evaporated to dryness and then purified by column chromatography to obtain the crizotinib-PEG4 intermediate.

[0046] (3) Crizotinib-PEG4 intermediate (100 mg, 1.0 eq), S0456-Der-01 (160 mg, 1.1 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (110 mg, 2.0 eq), N,N-diisopropylethylamine (56 mg, 3.0 eq), and DMSO (1 mL, 93.3 eq) were mixed and stirred, and reacted at room temperature in the dark for 1 hour. The reaction was monitored by liquid chromatography (HPLC). After the reaction was complete, a mixture was obtained.

[0047] (4) The mixture is purified by liquid phase to obtain a target fraction solution, which is lyophilized to obtain a green solid, namely the near-infrared fluorescent probe NY-cMet-02.

[0048] The synthetic route of the near-infrared fluorescent probe NY-cMet-02 is as follows:

[0049] (5) The structure of NY-cMet-02 was characterized by mass spectrometry, H NMR spectrum, and C NMR spectrum. The results of the structure determination are as follows: [M-2H] / 2-=845.8; 1H NMR (600MHz, DMSO-d6) δ8.16 (s, 1H), 8.05 (s, 2H), 7.87 (t, J=5.5Hz, 1H), 7.77 (d, J=14.0Hz, 2H), 7.71 (s, 1H), 7.60 (ddd, J=14.1, 11.0, 7.3Hz, 6H), 7.46 (t, J =8.7Hz, 1H), 7.31 (d, J = 8.3Hz, 2H), 7.20 (d, J = 8.6Hz, 2H), 7.14 (d, J = 0.9Hz, 1 H), 6.99 (d, J=6.7Hz, 2H), 6.29 (q, J=6.7Hz, 1H), 6.20 (d, J=14.2Hz, 2H), 4.45 ( d, J=10.9Hz, 2H), 4.12 (s, 4H), 4.00 (d, J=12.9Hz, 1H), 3.67–3.59 (m, 2H), 3.4 9(s, 4H), 3.46–3.40(m, 8H), 3.33(t, J=5.8Hz, 2H), 3.19(t, J=12.7Hz, 1H), 3.1 2(q, J=5.6Hz, 2H), 2.79–2.66(m, 7H), 2.65–2.54(m, 6H), 2.29–2.21(m, 2H), 2. 09–1.98 (m, 2H), 1.94–1.81 (m, 6H), 1.81–1.66 (m, 9H), 1.23 (d, J=7.1Hz, 12H); 13 C NMR (151MHz, DMSO-d6) δ172.05, 171.74, 169.30, 163.50, 158.97, 158.72, 158.46, 146.54, 145.41, 142.58, 141.4 9, 141.31, 140.73, 135.88, 135.65, 135.61, 130.54, 129.27, 126.62, 125.73, 122.51, 122.13, 120.08, 118.58, 11 ,8.51,118.43,117.98,116.70,114.82,110.84,101.08,74.00,70.26,70.21,70.17,69.99,69.53,67.39,58.69,51.11,48.94,44.28,44.04,38.97,37.68,33.28,33.05,32.11,30.69,27.56,26.32,24.19,22.74,21.18,18.91.

[0050] Example 3

[0051] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0052] The difference from Example 2 is that BOC-GLY-GLY-GLY-OH is used to replace the BOC-NH-PEG4 acid in Example 2 for the reaction.

[0053] The structural formula of the infrared fluorescent probe NY-cMet-03 is as follows:

[0054] The structure of NY-cMet-03 was characterized by mass spectrometry, H NMR spectrum, and C NMR spectrum. The results of the structure determination are as follows: [M-3H] / 3-=539.5; 1 H NMR (600MHz, DMSO-d6) δ8.24-8.15 (m, 3H), 8.02 (s, 2H), 7.91-7.86 (m, 1H), 7.79 (t, J=15.1Hz, 2H), 7.69 (dd, J=23.8, 12.6 Hz, 1H), 7.64-7.55 (m, 6H), 7.46 (q, J=8.7Hz, 1H), 7.31 (dd, J=17.3, 9.0Hz, 2H), 7.22 (d, J=8.6Hz, 2H), 7.15 (s, 1H), 7.03 ( dd, J=22.8, 7.1Hz, 2H), 6.37–6.27 (m, 1H), 6.20 (d, J=14.2Hz, 2H), 4.21–3.86 (m, 8H), 3.78–3.60 (m, 4H), 3.19 (t, J=12.5H z, 1H), 2.94–2.54 (m, 12H), 2.35 (s, 2H), 2.07–1.96 (m, 2H), 1.87 (t, J=12.4Hz, 6H), 1.79–1.63 (m, 9H), 1.28–1.13 (m, 12H); 13 C NMR (151MHz, DMSO-d6) δ172.07, 169.38, 167.08, 163.48, 158.96, 146.51, 145.3 5, 142.60, 141.36, 140.73, 135.65, 130.63, 126.60, 125.69, 122.49, 120.08, 118 .02,116.78,116.51,114.81,114.59,110.79,101.08,73.99,58.64,51.11,48.91,44.06,42.50,37.44,27.57,27.54,27.50,26.33,24.17,22.78,21.19,18.91.

[0055] Example 4

[0056] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0057] The difference from Example 2 is that BOC-NH-PEG4 acid in Example 2 is replaced by BOC-GLY-GLY-GLY-OH for the reaction, and S0456-Der-02 is replaced by S0456-Der-01 for the reaction.

[0058] The structural formula of the infrared fluorescent probe NY-cMet-04 is as follows:

[0059] The structure of NY-cMet-04 was characterized by mass spectrometry, H NMR spectrum, and C NMR spectrum. The results of the structure determination are as follows: [M-2H] / 2-=764.9; 1 H NMR (600MHz, DMSO-d6) δ8.70 (d, J=13.9Hz, 2H), 8.28 (t, J=5.7Hz, 1H), 8.19 (d, J=8.4Hz, 2H), 7.99 (s, 2H), 7.88 (t, J=5.2Hz, 1H), 7.76 (dd, J=11.7, 1.1Hz, 3H), 7.63 (ddd, J=13.9, 8.2, 3.0Hz, 4H), 7.48 (t, J=8.7Hz, 1H), 7.41 (d, J=8.4Hz, 2H), 7.16 (d, J=0.9Hz, 1H), 6.50 (d, J=14.1Hz, 2H), 6.30 (q, J=6.7Hz, 1H), 4 .49–4.26(m, 6H), 4.02(dd, J=16.5, 5.0Hz, 1H), 3.88(dd, J=16.3, 4.9Hz, 2H), 3.77–3.65 (m, 4H), 3.14 (t, J = 12.3Hz, 1H), 3.00 (t, J = 7.1Hz, 2H), 2.69 (dt, J = 13.6, 9.5Hz, 9H), 2.4 4(t, J=7.3Hz, 2H), 2.07–1.97(m, 6H), 1.86(d, J=6.6Hz, 3H), 1.79(s, 3H), 1.68(s, 12H); 13C NMR (151MHz, DMSO-d6) δ172.48, 170.70, 169.73, 169.30, 167.04, 158.98, 158.72, 156.59, 155.62, 146 .49, 145.36, 142.82, 141.58, 140.92, 135.61, 135.53, 134.37, 131.30, 129.29, 126.61, 125.56, 122.2 8, 121.66, 121.53, 120.27, 118.62, 118.52, 118.02, 116.75, 110.74, 102.47, 74.06, 58.61, 49.23, 48.32, 43.34, 43.25, 42.61, 42.40, 40.90, 35.93, 33.03, 32.83, 32.16, 27.85, 26.32, 23.71, 21.07, 18.90.

[0060] Example 5

[0061] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0062] The difference from Example 2 is that the BOC-NH-PEG4 acid in Example 2 is replaced by BOC-GLY-GLY-GLY-OH, and S0456-Der-03 is replaced by S0456-Der-01 for the reaction.

[0063] The structural formula of the infrared fluorescent probe NY-cMet-05 is as follows:

[0064] The structure of NY-cMet-05 was characterized by mass spectrometry, H NMR spectrum, and C NMR spectrum. The results of the structure determination are as follows: [M-2H] / 2-=779.2; 1H NMR (600MHz, DMSO-d6) δ8.68 (d, J=13.8Hz, 2H), 8.29 (t, J=5.3Hz, 1H), 8.19 (d, J=7.9Hz, 2H), 8.00 (s, 2H), 7.90 (d, J=5.2Hz, 1H), 7.78-7.70 (m, 3 H), 7.67-7.57 (m, 4H), 7.47 (t, J=8.6Hz, 1H), 7.37 (t, J=7.1Hz, 2H), 7.15 (d, J=9.8Hz, 1H), 6.40–6.26 (m, 3H), 4.43 (ddd, J=15.1, 11.3, 3.8Hz, 1H ), 4.18 (s, 4H), 4.04 (d, J = 13.7Hz, 1H), 3.87 (d, J = 12.4Hz, 2H), 3.76–3.64 (m, 4H), 3.15 (t, J = 12.5Hz, 1H), 3.00 (dd, J = 16.2, 9.1Hz, 2H), 2.75 (t, J=11.9Hz, 1H), 2.70–2.56 (m, 8H), 2.44 (t, J=7.2Hz, 2H), 2.02 (s, 2H), 1.86 (d, J=6.6Hz, 3H), 1.84–1.72 (m, 11H), 1.66 (dd, J=10.2, 6.6Hz, 12H); 13 C NMR (151MHz, DMSO-d6) δ172.39, 170.71, 169.73, 169.28, 167.07, 158.96, 158.71, 155.55, 146.4 9, 145.49, 145.21, 142.75, 141.52, 140.81, 135.65, 135.56, 134.06, 129.28, 126.65, 125.54, 12 2.20, 121.55, 120.23, 118.58, 118.02, 116.78, 110.86, 102.23, 74.02, 58.60, 51.15, 49.24, 44.16, 43.38, 42.63, 42.40, 40.97, 35.82, 32.83, 27.86, 27.82, 26.41, 26.21, 22.82, 20.99, 18.91.

[0065] Example 6

[0066] A method for preparing a near-infrared fluorescent probe comprises the following steps:

[0067] The difference from Example 2 is that the BOC-NH-PEG4 acid in Example 2 is replaced by BOC-GLY-GLY-GLY-OH, and S0456-Der-04 is replaced by S0456-Der-01 for the reaction.

[0068] The structural formula of the infrared fluorescent probe NY-cMet-06 is as follows:

[0069] The structure of NY-cMet-06 was characterized by mass spectrometry, H-NMR spectrum, and C-NMR spectrum. The results of structure determination are as follows: [MH]- = 1590.3; 1 H NMR (600MHz, DMSO-d6) δ8.21 (s, 1H), 8.15 (dd, J=14.4, 8.4Hz, 2H), 7.99 (s, 2H), 7. 87 (t, J=5.2Hz, 1H), 7.80 (d, J=13.9Hz, 2H), 7.75 (d, J=4.1Hz, 1H), 7.60 (dd, J=19.0 , 8.8Hz, 6H), 7.48 (td, J=8.7, 3.6Hz, 1H), 7.35 (d, J=8.3Hz, 2H), 7.23 (d, J=8.6Hz, 2H), 7.16 (d, J=1.1Hz, 1H), 7.04 (d, J=8.5Hz, 2H), 6.36 (d, J=14.1Hz, 2H), 6.30 (q, J =6.6Hz, 1H), 4.44(td, J=11.4, 5.7Hz, 1H), 4.38(s, 1H), 4.28(s, 4H), 4.06–3.85(m , 3H), 3.71 (d, J=5.7Hz, 2H), 3.64 (d, J=5.2Hz, 2H), 3.17 (dd, J=15.0, 9.3Hz, 1H), 2. 82–2.65(m, 7H), 2.60(t, J=6.7Hz, 4H), 2.36(dt, J=15.4, 4.7Hz, 2H), 2.08–1.93(m , 6H), 1.86 (d, J=6.6Hz, 6H), 1.67 (dd, J=14.4, 5.8Hz, 1H), 1.26 (d, J=10.0Hz, 12H); 13C NMR (151MHz, DMSO-d6) δ172.12, 169.90, 169.36, 167.06, 163.53, 158.97, 158.71, 158.55, 146.4 9, 145.31, 142.63, 141.57, 140.84, 135.84, 135.62, 135.51, 130.65, 129.30, 126.57, 125.55, 122 .80, 122.28, 121.67, 121.54, 120.10, 118.61, 118.03, 116.77, 114.87, 110.71, 101.36, 74.05, 58.68, 48.90, 48.26, 43.38, 43.12, 42.73, 42.49, 37.54, 30.39, 27.57, 24.29, 23.65, 21.30, 18.90.

[0070] Test Example 1 Spectral Test

[0071] The NY-cMet-01 to 06 series probes were prepared into approximately 1 nmol aqueous solutions. The absorption spectra of each probe in the range of 500-900 nm were first detected using an ultraviolet spectrophotometer (HITACHI, 3J1-0015). The results are shown in Figure 1. The maximum absorption of NY-cMet-01, NY-cMet-02, NY-cMet-03, and NY-cMet-05 is approximately 775 nm, and that of NY-cMet-04 and NY-cMet-06 is approximately 1 nmol. The maximum absorption is approximately 790 nm, and the fluorescence emission spectra of each probe in the range of 750-850 nm are detected on a microplate reader (Molecule desevices, D1524R). The results are shown in Figure 2. The maximum emission spectra of NY-cMet-01, NY-cMet-02, NY-cMet-03, and NY-cMet-05 are approximately 800 nm, and the maximum emission spectra of NY-cMet-04 and NY-cMet-06 are approximately 820 nm.

[0072] Test Example 2: In vivo imaging of subcutaneous HepG2 tumor-bearing mouse model (human liver cancer cells)

[0073] In the subcutaneous HepG2 tumor-bearing mouse model, NY-cMet-01-05 series probes (5 nmol / mouse, 100 μL of glucose injection) were administered via the tail vein, with indocyanine green (ICG, 1.0 mg / kg, 100 μL of water for injection) used as a control. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) at 0 h (before administration), 6 h, 12 h, 24 h, and 48 h after administration. The results are shown in Figure 3. As can be seen from the figure, in mice bearing subcutaneous HepG2 tumors, NY-cMet-02~05 have stronger tumor fluorescence signals compared to ICG, while NY-cMet-01 has a weaker fluorescence signal at the tumor site; NY-cMet-02 and NY-cMet-03 remain in the liver for up to 48 hours after administration; NY-cMet-04 and NY-cMet-05 not only exhibit good in vivo metabolic properties (renal excretion), but also have good tumor targeting capabilities. 24 hours after administration, only the tumor remains with fluorescence signals, and imaging lasts for more than 48 hours. NY-cMet-02~05 probes have potential for liver cancer tumor imaging and are worthy of further research and development with the goal of applying them to clinical fluorescence-guided tumor resection.

[0074] Test Example 3: In vivo imaging of a colorectal cancer (HCT116) tumor-bearing mouse model

[0075] In a colorectal cancer (HCT116) tumor-bearing mouse model, the probes NY-cMet-02 and NY-cMet-04 (5 nmol / mouse, in 100 μL of glucose injection) were administered via the tail vein. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) at 0 h (before administration), 6 h, 12 h, 24 h, and 48 h after administration. The results are shown in Figure 4. As shown in this figure, the probes NY-cMet-02 and NY-cMet-04 also exhibited good tumor targeting ability in colorectal cancer (HCT116) tumor-bearing mice, demonstrating potential clinical application prospects and warranting further research and development for application in clinical surgery.

[0076] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe, characterized in that: It is a compound represented by formula (I): In formula (I), X is a linker molecule selected from any one of: n-terminal, PEG, and polyglycine chains; Y is a dye molecule having fluorescence excitation and emission spectra in the near-infrared range; the structural formula of Y is: In the structural formula, R1 and R2 are selected from (CH2)nSO3H, n=3 or 4; Z is selected from 3-mercaptopropionic acid or 4-hydroxyphenylpropionic acid.

2. A method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: The following steps are involved: S1: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, and a polar solvent are mixed evenly, crizotinib and a linker molecule are added, and the mixture is reacted at room temperature for 1-2 hours to obtain product a, which is then added dropwise to water to form a suspension; S2: Ethyl acetate was added to the suspension for extraction, the extract was dried and concentrated, and trifluoroacetic acid was added to react at room temperature for 30-50 minutes. After column chromatography, intermediate b was obtained; S3: adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, a dye molecule having a fluorescence excitation and emission spectrum in the near-infrared range, and a polar solvent to the intermediate b, and stirring at room temperature for 1 to 2 hours to obtain a mixture c; S4: Purify the mixture c to obtain a near-infrared fluorescent probe.

3. The method for preparing a near-infrared fluorescent probe according to claim 2, wherein: The molar ratio of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, base, crizotinib, linker molecule, and polar solvent in S1 is (1-2):(1-3):1:1.2:70; the volume ratio of ethyl acetate and trifluoroacetic acid in S2 is 1:(1-2).

4. The method for preparing a near-infrared fluorescent probe according to claim 2, wherein: The molar ratio of the intermediate b, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, base, dye molecule with fluorescence excitation and emission spectrum in the near-infrared range, and polar solvent in S3 is (1-2):2:(1-3):1.1:(90-95).

5. The method for preparing a near-infrared fluorescent probe according to claim 2, wherein: The base is any one or more of triethylamine and diisopropylethylamine; the polar solvent is any one or more of dimethylformamide, dimethyl sulfoxide and N-methylpyrrolidone.

6. Use of the near-infrared fluorescent probe according to claim 1 in the preparation of a fluorescent contrast agent.

7. Use of the near-infrared fluorescent probe according to claim 1 in the preparation of tumor diagnostic drugs.

8. The use according to claim 7, characterized in that The tumor is liver cancer or colorectal cancer.

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