Near-infrared fluorescent probe for active targeting detection of epidermal growth factor receptor, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of specific molecular targeted diagnostic reagents. Provided are a near-infrared fluorescent probe for active targeting detection of an epidermal growth factor receptor, and a preparation method therefor and the use thereof. The preparation method comprises: mixing N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-yloxy)quinazolin-4-amine and X in the presence of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base and a polar solvent; adding the product dropwise to water; extracting the suspension with an organic solvent, followed by drying and concentration; removing the protecting group from the concentrate, followed by column chromatography to obtain an intermediate compound; and adding the intermediate compound to 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, Y and a polar solvent, followed by purification to obtain the near-infrared fluorescent probe. The prepared near-infrared fluorescent probe can achieve the effect of in-vivo diagnosis.

Description

A near-infrared fluorescent probe for active targeting of epidermal growth factor receptor, its preparation method and application Technical Field

[0001] This invention relates to the field of specific molecular targeted diagnostic reagent technology, specifically a near-infrared fluorescent probe for active targeting of epidermal growth factor receptor detection, its preparation method, and its application. Background Technology

[0002] With rapid economic and social development, people's living environment and habits have undergone tremendous changes. The faster pace of life, increased work pressure, and altered high-oil and high-fat diets have directly impacted people's health. The causes of cancer are closely related to lifestyle and living environment, and cancer is currently incurable, seriously endangering human life. In every country in the world, cancer is a leading cause of death and a significant obstacle to improving life expectancy.

[0003] Currently, the main treatments for cancer patients are chemotherapy and surgical resection. Chemotherapy often causes severe adverse reactions in cancer patients, such as vomiting, diarrhea, and pain, which can cause great suffering. Compared to chemotherapy, for most solid tumors, surgical treatment is more effective in removing lesions and improving patient survival. Traditional surgery relies entirely on the surgeon's experience and tactile sense, lacking a basis for determining tumor boundaries, thus often resulting in the removal of as much tissue as possible to avoid tumor recurrence. With the development of molecular imaging, the emergence of SPECT / CT, PET / CT, MRI, ultrasound imaging (USI), and fluorescence imaging has not only compensated for the shortcomings of traditional surgery in tumor localization but also greatly improved the success rate of surgery and extended patient survival. In particular, the real-time dynamic imaging of near-infrared fluorescence imaging, when applied to clinical surgery, provides surgeons with a clearer field of vision, accurately distinguishes the boundaries between tumors and normal tissues, and minimizes the removal of normal tissue, thus minimizing the impact of surgery. Commercially available fluorescence surgical navigation imaging systems are already in clinical use and have great potential for future applications in surgery, playing a significant role in the accurate diagnosis and resection of tumors.

[0004] Near-infrared fluorescent probes (650–1000 nm) for active targeting of epidermal growth factor receptors provide high-resolution images of tissues and organs, exhibit low biotoxicity and low autofluorescence, which helps minimize background interference. Indocyanine green (ICG), the first-generation passively targeted fluorescent contrast agent approved by the FDA, is widely used in fluorescence-guided tumor resection for tumors such as liver cancer, sentinel lymph node cancer, and esophageal cancer. It effectively improves tumor resection success rates, reduces tumor recurrence rates, and prolongs effective survival. However, its high false positive rate (66%) and insufficient tumor targeting ability severely limit its clinical application. In 2021, the first active folate receptor-targeting probe, OTL38, was approved. It specifically targets lung and ovarian cancers with high folate receptor expression, with a false positive rate of 24.8%, significantly reducing the false positive rate and improving patients' quality of life. The emergence of tumor-specific targeting probes can greatly improve the success rate of tumor surgery and reduce postoperative recurrence rates, which has significant clinical implications.

[0005] Human epidermal growth factor receptors (EGFR, HER2, HER3, HER4) are overexpressed to varying degrees in various tumors, including liver cancer, ovarian cancer, and lung cancer. Designing a specific near-infrared fluorescent contrast agent targeting EGFR has significant clinical value. Quinazoline small molecule inhibitors are also important in clinical oncology treatment. Therefore, designing and synthesizing specific tumor-targeting fluorescent probes using quinazoline small molecule compounds as targeting ligands also has significant development value for application in fluorescence-guided tumor resection in clinical oncology surgery.

[0006] In conclusion, with the development of medical imaging technology, near-infrared fluorescence imaging technology has broad application prospects in tumor surgery. The development of specific tumor-targeting fluorescent probes will further improve the success rate of tumor surgery, reduce postoperative recurrence rate, and prolong patient survival. Specific tumor-targeting fluorescent probes designed and synthesized using quinazoline small molecule compounds as targeting ligands have significant clinical application value.

[0007] Summary of the Invention

[0008] The purpose of this invention is to provide a near-infrared fluorescent probe for active targeting of epidermal growth factor receptor (EGFR) detection, its preparation method, and its application. The prepared near-infrared fluorescent probe for active targeting of EGFR detection can be rapidly cleared in normal tissues but remains for a long time in tumor sites, thereby achieving the function of in vivo diagnosis and having certain clinical application prospects, which can be applied to clinical intraoperative navigation.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] A near-infrared fluorescent probe for active targeting of epidermal growth factor receptor detection.

[0011] Compounds of formula (I) or pharmaceutically acceptable salts thereof:

[0012] in:

[0013] X is a linker molecule, selected from non-chain, polyethylene glycol, and polyglycine chains, and

[0014] Y is a dye molecule that has fluorescence excitation and emission spectra in the near-infrared range;

[0015] Compound Y has the structure of formula (II):

[0016] in:

[0017] R1 and R2 are selected from (CH2) n SO3H, n = 3 or 4;

[0018] Z is selected from -S-CH2CH2CO- or -O-Ph-CH2CH2CO-.

[0019] The method for preparing the near-infrared fluorescent probe for active targeting of epidermal growth factor receptor as described above includes the following steps:

[0020] (1) N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidine-4-oxo)quinazolin-4-amine and X were mixed in the presence of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, a base and a polar solvent;

[0021] (2) Drop the product obtained in step (1) into water to form a suspension;

[0022] (3) Extract the suspension obtained in step (2) with an organic solvent, dry it and concentrate it, and recover the concentrate;

[0023] (4) The concentrate obtained in step (3) is deprotected and then subjected to column chromatography to obtain the intermediate compound;

[0024] (5) The intermediate compound obtained in step (4) is added to 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, base, Y and polar solvent, and stirred at room temperature to obtain the resulting mixture;

[0025] (6) After purifying the mixture obtained in step (5), a near-infrared fluorescent probe for active targeting of epidermal growth factor receptor is obtained.

[0026] Preferably, in step (1), the molar ratio of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, base, N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-oxo)quinazolin-4-amine, X, and polar solvent is 3:5:1:1.2:10.

[0027] Preferably, the molar ratio of the intermediate compound, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, base, Y and polar solvent in step (5) is 1:3:5:1.2:10.

[0028] Preferably, the polar solvent is one or more of dimethylformamide, dimethyl sulfoxide, and N-methylpyrrolidone.

[0029] Preferably, the base is one or both of triethylamine and diisopropylethylamine.

[0030] The application of the near-infrared fluorescent probe for active targeting of epidermal growth factor receptor detection, as described above, in the preparation of fluorescent contrast agents.

[0031] Compared with the prior art, the beneficial effects of the present invention are:

[0032] The near-infrared fluorescent probe for active targeting of epidermal growth factor receptor (EGFR) provided by this invention can be rapidly prepared through a simple chemical synthesis method. Specifically, the reactants N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-oxo)quinazoline-4-amine and X undergo a nucleophilic substitution reaction in the presence of the organic synthesis reagent 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and a base, and under the dissolution and mediation of a polar solvent. The 2-(azabenzotriazole) structure acts as an activator or catalyst to promote the reaction process. Testing has shown that the probe actively targeting EGFR possesses advantages such as active tumor targeting capability, good water solubility, long tumor retention time, and no accumulation in normal tissue, demonstrating potential applications in clinical surgical procedures such as fluorescence-guided tumor resection. For example, in the diagnosis of colorectal cancer: In the diagnosis of colorectal cancer, doctors can use the near-infrared fluorescent probe provided by this invention for active targeting of epidermal growth factor receptor to detect the expression level of epidermal growth factor receptor in colorectal cancer tissue. Through the detection results, doctors can determine the nature and malignancy of colorectal cancer, thereby providing a basis for subsequent treatment. Attached Figure Description

[0033] Figure 1 is a synthetic route diagram for the product prepared in Example 1;

[0034] Figure 2 is a synthetic route diagram of the product prepared in Example 2;

[0035] Figure 3 is a synthetic route diagram of the product prepared in Example 3;

[0036] Figure 4 is a synthetic route diagram of the product prepared in Example 4;

[0037] Figure 5 shows the absorption spectra of the prepared NY-EGF-02, NY-EGF-03, NY-EGF-05 and NY-EGF-06;

[0038] Figure 6 shows the fluorescence spectra of the prepared NY-EGF-02, NY-EGF-03, NY-EGF-05 and NY-EGF-06;

[0039] Figure 7 shows in vivo imaging of the prepared NY-EGF-02, NY-EGF-03, NY-EGF-05 and NY-EGF-06 in HepG2 liver cancer-bearing mice.

[0040] Figure 8 shows the in vivo imaging of the prepared NY-EGF-05 in HCT116 colorectal cancer-bearing mice. Detailed Implementation

[0041] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0042] The materials used and their sources in the embodiments are shown in Table 1 below:

[0043] Table 1 Materials and Sources

[0044] Example 1

[0045] Synthesis of NY-EGF-02

[0046] As shown in Figure 1, N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-yloxy)quinazolin-4-amine (10 mg, 1.0 eq), 5,8,11,14-tetraoxa-2-azaheptadecanoic acid-1-tert-butyl ester (10.9 mg, 1.2 eq), HATU (18.90 mg, 3.0 eq), N,N-diisopropylethylamine (12.99 μL, 5.0 eq), and DMSO (100 μL) were reacted at room temperature for 1 h. The reaction was monitored by TLC (DCM:MeOH = 15:1). After the reaction was complete, the reaction solution was added dropwise to water (10 mL), and then extracted with ethyl acetate (7 mL × 3). The organic phases were combined, concentrated under reduced pressure, and then added with triethyl acetate. The tert-butyloxycarbonyl group of fluoroacetic acid was removed (Boc protecting group). The reaction was monitored by TLC. After the reaction was complete, the mixture was concentrated and purified under reduced pressure to obtain intermediate 3 (12 mg). Then, HATU (21 mg, 3.0 eq), SO456-Der-01 (22 mg, 1.2 eq), and N,N-diisopropylethylamine (12 mg, 5.0 eq) were added. After reacting at 30 °C for 1 h, HPLC monitoring showed that the reaction was complete. After purification and separation by preparative liquid chromatography, the target fraction was lyophilized to obtain the target compound NY-EGF-02. The green solid structure was confirmed by mass spectrometry, 1H NMR, and 1C NMR. The [M-2H] / 2- = 823.8, and the 1H NMR (600 MHz, DMSO) δ 11.41. (s,1H),8.87(s,1H),8.18-8.16(m,1H),7.87(t,J=5.5Hz,1H),7.79(d,J=13 .9Hz,2H),7.65(t,J=7.5Hz,1H),7.63(s,2H),7.58(t,J=7.4Hz,3H),7.44(s, 1H),7.36(t,J=8.1Hz,1H),7.31(d,J=8.4Hz,2H),7.22(d,J=8.6Hz,2H),7.04 (d,J=8.5Hz,2H),6.21(d,J=14.2Hz,2H),4.95-4.93(m,1H),4.77(d,J=8.5Hz ,12H),4.11(s,4H),3.98(s,3H),3.88(dd,J=16.5,10.4Hz,1H),3.78-3.71( m,1H),3.62(t,J=6.6Hz,2H),3.47-3.45(m,2H),3.44-3.41(m,2H),3.33(t,J =5.8Hz,2H),3.13(q,J=5.6Hz,2H),2.76-2.70(m,2H),2.67(s,4H),2.63-2.5 8(m,4H),2.27(t,J=7.9Hz,2H),2.11-1.98(m,2H),1.85(s,2H),1.74(d,J=2.7Hz, 10H), 1.25s, 12H. 13 C NMR (151MHz, DMSO) δ172.02,171.72,169.23,163.50,159.57,158.46,158.15,154.01,152.35,149.49,148.51,14 5.31,142.56,141.31,140.74,135.76,130.55,130.15,128.17,126.61,126.32,125.89,122.52,120.95,120.84, 120.06,114.86,110.83,107.21,106.01,101.08,100.68,74.19,70.26,70.20,70.16,69.99,69.51,67.39,57.25,51.08,48.94,44.03,42.69,38.97,38.64,37.68,33.25,31.06,30.69,30.33,27.57,26.28,24.17,22.74,21.17.

[0047] Example 2

[0048] Synthesis of NY-EGF-03

[0049] As shown in Figure 2, the synthesis method is the same as that in NY-EGF-02, except that 5,8,11,14-tetraoxa-2-azaheptadecanoic acid-1-tert-butyl ester is replaced with (tert-butyloxycarbonyl)glycylglycylglycine.

[0050] The structure was confirmed by mass spectrometry, hydrogen NMR, and carbon NMR, with [M-2H] / 2- = 785.9. 11H NMR (600 MHz, DMSO) δ 11.44 (s, 1H), 8.88 (s, 1H), 8.21 (s, 2H), 8.12 (t, J = 5.5 Hz, 1H), 7.90 (s, 1H), 7.79 (d, J = 13.9 Hz, 2H), 7.67 (t, J = 7.4 Hz, 1H), 7.63 (s, 2H), 7.60 - 7.53 (m, 3H), 7.39 (dd, J = 16.1, 8.0 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.25 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 8.2 Hz, 2H), 6.21 (d, J = 14.1 Hz, 2H), 4.96 (s, 1H), 4.11 (s, 4H), 4.01 (d, J = 3.2 Hz, 1H), 3.99 (s, 3H), 3.94 (d, J = 4.1 Hz, 1H), 3.81 (s, 2H), 3.71 (m, 6H), 3.42 - 3.34 (m, 2H), 2.78 - 2.73 (m, 2H), 2.67 (s, 4H), 2.55 (s, 4H), 2.37 (t, J = 7.6 Hz, 2H), 2.04 (d, J = 46.8 Hz, 2H), 1.86 (s, 2H), 1.73 (s, 10H), 1.26 (s, 12H). 13 13C NMR (151 MHz, DMSO) δ 172.47, 172.03, 169.87, 169.86, 169.33, 169.27, 167.13, 167.09, 167.08, 163.47, 163.41, 158.49, 158.14, 148.46, 145.41, 142.70, 142.66, 142.55, 142.48, 142.46, 142.31, 142.25, 141.35, 140.75, 135.87, 132.63, 130.61, 130.25, 130.16, 122.49, 122.45, 120.18, 120.07, 119.58, 114.89, 114.85, 110.83, 110.77, 107.24, 107.19, 107.18, 106.15, 106.09, 106.08, 101.05, 101.00, 74.01, 73.97, 73.90, 72.35, 57.24, 51.26, 51.18, 48.93, 44.10, 42.79, 42.77, 42.45, 37.60, 37.55, 37.49, 30.19, 27.58, 26.36, 24.15, 24.03, 22.87.

[0051] Example 3

[0052] Synthesis of NY-EGF-05

[0053] As shown in Figure 3, the synthesis method is the same as that of NY-EGF-02, except that SO456-Der-02 was used instead of SO456-Der-01. The structure was confirmed by mass spectrometry, 1H NMR, and 1C NMR, with [M-2H] / 2- = 779.7. 1 H NMR (600MHz, DMSO) δ11.41 (s, 1H), 8.88 (s, 1H), 8.67 (d, J = 14.0Hz, 2H), 8.19 (s,1H),8.00(t,J=5.5Hz,1H),7.76(s,2H),7.62(dd,J=12.1,8.8Hz,3H),7.5 7(t,J=7.4Hz,1H),7.41(s,1H),7.38(d,J=8.4Hz,2H),7.35-7.31(m,1H),6.4 8(d,J=14.2Hz,2H),4.95(d,J=11.1Hz,12H),4.32(s,4H),4.00(s,3H),3.94- 3.89(m,1H),3.78-3.71(m,1H),3.61(dd,J=12.0,5.4Hz,2H),3.50(d,J=13. 3Hz,2H),3.47(s,4H),3.36(t,J=5.7Hz,2H),3.15(q,J=5.6Hz,2H),2.97(t,J =7.2Hz,2H),2.64(dd,J=14.2,7.3Hz,4H),2.59(dd,J=10.4,6.1Hz,4H),2.35 (s,2H),2.07(d,J=8.0Hz,1H),2.04-1.96(m,4H),1.69(s,4H),1.67(s,12H). 13 C NMR(151MHz,DMSO)δ172.01,169.59,163.53,159.45,157.95,152.31,148.4 7,144.79,142.75,141.55,140.80,136.52,131.50,129.87,127.99,126.65, 122.51,120.69,119.92,114.59,111.08,107.30,105.97,101.13,100.37,73.86,57.12,51.10,48.84,44.03,34.74,27.56,27.53,26.31,24.21,22.75.

[0054] Example 4

[0055] Synthesis of NY-EGF-06

[0056] As shown in Figure 4, the synthesis method is the same as that of NY-EGF-03, except that SO456-Der-02 is used instead of SO456-Der-01 in Example 2.

[0057] The structure was confirmed by mass spectrometry, proton NMR, and carbon NMR. [M-2H] / 2- = 741.4. 1 H NMR (600MHz, DMSO) δ11.43(s,1H),8.88(s,1H),8.71(d,J=13.8Hz,2H),8.33(t,J=5.6Hz,1H),8.18(s,2H),7.87(t,J=5.1Hz,1H), 7.78(s,2H),7.64(ddd,J=11.7,9.5,6.9Hz,4H),7.44-7.31(m,4H),6.50(d,J=14.1Hz,2H),4.90(s,1H),4.32(s,6H)4.01(s,3H),3 .97(d,J=4.6Hz,1H),3.91(d,J=4.4Hz,1H),3.83-3.75(m,1H),3.69(dd,J=13.7,6.5Hz,5H),3.36-3.28(m,1H),3.23(t,J=8.6Hz,1 H),3.01(t,J=7.2Hz,2H),2.64(dd,J=16.6,9.9Hz,8H),2.45(t,J=7.3Hz,2H),2.01(t,J=18z.2Hz,6H),1.79(s,2H),1.69(s,12H). 13 C NMR (151MHz, DMSO) δ172.45,170.80,169.69,169.29,167.00,159.58,158.13,155.47,152.37 ,149.52,148.46,145.28,142.81,140.88,135.75,134.42,130.15,128.18,126.62,126.33,1 25.90,120.95,120.84,120.24,110.75,107.21,106.09,102.49,100.57,73.99,57.21,49.22,48.33,43.25,42.74,42.42,41.71,36.11,32.96,30.78,30.19,27.84,26.33,23.74,21.06.

[0058] Example 5

[0059] Four compounds, NY-EGF-02, NY-EGF-03, NY-EGF-05, and NY-EGF-06, were prepared into approximately 1 nmol aqueous solutions. The absorption spectra of each probe in the range of 500-900 nm were measured using a UV spectrophotometer (HITACHI, 3J1-0015). The maximum absorption of compounds NY-EGF-02 and NY-EGF-03 was approximately 775 nm, and that of compounds NY-EGF-05 and NY-EGF-06 was approximately 790 nm. Subsequently, the fluorescence emission spectra of each probe in the range of 750-850 nm were measured using a microplate reader (Moleculedevices, D1524R). The maximum emission spectra of compounds NY-EGF-02 and NY-EGF-03 were approximately 800 nm, and that of compounds NY-EGF-05 and NY-EGF-06 was approximately 820 nm, as shown in Figures 5 and 6.

[0060] Example 6

[0061] In vivo imaging of a subcutaneous tumor HepG2 tumor-bearing mouse model (human liver cancer cells)

[0062] In the subcutaneous tumor HepG2 tumor-bearing mouse model, NY-EGF series probes (probes prepared in Examples 1-4, 5 nmol / vial, 100 μL glucose injection) were administered via tail vein, with indocyanine green (ICG, 1.0 mg / kg, 100 μL water for injection) as a control. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) after administration at 0 h (before administration), 6 h, 12 h, 24 h, and 48 h.

[0063] The results showed (as shown in Figure 7): (1) In HepG2 subcutaneous tumor-bearing mice, compared with ICG, the four probes NY-EGF-02 / NY-EGF-03 / NY-EGF-05 / NY-EGF-06 had stronger tumor fluorescence signals; (2) The two probes NY-EGF-02 and NY-EGF-03 had certain liver uptake and were basically cleared 24 hours after administration, with a tumor imaging window of more than 48 hours; (3) The two probes NY-EGF-05 and NY-EGF-06 had better in vivo metabolic characteristics, with basically no liver accumulation, and showed obvious tumor fluorescence signals 12 hours after administration. The tumor fluorescence signal of the NY-EGF-05 probe was stronger and lasted longer; (4) The NY-EGF-02 series of probes have potential liver cancer tumor imaging capabilities and can be further studied and developed for clinical fluorescence-guided tumor surgical resection.

[0064] Based on this, as shown in Figure 8, the present invention validated the targeting of the NY-EGF-05 probe in colorectal cancer (HCT116) tumor-bearing mice. It was found that the NY-EGF-05 probe also has good tumor targeting ability in colorectal cancer (HCT116) tumor-bearing mice, and has potential clinical application prospects. Further research and development are needed for its application in clinical surgery.

[0065] The preferred embodiments of this patent have been described in detail above. However, this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this patent.

Claims

1. A near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection, characterized in that: wherein: X is a linking molecule selected from the group consisting of nothing, polyethylene glycol, polyglycine chain, and Y is a dye molecule having fluorescent excitation and emission spectra in the near-infrared range; wherein: Z is selected from the group consisting of -S-CH2CH2CO- or -O-Ph-CH2CH2CO-. A compound as shown in formula (I) or a pharmaceutically acceptable salt thereof:

2. A method for preparing the near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection according to claim 1, characterized in that: comprising the following steps: (1) mixing N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-yloxy)quinazolin-4-amine and X in the presence of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base and a polar solvent; (2) dropping the product obtained in step (1) into water to form a suspension; (3) extracting the suspension obtained in step (2) with an organic solvent, drying and concentrating to recover the concentrate; (4) deprotecting the concentrate obtained in step (3) and then purifying by column chromatography to obtain an intermediate compound; (5) adding the intermediate compound obtained in step (4) to 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, Y and a polar solvent, and stirring at room temperature to obtain a resulting mixture; (6) purifying the mixture obtained in step (5) to obtain the near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection. The molar ratio of 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, N-(3-chloro-2-fluorophenyl)-7-methoxy-6-(piperidin-4-yloxy)quinazolin-4-amine, X, a polar solvent in step (1) is 3:5:1:1.2:

10. The molar ratio of the intermediate compound, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, a base, Y and a polar solvent in step (5) is 1:3:5:1.2:

10. wherein compound Y has the structure of formula (II):

5. The method for preparing the near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection according to claim 2, characterized in that: wherein the polar solvent is one or more of dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone. R1, R2are selected from (CH2) n SO3H, n = 3 or 4; 6. The method for preparing the near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection according to claim 2, characterized in that: wherein the base is one or both of triethylamine and diisopropylethylamine.

7. Use of the near-infrared fluorescent probe for active targeting epidermal growth factor receptor detection according to claim 1 in the preparation of a fluorescent contrast agent. ​ ​ ​ ​ ​ ​ ​ 3. The method for preparing near-infrared fluorescent probes for active targeting of epidermal growth factor receptor detection according to claim 2, characterized in that: ​ 4. The method for preparing near-infrared fluorescent probes for active targeting of epidermal growth factor receptor detection according to claim 2, characterized in that: ​ ​ ​ ​ ​ ​

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