Near-infrared fluorescent probe for actively targeting vascular endothelial growth factor receptor testing, preparation method therefor and use thereof

Abstract

The present disclosure relates to the field of organic fluorescent probe molecules, and provides a near-infrared fluorescent probe for actively targeting vascular endothelial growth factor receptor testing, a preparation method therefor and a use thereof. The probe provided by the present disclosure can actively target a vascular endothelial growth factor receptor, can be rapidly cleared from normal tissues while remaining at tumor sites for an extended period, thereby achieving the effect of preparing in vivo diagnostic agents; thus, the probe exhibits promising clinical application prospects and can be developed for use in clinical fluorescence‑guided intraoperative navigation.

Description

Near-infrared fluorescent probes for active targeting of vascular epidermal growth factor receptor, their preparation methods and applications

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 2024117572251, filed on December 3, 2024, entitled “Near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor and its preparation method and application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to the field of organic fluorescent probe molecules, and in particular to a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, its preparation method, and its application. Background Technology

[0004] Cancer is currently the leading cause of death worldwide and is projected to be the biggest obstacle to life expectancy growth in the 21st century. Although modern medicine offers a plethora of cancer treatments, such as immunotherapy and gene therapy, traditional treatments remain the most widely used in clinical practice: surgical resection, chemotherapy, and radiation therapy. For solid tumors, surgical resection remains the preferred treatment. Precision medicine, aiming to minimize medical harm and maximize disease control through improved diagnostic and treatment protocols, has become a key research focus in the medical field.

[0005] To achieve precise tumor identification and surgical navigation, technologies such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound (US) have been used for imaging and diagnosis of cancerous tissues. However, inherent limitations such as low spatial resolution or sensitivity affect their ability to accurately identify lesion boundaries. Optical molecular imaging surgical navigation technology, characterized by real-time, non-invasive, and high resolution, provides a novel imaging aid for precise tumor surgery. Near-infrared fluorescence imaging diagnosis has advantages in tumor diagnosis (early, middle, and late stages), intraoperative navigation, prognosis, and recurrence monitoring. This diagnostic method mainly uses near-infrared fluorescent probes (650–1000 nm) for specific detection, providing high-resolution images of tissues and organs. It has the advantages of low biotoxicity and low autofluorescence, which helps to minimize background interference.

[0006] Indocyanine green (ICG) is an FDA-approved near-infrared fluorescent dye. After intravenous injection, it readily binds to plasma proteins, is metabolized by the liver into bile, and can remain in tumor tissue via the EPR effect. However, ICG has poor specificity, making it difficult to distinguish between tumor tissue and peritumoral inflammatory tissue, benign nodules, etc., easily leading to false positive results. Near-infrared fluorescent probes with specific targeting and renal excretion can effectively avoid false positive results, significantly improve the tumor-to-background ratio, enabling surgeons to more accurately identify tumor boundaries during surgery, completely remove residual tumor lesions, maximize patient survival, and improve patient quality of life.

[0007] Vascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and angiogenesis. Under normal physiological conditions, the vascular system remains quiescent after an organism reaches maturity, with transient angiogenesis only occurring when the body is injured. Numerous studies have shown that angiogenesis is a characteristic feature of malignant tumor development, as the abnormal structure and high levels of tumor blood vessel formation promote tumor proliferation, invasion, and metastasis. VEGF and its tyrosine kinase receptors (VEGFRs) can induce a broad-spectrum signal transduction cascade during tumor angiogenesis, acting as key mediators of tumor angiogenesis. Therefore, focusing on the VEGF / VEGFR signaling pathway as a target in studying tumor development has significant theoretical research value and application prospects.

[0008] Vandetanib is a small-molecule inhibitor targeting VEGFR-1 and VEGFR-2, approved by the FDA on April 6, 2011, and can also selectively inhibit other tyrosine kinases. Clinically, it is used to treat unresectable locally advanced or metastatic medullary thyroid carcinoma (MTC) and non-small cell lung cancer (NSCLC). Compared with traditional chemotherapy drugs, vandetanib has significantly reduced non-specific toxicity and hematologic toxicity, and its clinical application has been expanding in recent years. Its use in treating locally advanced or metastatic pancreatic cancer, advanced or metastatic breast cancer, and metastatic colorectal cancer is currently in Phase I or II clinical trials. Vandetanib's high specificity against various tumors and low toxicity make it valuable for developing tumor-targeting fluorescent probes, thereby further expanding its application value for surgical navigation and tumor resection.

[0009] In view of this, this disclosure is hereby made. Summary of the Invention

[0010] The purpose of this disclosure is to provide a near-infrared fluorescent probe for active targeting of vascular endothelial growth factor receptor (VEGF) and its preparation method and application. The near-infrared fluorescent probe described in this disclosure can actively target VEGF receptors, is rapidly cleared in normal tissues, and remains for a long time in tumor sites, thereby achieving in vivo diagnostic capabilities. It has good clinical application prospects and can be developed for use in clinical fluorescence-guided intraoperative navigation.

[0011] In order to achieve the above-mentioned objectives of this disclosure, the following technical solution is adopted:

[0012] In a first aspect, this disclosure provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, the near-infrared fluorescent probe comprising a compound as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0013] Wherein, the L group is a linking group, and the L group is selected from polyethylene glycol chain and / or polyglycine chain, and m is 0 or 1; the Y group is a dye molecule group with fluorescence excitation and emission spectra in the near infrared range.

[0014] In one embodiment, the Y group has a structure as shown in Formula II:

[0015] The R1 group and the R2 group are each independently selected from... n is 3 or 4;

[0016] Wherein, the Z group is selected from Furthermore, the S or O atom in the Z group is connected to the cyclohexene ring on one side of the Y group, and the carbonyl side of the Z group is connected to the L group or directly connected to the piperidine ring of the compound shown in Formula I.

[0017] In one embodiment, the L group has a structure as shown in formula L1 or L2:

[0018] In this context, p and q are each independently selected from integers between 1 and 4; and the carbonyl side of the L group is connected to the piperidine ring of the compound shown in Formula I, while the amino side is connected to the Y group.

[0019] Secondly, this disclosure provides a method for preparing a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor as described in the first aspect, the preparation method comprising method one and method two:

[0020] Method 1: When m is 1, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection includes the following steps:

[0021] (1) In the presence of a condensing agent and a base, N-demethylvandetanil and a compound having an L group and an amino protecting group undergo a condensation reaction to give intermediate A;

[0022] (2) Intermediate A undergoes a deprotection reaction to obtain intermediate B;

[0023] (3) In the presence of a condensing agent and a base, intermediate B and a compound with a Y group undergo a condensation reaction to obtain the near-infrared fluorescent probe of Formula I for active targeting of vascular epidermal growth factor receptor detection.

[0024] Method 2: When m is 0, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor includes the following steps:

[0025] In the presence of a condensing agent and a base, N-demethylvandetanil and a compound with a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe of formula I for active targeting of vascular epidermal growth factor receptor detection.

[0026] In one embodiment, in step (1) of method one, the compound having the L group has a structure as shown in formula L10 or L20:

[0027] Where X represents the protecting group of an amino group, and p and q are each independently selected from integers between 1 and 4.

[0028] In one embodiment, in step (1) of method one, the condensing agent is HATU.

[0029] In one embodiment, in step (1) of method one, the base is selected from triethylamine and / or diisopropylethylamine.

[0030] In one embodiment, in step (1) of method one, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

[0031] In one embodiment, in step (1) of method one, the molar ratio of N-demethylvandetanil, the compound having an L group and an amino protecting group, the condensing agent, the base and the polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20).

[0032] In one embodiment, in step (1) of method one, the temperature of the condensation reaction is 20-30°C, and the time of the condensation reaction is 0.5-2h.

[0033] In one embodiment, step (1) of method one further includes the following post-processing step after the condensation reaction:

[0034] The reaction solution obtained after the condensation reaction was added dropwise to water to obtain a suspension; the suspension was then extracted with an organic solvent, dried and concentrated to obtain intermediate A.

[0035] In one embodiment, in step (2) of method one, the deprotection reaction is carried out in the presence of an acid.

[0036] In one embodiment, in step (2) of method one, the acid is trifluoroacetic acid.

[0037] In one embodiment, in step (2) of method one, the molar ratio of intermediate A to acid is 1:(1-10).

[0038] In one embodiment, in step (2) of method one, the temperature of the deprotection reaction is 20-30°C and the time of the deprotection reaction is 20-40 min.

[0039] In one embodiment, in step (3) of method one, the condensing agent is HATU.

[0040] In one embodiment, in step (3) of method one, the base is selected from triethylamine and / or diisopropylethylamine.

[0041] In one embodiment, in step (3) of method one, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

[0042] In one embodiment, in step (3) of method one, the molar ratio of intermediate B, compound having a Y group, condensing agent, base and polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20).

[0043] In one embodiment, in step (3) of method one, the temperature of the condensation reaction is 20-30°C, and the time of the condensation reaction is 0.5-2h.

[0044] In one embodiment, in method two, the condensing agent is HATU.

[0045] In one embodiment, in method two, the base is selected from triethylamine and / or diisopropylethylamine.

[0046] In one embodiment, in method two, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

[0047] In one embodiment, in method two, the molar ratio of N-demethylvandetanil, the compound having a Y group, the condensing agent, the base, and the polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20).

[0048] In one embodiment, in method two, the temperature of the condensation reaction is 20–30°C, and the time of the condensation reaction is 0.5–2 hours.

[0049] Thirdly, this disclosure provides the application of a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection as described in the first aspect in the preparation of fluorescent contrast agents.

[0050] In one embodiment, the fluorescent contrast agent includes a fluorescent contrast agent for tumor diagnosis.

[0051] In one embodiment, the tumor is selected from any one of liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small bowel cancer, and anal cancer.

[0052] Fourthly, this disclosure provides a fluorescent contrast agent comprising the above-described near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection or a near-infrared fluorescent probe prepared by the above-described preparation method.

[0053] Fifthly, this disclosure provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, or a near-infrared fluorescent probe prepared by the above-described preparation method, for use in fluorescence imaging or tumor diagnosis.

[0054] In one embodiment, the tumor is selected from any one of liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small bowel cancer, and anal cancer.

[0055] Compared with the prior art, this disclosure has the following beneficial effects:

[0056] (1) This disclosure connects N-demethylvandetanib and dye molecules through a linker molecule. Vandetanib is a small molecule multi-target tyrosine kinase inhibitor (TKI) that can act on tumor cells VEGFR-1 and VEGFR-2 simultaneously. It can specifically target tumor tissue. The drug inhibits the VEGFR signaling pathway and blocks angiogenesis, thereby slowing down tumor growth.

[0057] (2) This invention connects N-demethylvandetanil and dye molecules through a linker molecule. Under the action of a condensing agent, the carboxyl and amino groups in these three compounds react to form amide bonds and are coupled together, thereby preparing a near-infrared fluorescent probe. This further expands the application value of the drug. This probe can be used for surgical navigation to remove tumors.

[0058] (3) The near-infrared fluorescent probe prepared in this disclosure can actively target vascular endothelial growth factor receptor. It has the advantages of active tumor targeting ability, good water solubility, long tumor retention time and no accumulation in normal tissue. It can not only be used to prepare fluorescent contrast agents or tumor diagnostic drugs, but also has the potential for application in clinical surgery, such as fluorescent-guided tumor resection. Attached Figure Description

[0059] Figure 1 shows the absorption spectra of the near-infrared fluorescent probes prepared in Examples 1-4 for the active targeting of vascular epidermal growth factor receptor detection.

[0060] Figure 2 shows the fluorescence spectra of the near-infrared fluorescent probes prepared in Examples 1-4 for the active targeting of vascular epidermal growth factor receptor detection.

[0061] Figure 3 shows the in vivo imaging of the near-infrared fluorescent probes prepared in Examples 1-4 for active targeting of vascular epidermal growth factor receptor detection in lung cancer HCC827 tumor-bearing mice.

[0062] Figure 4 shows in vivo imaging of NY-VEGF-21, NY-VEGF-22, NY-VEGF-23, and NY-VEGF-24 prepared in Examples 1-4 in HepG2 hepatocellular carcinoma-bearing mice.

[0063] Figure 5 shows an in vivo imaging image of NY-VEGF-021 prepared in Example 1 in HCT116 colorectal cancer-bearing mice.

[0064] Figure 6 shows in vivo imaging of NY-VEGF-21, NY-VEGF-22, NY-VEGF-23, and NY-VEGF-24 prepared in Examples 1-4 in pancreatic cancer PANC-1 tumor-bearing mice.

[0065] Figure 7 shows in vivo imaging of NY-VEGF-22 prepared in Example 2 and NY-VEGF-23 prepared in Example 3 in ASPC-1 pancreatic cancer-bearing mice. Detailed Implementation

[0066] In a first aspect, this disclosure provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, the near-infrared fluorescent probe comprising a compound as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0067] Wherein, the L group is a linking group, and the L group is selected from polyethylene glycol chain and / or polyglycine chain, and m is 0 or 1; the Y group is a dye molecule group with fluorescence excitation and emission spectra in the near infrared range.

[0068] It should be noted that when m is 0, it means that there is no connecting group L, and the Y group is directly attached to the N atom. The structural formula of the compound shown in Formula I is as follows:

[0069] It should be noted that when m is 1, it indicates the presence of a linking group L. The N-demethylvandetanil small molecule group is connected to the dye molecule group through the linking group L. The structural formula of the compound shown in Formula I is as follows:

[0070] In this disclosure, N-desmethylvandetanib and a dye molecule are linked by a linker molecule to prepare a near-infrared fluorescent probe for active targeting of vascular endothelial growth factor receptor (VEGF receptor). Vandetanib is a small-molecule multi-target tyrosine kinase inhibitor (TKI) that can simultaneously act on VEGFR-1 and VEGFR-2 in tumor cells. It can specifically target tumor tissue, and the drug inhibits the VEGFR signaling pathway, blocking angiogenesis and thus slowing tumor growth.

[0071] It should be noted that a "pharmaceutically acceptable salt" refers to any organic or inorganic addition salt of a compound represented by Formula I, which is relatively non-toxic and harmless to the patient, and whose side effects do not impair the beneficial effects of the compound. Pharmaceutically acceptable metal salts can be prepared using alkalis. Alkali metal salts or alkaline earth metal salts are obtained by dissolving the compound in an excess of an alkali metal or alkaline earth metal compound solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, sodium, potassium, or calcium salts are prepared as metal salts suitable for pharmaceutical use, but this disclosure is not limited thereto.

[0072] As an optional implementation, the Y group has a structure as shown in Formula II:

[0073] The R1 group and the R2 group are each independently selected from... n is 3 or 4;

[0074] Wherein, the Z group is selected from Furthermore, the S or O atom in the Z group is connected to the cyclohexene ring on one side of the Y group, and the carbonyl side of the Z group is connected to the L group or directly connected to the piperidine ring of the compound shown in Formula I.

[0075] As an optional embodiment, the Y group has a structure as shown in Formula II-1 or Formula II-2:

[0076] As an optional implementation, the L group has a structure as shown in formula L1 or L2:

[0077] Wherein, p and q are each independently selected from integers between 1 and 4, for example, they can be 1, 2, 3, 4; and the carbonyl side of the L group is connected to the piperidine ring of the compound shown in Formula I, and the amino side is connected to the Y group.

[0078] In a second aspect, this disclosure provides a method for preparing a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection as described in the first aspect, wherein the preparation method includes any one of the following methods one and two.

[0079] As an optional implementation method, Method 1 refers to the case where m=1, and its specific implementation is as follows:

[0080] Method 1: When m is 1, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection includes the following steps:

[0081] (1) In the presence of a condensing agent and a base, N-demethylvandetanil and a compound having an L group and an amino protecting group undergo a condensation reaction to give intermediate A;

[0082] (2) Intermediate A undergoes a deprotection reaction to obtain intermediate B;

[0083] (3) In the presence of a condensing agent and a base, intermediate B and a compound with a Y group undergo a condensation reaction to obtain the near-infrared fluorescent probe of Formula I for active targeting of vascular epidermal growth factor receptor detection.

[0084] As an optional implementation, in step (1) of method one, the compound with the L group has a structure as shown in formula L10 or L20:

[0085] Where X represents the protecting group of the amino group, and p and q are each independently selected from integers between 1 and 4, for example, they can be 1, 2, 3, or 4.

[0086] As an optional implementation, in step (1) of method one, the reaction formula of the condensation reaction is as follows:

[0087] As an optional implementation, in step (1) of method one, the condensing agent is HATU.

[0088] As an optional implementation, in step (1) of method one, the base is selected from triethylamine and / or diisopropylethylamine.

[0089] As an optional implementation, in step (1) of method one, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

[0090] As an optional implementation, in step (1) of method one, the molar ratio of N-demethylvandetanil, the compound having an L group and an amino protecting group, the condensing agent, the base and the polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20).

[0091] Among them, "1 to 1.2" can be, for example, 1, 1.05, 1.1, 1.15, 1.2, etc.;

[0092] Among them, "1 to 4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.;

[0093] Among them, "2 to 5" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0094] Among them, "5 to 20" can be, for example, 5, 7, 10, 12, 14, 15, 16, 18, 20, etc.

[0095] As an optional implementation, in step (1) of method one, the temperature of the condensation reaction is 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the condensation reaction is 0.5-2h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0096] As an optional implementation, in step (1) of method one, the following post-processing step is further included after the condensation reaction:

[0097] The reaction solution obtained after the condensation reaction was added dropwise to water to obtain a suspension; the suspension was then extracted with an organic solvent, dried and concentrated to obtain intermediate A.

[0098] As an optional implementation, in step (2) of method one, the reaction formula for the deprotection reaction is as follows:

[0099] As an optional implementation, in step (2) of method one, the deprotection reaction is carried out in the presence of an acid.

[0100] As an optional implementation, in step (2) of method one, the acid is trifluoroacetic acid.

[0101] As an optional implementation, in step (2) of method one, the molar ratio of intermediate A to acid is 1:(1 to 10), for example, it can be 1:1, 1:2, 1:4, 1:5, 1:6, 1:10, etc.

[0102] As an optional implementation, in step (2) of method one, the temperature of the deprotection reaction is 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the deprotection reaction is 20-40 min, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.

[0103] As an optional implementation, in step (3) of method one, the condensing agent is HATU.

[0104] As an optional implementation, in step (3) of method one, the base is selected from triethylamine and / or diisopropylethylamine.

[0105] As an optional implementation, in step (3) of method one, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

[0106] As an optional implementation, in step (3) of method one, the molar ratio of intermediate B, compound having Y group, condensing agent, base and polar solvent is 1:(1~1.2):(1~4):(2~5):(5~20);

[0107] Among them, "1 to 1.2" can be, for example, 1, 1.05, 1.1, 1.15, 1.2, etc.;

[0108] Among them, "1 to 4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.;

[0109] Among them, "2 to 5" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0110] Among them, "5 to 20" can be, for example, 5, 7, 10, 12, 14, 15, 16, 18, 20, etc.

[0111] As an optional implementation, in step (3) of method one, the temperature of the condensation reaction is 20-30°C, for example, 20°C, 22°C, 24°C, 25°C, 26°C, 28°C, 30°C, etc., and the time of the condensation reaction is 0.5-2h, for example, 0.5h, 0.6h, 0.8h, 1h, 1.2h, 1.4h, 1.6h, 1.8h, 2h, etc.

[0112] As an optional implementation method, method two refers to the case where m = 0, as detailed below:

[0113] Method 2: When m is 0, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor includes the following steps:

[0114] In the presence of a condensing agent and a base, N-demethylvandetanil and a compound with a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe of formula I for active targeting of vascular epidermal growth factor receptor detection.

[0115] As an optional implementation method, the flow of method two is as follows:

[0116] Thirdly, this disclosure provides the application of a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection as described in the first aspect in the preparation of fluorescent contrast agents.

[0117] As an optional implementation, the fluorescent contrast agent includes a fluorescent contrast agent for tumor diagnosis.

[0118] As an optional implementation, the tumor is selected from any one of liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small bowel cancer, and anal cancer.

[0119] Fourthly, this disclosure provides a fluorescent contrast agent comprising the above-described near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection or a near-infrared fluorescent probe prepared by the above-described preparation method.

[0120] Fifthly, this disclosure provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, or a near-infrared fluorescent probe prepared by the above-described preparation method, for use in fluorescence imaging or tumor diagnosis.

[0121] In one embodiment, the tumor is selected from any one of liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small bowel cancer, and anal cancer.

[0122] The present disclosure will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0123] The materials used and their sources in the embodiments are shown in Table 1 below:

[0124] Table 1

[0125] Example 1

[0126] This embodiment provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-21) has the following structure:

[0127] The synthetic route for the compound NY-VEGF-21 is shown below:

[0128] The preparation method of the compound NY-VEGF-21 is as follows:

[0129] (1) N-demethylvandetanil (100 mg, 1.0 eq), BOC-NH-PEG4-COOH acid (95 mg, 1.2 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (165 mg, 2.0 eq), N,N-diisopropylethylamine (85 mg, 3.0 eq), and DMSO (1 mL, 70.0 eq) were reacted at room temperature for 1 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was added dropwise to water to form a suspension. 10 mL of ethyl acetate was added to the suspension for extraction. After the organic layers were combined and concentrated, 10 mL of trifluoroacetic acid was added and reacted at room temperature for 30 min. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solution was evaporated to dryness and then purified by column chromatography to obtain vandetanil-PEG4 intermediate 1.

[0130] (2) Vandetanib-PEG4 intermediate 1 (100 mg, 1.0 eq), SO456-Der-01 (158 mg, 1.1 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (108 mg, 2.0 eq), N,N-diisopropylethylamine (55 mg, 3.0 eq), and DMSO (1 mL, 91 e q) 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, the mixture was purified by preparative liquid chromatography and lyophilized to obtain the green solid NY-VEGF-21.

[0131] Structural characterization was performed using 1H NMR and 1C NMR spectra, and the results are as follows:

[0132] [M-2H] / 2-=851.7;

[0133] 1 H NMR(600MHz,DMSO-d6)δ11.30(d,J=14.3Hz,1H),8.85(s,1H),8.08(s,1H) ,7.91-7.72(m,4H),7.69-7.44(m,7H),7.25(d,J=52.9Hz,4H),7.03(s,2H) ,6.20(s,2H),4.41(d,J=12.5Hz,1H),4.21-4.03(m,6H),3.99(d,J=4.9Hz, 4H),3.93(d,J=13.0Hz,2H),3.50-3.44(m,13H),3.44-3.41(m,2H),3.37(t ,J=5.9Hz,1H),3.33(t,J=5.8Hz,2H),3.15(dq,J=29.0,5.8Hz,3H),3.00(t ,J=12.4Hz,1H),2.72(s,2H),2.65(s,2H),2.61(p,J=1.9Hz,1H),2.59-2.5 3(m,7H),2.38(p,J=1.8Hz,0H),2.28(dt,J=15.8,7.1Hz,2H),2.15-2.07(m ,1H),2.00-1.55(m,12H),1.25(s,13H),1.12(dq,J=16.2,6.3,4.1Hz,1H);

[0134] 13C NMR (151MHz, DMSO) δ171.60,171.30,168.60,163.08,159.07,158.55,158.26,158.02,156.13,150.68,148. 91,144.93,142.14,140.89,140.32,135.39,130.16,128.15,126.21,124.04,122.11,120.50,119.70,114. 44,114.13,110.41,106.82,103.43,100.65,100.41,73.25,69.79,69.55,69.07,66.93,56.76,50.63,48.51,44.71,43.61,39.66,39.46,37.23,34.97,32.93,30.25,28.84,28.03,27.15,25.82,23.75,22.31,20.74.

[0135] Example 2

[0136] This embodiment provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-22) has the following structure:

[0137] The synthetic route for the compound NY-VEGF-22 is shown below:

[0138] The preparation method of the compound NY-VEGF-22 differs from that of Example 1 only in that (tert-butoxycarbonyl)glycylglycylglycine is used instead of BOC-NH-PEG4-COOH for the reaction, and then the green solid NY-VEGF-22 is obtained after preparation and freeze-drying.

[0139] Structural characterization was performed using 1H NMR and 1C NMR spectra, and the results are as follows:

[0140] [M-2H] / 2-=813.7;

[0141] 1H NMR (600MHz, DMSO-d6) δ11.27(s,1H),8.85(s,1H),8.20(t,J=5.8Hz,1H),8.11(t,J=6.0Hz,1H),8.07(s,1H),7.82(dd,J=11.6,7.3 Hz,4H),7.67-7.54(m,6H),7.51(s,1H),7.27(d,J=38.7Hz,4H),7.04(s,2H),6.21(s,2H),4.35(d,J=12.4Hz,1H),4.15-4.04(m,6H) ,3.99(s,5H),3.90(dd,J=16.5,5.2Hz,1H),3.83(d,J=13.0Hz,1H),3.70(d,J=5.9Hz,3H),3.64(d,J=5.6Hz,3H),3.02(t,J=12.7Hz ,1H),2.83-2.58(m,9H),2.40-2.33(m,3H),2.12(d,J=11.9Hz,1H),1.91-1.66(m,12H),1.25(s,15H),1.15(dd,J=15.4,9.1Hz,1H);

[0142] 13 C NMR(151MHz,DMSO-d6)δ172.40,172.06,169.87,169.31,166.83,163.52,159.52,158.96,158.71,158.5 0,158.11,156.61,151.12,149.37,145.29,142.61,141.37,140.79,135.84,130.64,128.59,126.64,124 .45,122.54,120.96,120.39,120.24,120.09,116.47,114.88,110.89,107.27,103.85,101.11,100.91,73.70,57.19,51.09,48.94,44.20,44.04,35.32,30.42,29.07,28.40,27.58,26.28,24.18,22.75,21.19.

[0143] Example 3

[0144] This embodiment provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-23) has the following structure:

[0145] The synthetic route for the compound NY-VEGF-23 is shown below:

[0146] The preparation method of the compound NY-VEGF-23 is as follows:

[0147] N-Desmethylvandetanil (100 mg, 1.0 eq), SO456-Der-01 (252 mg, 1.1 eq), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 165 mg, 2.0 eq), N,N-diisopropylethylamine (85 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 h. The reaction was monitored by high-performance liquid chromatography (HPLC). After the reaction was complete, the mixture was purified, separated, and lyophilized to obtain the green solid NY-VEGF-23.

[0148] Structural characterization was performed using 1H NMR and 1C NMR spectra, and the results are as follows:

[0149] [M-2H] / 2-=728.1;

[0150] 1 H NMR (600MHz, DMSO-d6) δ11.22(s,1H),8.87(s,1H),8.03(s,1H),7.82(d,J=1.9Hz,1H),7.81(s,2H),7.69-7.46(m,7H),7.27(d,J=8.0 Hz,4H),7.05(d,J=8.0Hz,2H),6.21(s,2H),4.31(d,J=13.0Hz,1H),4.10(s,4H),3.97(s,5H),3.87(dd,J=10.2,7.3Hz,4H),2.90(t,J =12.4Hz,1H),2.82(s,1H),2.76-2.67(m,4H),2.61(t,J=1.9Hz,1H),2.58-2.53(m,5H),2.53-2.51(m,1H),2.48-2.45(m,1H),2.05-1 .97(m,1H),1.93-1.86(m,2H),1.80-1.59(m,10H),1.25(d,J=6.2Hz,13H),1.04(dd,J=12.3,3.7Hz,1H),0.95(dd,J=12.0,3.9Hz,1H);

[0151] 13C NMR(151MHz,DMSO-d6)δ172.01,169.82,163.54,159.52,158.49,158.11,156.60,156.44,151.0 9,149.32,145.17,142.66,141.38,140.88,136.32,135.81,131.05,130.70,128.58,126.55,124 .48,122.59,120.94,120.39,120.24,120.07,114.78,110.82,107.23,103.76,101.13,73.77,57.12,51.10,48.94,44.82,44.05,35.09,30.03,28.82,28.64,27.59,26.31,24.20,22.74,21.21.

[0152] Example 4

[0153] This embodiment provides a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-24) has the following structure:

[0154] The synthetic route for the compound NY-VEGF-24 is shown below:

[0155] The synthesis of the compound NY-VEGF-24 was carried out in accordance with Example 2, except that SO456-Der-01 was replaced with SO456-Der-02 in the reaction. After preparation, purification and freeze-drying, green solid NY-VEGF-24 was obtained.

[0156] Structural characterization was performed using 1H NMR and 1C NMR spectra, and the results are as follows:

[0157] [M-2H] / 2-=769.6;

[0158] 1H NMR (600MHz, DMSO-d6) δ11.24(s,1H),8.86(d,J=7.3Hz,1H),8.71(d,J=14.0Hz,2H),8.33(t,J=5.8Hz,1H),8.21(t,J=6.0Hz,1H),8.05(d,J =4.5Hz,2H),7.86-7.72(m,4H),7.67-7.53(m,4H),7.49(s,1H),7.39(d,J=8.4Hz,2H),6.50(d,J=14.1Hz,2H),4.33(t,J=8.0Hz,5H),4.06(t d,J=11.8,9.8,6.8Hz,3H),3.99(s,4H),3.94(d,J=5.3Hz,1H),3.86(dd,J=16.6,5.3Hz,3H),3.00(q,J=7.6Hz,3H),2.71-2.57(m,9H),2.48- 2.36(m,3H),2.16-2.08(m,1H),2.02(t,J=7.5Hz,4H),1.93-1.72(m,5H),1.68(s,13H),1.22(d,J=9.7Hz,2H),1.10(dd,J=12.2,4.0Hz,1H);

[0159] 13 C NMR(151MHz,DMSO-d6)δ172.47,170.79,169.74,169.26,166.77,159.52,158.97,158.71,158.10,15 6.59,156.42,155.58,151.11,149.38,145.39,142.79,140.90,135.77,134.37,130.69,128.59,126 .60,124.44,120.98,120.39,120.25,116.47,114.55,110.72,107.27,103.81,102.47,100.92,73.70,57.17,49.23,48.32,44.19,43.25,35.99,35.27,33.03,29.09,28.38,27.85,26.32,23.74,21.05.

[0160] Test Example 1

[0161] UV and fluorescence tests

[0162] Test samples: four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0163] Test method:

[0164] (1) Prepare an aqueous solution of about 10 nmol of four compounds, NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24. First, use an enzyme-linked immunosorbent assay (ELISA) reader (Thermo Fisher Scientific, Varioskan LUX) to measure the absorption spectrum of each probe in the range of 500-1000 nm.

[0165] (2) The fluorescence emission spectra of each probe in the range of 8800-900nm were measured using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B).

[0166] Test results:

[0167] As shown in Figure 1, compounds NY-VEGF-21, NY-VEGF-22, and NY-VEGF-23 have maximum absorption wavelengths around 770 nm, while compound NY-VEGF-24 has maximum absorption at 790 nm.

[0168] As shown in Figure 2, compounds NY-VEGF-21, NY-VEGF-22, and NY-VEGF-23 have maximum absorption at approximately 820 nm, while compound NY-VEGF-24 has maximum emission at 840 nm.

[0169] Test Example 2

[0170] HCC827 (human non-small cell lung cancer cells) subcutaneous tumor-bearing mouse model in vivo imaging test

[0171] Test samples: four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0172] Test method: In the HCC827 subcutaneous tumor-bearing mouse model, NY-VEGF-21 to 24 series probes (10 nmol / mouse, 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), 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h.

[0173] Test Results: As shown in Figure 3, compared with ICG, the NY-VEGF-21 to NY-24 series probes all showed superior tumor imaging performance. Among them, NY-VEGF-22 and NY-VEGF-23 were metabolized more slowly and retained longer in the liver, while NY-VEGF-24 was metabolized rapidly in normal tissues, with almost no background signal 2 hours after administration. The duration of tumor fluorescence signal exceeded 72 hours, indicating that all four fluorescent probes have good tumor imaging capabilities and show promising prospects for development and application in intraoperative guided surgery.

[0174] Test Example 3

[0175] In vivo imaging of a subcutaneous tumor-bearing mouse model of HepG2 (human liver cancer cells)

[0176] Test samples: four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0177] Test method: In the HepG2 subcutaneous tumor-bearing mouse model, NY-VEGF-21 to 24 series probes (10 nmol / mouse, 100 μL glucose injection) were administered via tail vein. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) at 0 h (before administration), 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h.

[0178] Test Results: As shown in Figure 4, the NY-VEGF-21–24 series probes all exhibited excellent tumor imaging capabilities in HepG2 subcutaneous tumor-bearing mice. NY-VEGF-22 and NY-VEGF-23 showed slower tissue metabolism and relatively longer liver retention times compared to other probes. NY-VEGF-24 was metabolized rapidly, with tissue essentially cleared within 12 hours after administration. All four probes demonstrated good tumor imaging capabilities, with a visual window exceeding 72 hours, indicating promising prospects for clinical development and application.

[0179] Test Example 4

[0180] In vivo imaging of a subcutaneous mouse model bearing HCT116 (human colorectal cancer cells) tumor.

[0181] Test samples: four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0182] Test method: In the HCT116 subcutaneous tumor-bearing mouse model, NY-VEGF-21 probe (10 nmol / mouse, 100 μL glucose injection) was 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), 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h.

[0183] Test results: As shown in Figure 5, compared with ICG, the probe NY-VEGF-21 showed superior tumor imaging performance, with a tumor imaging time of over 72 hours, demonstrating good prospects for development and application.

[0184] Test Example 5

[0185] In vivo imaging of a subcutaneous mouse model bearing PANC-1 (human pancreatic cancer cells).

[0186] Test samples: four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0187] Test methods: In the PANC-1 subcutaneous tumor-bearing mouse model, NY-VEGF-21–24 series probes (10 nmol / mouse, 100 μL glucose injection) were administered via tail vein. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) at 0 h (before administration), 2 h, 4 h, 6 h, 9 h, 12 h, 24 h, 48 h, and 72 h. Further imaging experiments were conducted on ASPC-1 tumor-bearing mice with pancreatic cancer using probes NY-VEGF-22 and NY-VEGF-23.

[0188] Test results: As shown in Figure 6, the series of probes were basically completely metabolized in vivo 24 hours after administration, and the tumor morphology and edges were clearly visible. The imaging duration could reach 72 hours. The four probes NY-VEGF-21 to 24 have good tumor imaging capabilities and have good prospects for clinical development and application.

[0189] As shown in Figure 7, NY-VEGF-22 and NY-VEGF-23 also have good imaging ability and have potential for clinical intraoperative guided surgery applications.

[0190] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this disclosure, and are not intended to limit them. Although this disclosure has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this disclosure. Industrial applicability

[0191] This disclosure connects N-desmethylvandetanib and a dye molecule via a linker molecule. Vandetanib is a small-molecule multi-target tyrosine kinase inhibitor (TKI) that can simultaneously act on VEGFR-1 and VEGFR-2 in tumor cells. It can specifically target tumor tissue, and the drug slows tumor growth by inhibiting the VEGFR signaling pathway and blocking angiogenesis. This disclosure also provides a near-infrared fluorescent probe, which further expands the application value of the drug. This probe can be used for surgical navigation to remove tumors. The near-infrared fluorescent probe prepared by this disclosure can actively target vascular endothelial growth factor receptors. It has advantages such as active tumor targeting ability, good water solubility, long tumor retention time, and no accumulation in normal tissue. It can not only be used in the preparation of fluorescent contrast agents or tumor diagnostic drugs, but also has the potential for application in clinical surgical fields such as fluorescence-guided tumor resection.

Claims

1. A near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection, characterized in that, The near-infrared fluorescent probe comprises a compound as shown in Formula I or a pharmaceutically acceptable salt thereof; in, The L group is a linking group, and the L group is selected from polyethylene glycol chain and / or polyglycine chain, where m is 0 or 1; The Y group has a structure as shown in Formula II: The R1 group and the R2 group are each independently selected from... n is 3 or 4; The Z group is selected from Furthermore, the S or O atom in the Z group is connected to the cyclohexene ring on one side of the Y group, and the carbonyl side of the Z group is connected to the L group or directly connected to the piperidine ring of the compound shown in Formula I.

2. The near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 1, characterized in that, The L group has a structure as shown in formula L1 or L2: Wherein, p and q are each independently selected from integers between 1 and 4; and the carbonyl side of the L group is connected to the piperidine ring of the compound shown in Formula I, and the amino side is connected to the Y group.

3. A method for preparing a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 1 or 2, characterized in that, The preparation method includes Method 1 and Method 2: Method 1: When m is 1, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection includes the following steps: (1) In the presence of a condensing agent and a base, N-demethylvandetanil and a compound having an L group and an amino protecting group undergo a condensation reaction to give intermediate A; (2) Intermediate A undergoes a deprotection reaction to obtain intermediate B; (3) In the presence of a condensing agent and a base, intermediate B and a compound with a Y group undergo a condensation reaction to obtain the near-infrared fluorescent probe of Formula I for active targeting of vascular epidermal growth factor receptor detection. Method 2: When m is 0, the preparation method of the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor includes the following steps: In the presence of a condensing agent and a base, N-demethylvandetanil and a compound with a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe of formula I for active targeting of vascular epidermal growth factor receptor detection.

4. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (1) of method one, the compound having the L group has a structure as shown in formula L10 or L20: Where X represents the protecting group of an amino group, and p and q are each independently selected from integers between 1 and 4.

5. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (1) of Method 1, the condensing agent is HATU.

6. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (1) of Method 1, the base is selected from triethylamine and / or diisopropylethylamine.

7. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (1) of Method 1, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone.

8. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 7, characterized in that, In step (1) of Method 1, the molar ratio of N-demethylvandetanil, the compound having an L group and an amino protecting group, the condensing agent, the base and the polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20).

9. The method for preparing a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3 or 7, characterized in that, In step (1) of Method 1, the temperature of the condensation reaction is 20-30°C and the time of the condensation reaction is 0.5-2h.

10. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 9, characterized in that, In step (1) of Method 1, the condensation reaction further includes the following post-processing steps: The reaction solution obtained after the condensation reaction was added dropwise to water to obtain a suspension; the suspension was then extracted with an organic solvent, dried and concentrated to obtain intermediate A.

11. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (2) of Method 1, the deprotection reaction is carried out in the presence of an acid.

12. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 11, characterized in that, In step (2) of Method 1, the acid is trifluoroacetic acid.

13. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 11, characterized in that, In step (2) of method one, the molar ratio of intermediate A to acid is 1:(1~10); And / or, in step (2) of method one, the temperature of the deprotection reaction is 20-30°C and the time of the deprotection reaction is 20-40 min.

14. The method for preparing the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 3, characterized in that, In step (3) of method one, the condensing agent is HATU; And / or, in step (3) of method one, the base is selected from triethylamine and / or diisopropylethylamine; And / or, in step (3) of method one, the condensation reaction is carried out in the presence of a polar solvent selected from any one or at least a combination of two of dimethylformamide, dimethyl sulfoxide or N-methylpyrrolidone; And / or, in step (3) of method one, the molar ratio of intermediate B, compound having Y group, condensing agent, base and polar solvent is 1:(1~1.2):(1~4):(2~5):(5~20); And / or, in step (3) of method one, the temperature of the condensation reaction is 20-30°C, and the time of the condensation reaction is 0.5-2h; And / or, in method two, the condensing agent is HATU; And / or, in Method 2, the base is selected from triethylamine and / or diisopropylethylamine; And / or, in Method 2, the condensation reaction is carried out in the presence of a polar solvent selected from any one or a combination of at least two of dimethylformamide, dimethyl sulfoxide, or N-methylpyrrolidone; And / or, in Method 2, the molar ratio of N-demethylvandetanil, the compound having a Y group, the condensing agent, the base and the polar solvent is 1:(1-1.2):(1-4):(2-5):(5-20); And / or, in Method 2, the temperature of the condensation reaction is 20–30°C, and the time of the condensation reaction is 0.5–2 h.

15. The use of a near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection as described in claim 1 or 2, or a near-infrared fluorescent probe prepared by the preparation method according to any one of claims 3-14, in the preparation of a fluorescent contrast agent.

16. The application according to claim 15, characterized in that, The fluorescent contrast agent includes fluorescent contrast agents used for tumor diagnosis.

17. The application according to claim 16, characterized in that, The tumor is selected from any one of the following: liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small intestine cancer, and anal cancer.

18. A fluorescent contrast agent, characterized in that, It includes the near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection as described in claim 1 or 2, or the near-infrared fluorescent probe prepared by the preparation method described in any one of claims 3-14.

19. A near-infrared fluorescent probe for active targeting of vascular epidermal growth factor receptor detection according to claim 1 or 2, or a near-infrared fluorescent probe prepared by the preparation method according to any one of claims 3-14, characterized in that, Used for fluorescence imaging or tumor diagnosis.

20. The near-infrared fluorescent probe according to claim 19, characterized in that, The tumor is selected from any one of the following: liver cancer, colorectal cancer, lung cancer, pancreatic cancer, stomach cancer, colon cancer, rectal cancer, laryngeal cancer, breast cancer, ovarian cancer, uterine cancer, squamous cell carcinoma of the skin, esophageal cancer, oral squamous cell carcinoma, gallbladder cancer, bile duct cancer, small intestine cancer, and anal cancer.

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