Near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection and its preparation method and application

Abstract

The present invention provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection and its preparation method and application, which relates to the field of organic fluorescent probe molecules. The probe provided by the present invention can actively target vascular endothelial growth factor receptors, can be quickly cleared in normal tissues, and can be retained for a long time in tumor sites, thereby achieving the actual role of preparing living body diagnosis, having good clinical application prospects, and being developed and applied to clinical fluorescence-guided intraoperative navigation.

Description

Technical Field

[0001] The invention relates to the field of organic fluorescent probe molecules, and in particular to a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection, and a preparation method and application thereof. Background Art

[0002] Cancer is currently the leading cause of death in humans and is expected to become the main obstacle to the increase in life expectancy in the 21st century. Although modern medicine has more and more cancer treatments, such as immunotherapy and gene therapy, the most widely used clinical treatments are still traditional treatments: surgical resection, chemotherapy and radiotherapy. For solid tumors, surgical resection is still the preferred treatment method in clinical practice. Precision medicine aims to minimize medical damage and maximize disease control by improving diagnosis and treatment plans, and has become a research focus in the field of medicine today.

[0003] In order to achieve accurate tumor identification 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 auxiliary 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 (650~1000 nm) for specific detection, which 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.

[0004] Indocyanine green (ICG) is a near-infrared fluorescent dye approved by the FDA. After intravenous injection, it is easily bound to plasma proteins, metabolized by the liver into bile, and can be retained in tumor tissue through the EPR effect. However, ICG has poor specificity and is difficult to distinguish between tumor tissue and peritumoral inflammatory tissue, benign nodules, etc., 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-background ratio, allowing surgeons to more accurately identify tumor boundaries during surgery, completely remove residual tumor lesions, maximize patient survival, and improve patient quality of life.

[0005] Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell growth factor that promotes vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis. Under normal physiological conditions, when the organism matures, the vascular system will be in a quiescent state for a long time, and only when the body is damaged will transient angiogenesis occur. A large number of studies have shown that angiogenesis is one of the characteristics of the development of malignant tumors. The abnormal structure and high level of tumor blood vessels promote tumor proliferation, invasion and metastasis. Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors (VEGFRs) can induce a wide spectrum of signal transduction cascade reactions during tumor blood vessel growth and are key mediators of tumor angiogenesis. Therefore, focusing on the VEGF / VEGFR signaling pathway as a target to study the occurrence and development of tumors has certain theoretical research value and application prospects.

[0006] Vandetanib is a small molecule inhibitor of VEGFR-1, VEGFR-2 and other targets approved by the FDA on April 6, 2011. It can also selectively inhibit other tyrosine kinases. It is clinically used to treat unresectable locally advanced or metastatic medullary thyroid cancer (MTC) and non-small cell lung cancer (NSCLC). Compared with traditional chemotherapy drugs, vandetanib has significantly reduced non-specific toxicity and blood system toxicity. In recent years, its clinical application has been continuously expanded. Among them, the treatment of various cancers such as locally advanced or metastatic pancreatic cancer, advanced or metastatic breast cancer, and metastatic colorectal cancer is in the I or II clinical trial stage. The high specificity and low toxicity of vandetanib for various tumors make it valuable for the development of fluorescent probes with tumor targeting specificity, thereby further expanding the application value of the drug for surgical navigation and tumor removal.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection and its preparation method and application. The near-infrared fluorescent probe of the present invention can actively target vascular endothelial growth factor receptor, can be quickly cleared in normal tissues, and can be retained for a long time in tumor sites, thereby achieving the effect of in vivo diagnosis, having good clinical application prospects, and being developed and applied to clinical fluorescence-guided intraoperative navigation.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection, wherein the near-infrared fluorescent probe comprises a compound as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0011]

[0012] Formula I;

[0013] Among them, the L group is a connecting group, the L group is selected from a polyethylene glycol chain and / or a polyglycine chain, and m is 0 or 1; the Y group is a dye molecule group having a fluorescence excitation and emission spectrum in the near-infrared range.

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

[0015]

[0016] Formula II;

[0017] Among them, R 1 Group and R 2 The groups are each independently selected from , n is 3 or 4;

[0018] Wherein the Z group is selected from or ; and the S atom or O atom in the Z group is connected to the cyclohexene ring in one side of the Y group, and the carbonyl side in the Z group is connected to the L group or directly connected to the piperidine ring of the compound shown in formula I.

[0019] Preferably, the L group has a structure as shown in formula L1 or L2:

[0020] ;

[0021] 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.

[0022] In a second aspect, the present invention provides a method for preparing a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as described in the first aspect, wherein the preparation method comprises method one and method two:

[0023] Method 1: When m is 1, the method for preparing the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection comprises the following steps:

[0024] (1) In the presence of a condensing agent and a base, N -Demethyl vandetanib and a compound having an L group and an amino protecting group undergo a condensation reaction to obtain an intermediate A;

[0025] (2) intermediate A undergoes a deprotection reaction to obtain intermediate B;

[0026] (3) In the presence of a condensing agent and a base, the intermediate B and the compound having a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as shown in formula I.

[0027] Method 2: When m is 0, the method for preparing the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection comprises the following steps:

[0028] In the presence of a condensing agent and a base, N -Demethyl vandetanib and a compound having a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe of formula I for active targeted vascular epidermal growth factor receptor detection.

[0029] Preferably, in step (1) of method 1, the compound having an L group has a structure as shown in formula L10 or L20:

[0030]

[0031] Wherein, X represents a protecting group of an amino group, and p and q are each independently selected from integers between 1 and 4.

[0032] Preferably, in step (1) of method 1, the condensing agent is HATU.

[0033] Preferably, in step (1) of method 1, the base is selected from triethylamine and / or diisopropylethylamine.

[0034] Preferably, in step (1) of method 1, the condensation reaction is carried out in the presence of a polar solvent selected from dimethylformamide, dimethyl sulfoxide or N -Methylpyrrolidone, any one or a combination of at least two thereof.

[0035] Preferably, in step (1) of method 1, N The molar ratio of -desmethyl vandetanib, 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).

[0036] Preferably, in step (1) of method 1, the temperature of the condensation reaction is 20-30° C., and the time of the condensation reaction is 0.5-2 h.

[0037] Preferably, in step (1) of method 1, the condensation reaction further includes the following post-treatment steps:

[0038] The reaction solution obtained after the condensation reaction is dropped into water to obtain a suspension; the suspension is then extracted with an organic solvent, dried and concentrated to obtain an intermediate A.

[0039] Preferably, in step (2) of method 1, the deprotection reaction is carried out in the presence of an acid.

[0040] Preferably, in step (2) of method 1, the acid is trifluoroacetic acid.

[0041] Preferably, in step (2) of method 1, the molar ratio of the intermediate A to the acid is 1:(1-10).

[0042] Preferably, in step (2) of method 1, the temperature of the deprotection reaction is 20-30° C., and the time of the deprotection reaction is 20-40 min.

[0043] Preferably, in step (3) of method 1, the condensing agent is HATU.

[0044] Preferably, in step (3) of method 1, the base is selected from triethylamine and / or diisopropylethylamine.

[0045] Preferably, in step (3) of method 1, the condensation reaction is carried out in the presence of a polar solvent selected from dimethylformamide, dimethyl sulfoxide or N -Methylpyrrolidone, any one or a combination of at least two thereof.

[0046] Preferably, in step (3) of method 1, the molar ratio of the intermediate B, 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).

[0047] Preferably, in step (3) of method 1, the temperature of the condensation reaction is 20-30° C., and the time of the condensation reaction is 0.5-2 h.

[0048] Preferably, in method 2, the condensing agent is HATU.

[0049] Preferably, in method 2, the base is selected from triethylamine and / or diisopropylethylamine.

[0050] Preferably, in method 2, the condensation reaction is carried out in the presence of a polar solvent, and the polar solvent is selected from any one of dimethylformamide, dimethyl sulfoxide or N-methylpyrrolidone, or a combination of at least two thereof.

[0051] Preferably, in method 2, the NThe molar ratio of -desmethyl vandetanib, 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).

[0052] Preferably, 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.

[0053] In a third aspect, the present invention provides a use of a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as described in the first aspect in the preparation of a fluorescent contrast agent.

[0054] Preferably, the fluorescent contrast agent includes a fluorescent contrast agent for tumor diagnosis.

[0055] Preferably, the tumor includes any one of liver cancer, colorectal cancer, lung cancer, and pancreatic cancer.

[0056] Compared with the prior art, the present invention has the following beneficial effects:

[0057] (1) The present invention connects molecules N -Demethyl vandetanib is connected to the dye molecule, where vandetanib is a small molecule multi-target tyrosine kinase inhibitor (TKI) that can act on tumor cell VEGFR-1 and VEGFR-2 at the same time. It can specifically target tumor tissues. The drug inhibits the VEGFR signaling pathway and blocks angiogenesis, thereby slowing down tumor growth.

[0058] (2) The present invention connects molecules N -Demethyl vandetanib is connected to the dye molecule, and under the action of the condensing agent, the carboxyl and amino groups in the three compounds react to form amide bonds, which are coupled to prepare 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.

[0059] (3) The near-infrared fluorescent probe prepared by the present invention can actively target the 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 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

[0060] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 This is the absorption spectrum of the near-infrared fluorescent probe prepared in Examples 1 to 4 for active targeted vascular epidermal growth factor receptor detection.

[0062] Figure 2 This is a fluorescence spectrum diagram of the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection prepared in Examples 1 to 4.

[0063] Figure 3 This is an in vivo imaging diagram of the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection prepared in Examples 1 to 4 in lung cancer HCC827 tumor-bearing mice.

[0064] Figure 4 This is the in vivo imaging of NY-VEGF-021 prepared in Example 1 in mice bearing HepG2 liver cancer.

[0065] Figure 5 This is the in vivo imaging of NY-VEGF-021 prepared in Example 1 in mice bearing colorectal cancer HCT116.

[0066] Figure 6 This is the in vivo imaging of NY-VEGF-021 prepared in Example 1 in mice bearing pancreatic cancer PANC-1.

[0067] Figure 7 The in vivo imaging diagrams of NY-VEGF-022 prepared in Example 2 and NY-VEGF-023 prepared in Example 3 in mice bearing pancreatic cancer ASPC-1 are shown. DETAILED DESCRIPTION

[0068] Unless otherwise defined herein, scientific and technological terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0069] It should be noted that specific details are described in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in a variety of other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0070] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0071] In a first aspect, the present invention provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection, wherein the near-infrared fluorescent probe comprises a compound as shown in Formula I or a pharmaceutically acceptable salt thereof;

[0072]

[0073] Formula I;

[0074] Among them, the L group is a connecting group, the L group is selected from a polyethylene glycol chain and / or a polyglycine chain, and m is 0 or 1; the Y group is a dye molecule group having a fluorescence excitation and emission spectrum in the near-infrared range.

[0075] It should be noted that when m is 0, it means that there is no connecting group L, and the Y group is directly connected to the N atom. The structural formula of the compound shown in Formula I is as follows:

[0076] .

[0077] It should be noted that when m is 1, it represents the presence of a connecting group L. N The -desmethyl vandetanib small molecule group is connected to the dye molecule group through a linker group L. The structural formula of the compound shown in Formula I is as follows:

[0078] .

[0079] In the present invention, the present invention connects molecules N -Demethyl vandetanib is connected to a dye molecule to prepare a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection. Among them, vandetanib is a small molecule multi-target tyrosine kinase inhibitor (TKI) that can act on tumor cell VEGFR-1 and VEGFR-2 at the same time. It can specifically target tumor tissues. The drug inhibits the VEGFR signaling pathway and blocks angiogenesis, thereby slowing down tumor growth.

[0080] It should be noted that "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 the side effects caused by the salt do not impair the beneficial effects of the compound. Pharmaceutically acceptable metal salts can be prepared using a base. Alkali metal salts or alkaline earth metal salts are obtained by dissolving the compound in an excess of an alkali metal basic compound or an alkaline earth metal basic compound solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, the preparation of sodium salts, potassium salts or calcium salts as metal salts is suitable for pharmaceutical use, but the present invention is not limited thereto.

[0081] As an optional embodiment, the Y group has a structure as shown in Formula II:

[0082]

[0083] Formula II;

[0084] Among them, R 1 Group and R 2 The groups are each independently selected from , n is 3 or 4;

[0085] Wherein the Z group is selected from or ; and the S atom or O atom in the Z group is connected to the cyclohexene ring in one side of the Y group, and the carbonyl side in the Z group is connected to the L group or directly connected to the piperidine ring of the compound shown in formula I.

[0086] As an optional embodiment, the Y group has a structure as shown in Formula II-1 or Formula II-2:

[0087]

[0088] Formula II-1;

[0089]

[0090] Formula II-2.

[0091] As an optional embodiment, the L group has a structure as shown in formula L1 or L2:

[0092] ;

[0093] Wherein, p and q are each independently selected from integers between 1 and 4, for example, 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.

[0094] In a second aspect, the present invention provides a method for preparing a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as described in the first aspect, wherein the preparation method comprises any one of the following methods 1 and 2.

[0095] As an optional implementation, method 1 refers to the case where m=1, which is specifically as follows:

[0096] Method 1: When m is 1, the method for preparing the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection comprises the following steps:

[0097] (1) In the presence of a condensing agent and a base, N -Demethyl vandetanib and a compound having an L group and an amino protecting group undergo a condensation reaction to obtain an intermediate A;

[0098] (2) intermediate A undergoes a deprotection reaction to obtain intermediate B;

[0099] (3) In the presence of a condensing agent and a base, the intermediate B and the compound having a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as shown in formula I.

[0100] As an optional embodiment, in step (1) of method 1, the compound having an L group has a structure as shown in formula L10 or L20:

[0101]

[0102] Wherein, X represents a protecting group of an amino group, and p and q are each independently selected from integers between 1 and 4, for example, 1, 2, 3, or 4.

[0103] As an optional embodiment, in step (1) of method 1, the reaction formula of the condensation reaction is as follows:

[0104] .

[0105] As an optional embodiment, in step (1) of method 1, the condensing agent is HATU.

[0106] As an optional embodiment, in step (1) of method 1, the base is selected from triethylamine and / or diisopropylethylamine.

[0107] As an optional embodiment, in step (1) of method 1, the condensation reaction is carried out in the presence of a polar solvent, and the polar solvent is selected from dimethylformamide, dimethyl sulfoxide or N -Methylpyrrolidone, any one or a combination of at least two thereof.

[0108] As an optional implementation, in step (1) of method 1, the N - The molar ratio of demethyl vandetanib, 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);

[0109] Here, “1~1.2” can be, for example, 1, 1.05, 1.1, 1.15, 1.2, etc.;

[0110] Among them, "1~4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.;

[0111] Among them, "2~5" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0112] Among them, "5~20" can be, for example, 5, 7, 10, 12, 14, 15, 16, 18, 20, etc.

[0113] As an optional embodiment, 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 0.5-2 h, for example, 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.

[0114] As an optional embodiment, in step (1) of method 1, the condensation reaction further includes the following post-treatment steps:

[0115] The reaction solution obtained after the condensation reaction is dropped into water to obtain a suspension; the suspension is then extracted with an organic solvent, dried and concentrated to obtain an intermediate A.

[0116] As an optional embodiment, in step (2) of method 1, the reaction formula of the deprotection reaction is as follows:

[0117] .

[0118] As an optional embodiment, in step (2) of method 1, the deprotection reaction is carried out in the presence of an acid.

[0119] As an optional embodiment, in step (2) of method 1, the acid is trifluoroacetic acid.

[0120] As an optional embodiment, in step (2) of method one, the molar ratio of the intermediate A to the acid is 1:(1-10), for example, it can be 1:1, 1:2, 1:4, 1:5, 1:6, 1:10, etc.

[0121] As an optional embodiment, 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.

[0122] As an optional embodiment, in step (3) of method 1, the condensing agent is HATU.

[0123] As an optional embodiment, in step (3) of method 1, the base is selected from triethylamine and / or diisopropylethylamine.

[0124] As an optional embodiment, in step (3) of method 1, the condensation reaction is carried out in the presence of a polar solvent, and the polar solvent is selected from dimethylformamide, dimethyl sulfoxide or N -Methylpyrrolidone, any one or a combination of at least two thereof.

[0125] As an optional embodiment, in step (3) of method 1, the molar ratio of the intermediate B, 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);

[0126] Here, “1~1.2” can be, for example, 1, 1.05, 1.1, 1.15, 1.2, etc.;

[0127] Among them, "1~4" can be, for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, etc.;

[0128] Among them, "2~5" can be, for example, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.;

[0129] Among them, "5~20" can be, for example, 5, 7, 10, 12, 14, 15, 16, 18, 20, etc.

[0130] As an optional embodiment, 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 0.5-2 h, for example, 0.5 h, 0.6 h, 0.8 h, 1 h, 1.2 h, 1.4 h, 1.6 h, 1.8 h, 2 h, etc.

[0131] As an optional implementation, method 2 refers to the case where m=0, which is specifically as follows:

[0132] Method 2: When m is 0, the method for preparing the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection comprises the following steps:

[0133] In the presence of a condensing agent and a base, N -Demethyl vandetanib and a compound having a Y group undergo a condensation reaction to obtain a near-infrared fluorescent probe of formula I for active targeted vascular epidermal growth factor receptor detection.

[0134] As an optional implementation, the route flow of method 2 is as follows:

[0135] .

[0136] In a third aspect, the present invention provides a use of a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection as described in the first aspect in the preparation of a fluorescent contrast agent.

[0137] As an optional embodiment, the fluorescent contrast agent includes a fluorescent contrast agent for tumor diagnosis.

[0138] As an optional embodiment, the tumor includes any one of liver cancer, colorectal cancer, lung cancer, and pancreatic cancer.

[0139] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0140] The materials and sources used in the examples are shown in Table 1 below:

[0141] Table 1

[0142]

[0143] Example 1

[0144] This embodiment provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-21) has a structure as shown below:

[0145]

[0146] The synthetic route of the compound NY-VEGF-21 is as follows:

[0147]

[0148] The preparation method of the compound NY-VEGF-21 is as follows:

[0149] (1) N-demethyl vandetanib (100 mg, 1.0 eq), BOC-NH-PEG4-COOH acid (95 mg, 1.2 eq), 2-(7-azabenzotriazole)- N,N,N',N' -Tetramethyluronium hexafluorophosphate (165 mg, 2.0 eq), N,N -Diisopropylethylamine (85 mg, 3.0 eq), DMSO (1 mL, 70.0 eq), react at room temperature for 1 h, monitor the reaction using thin layer chromatography (TLC), after the reaction is complete, drop the reaction solution into water to form a suspension; add 10 mL of ethyl acetate to the suspension for extraction, combine the organic layers and concentrate, then add 10 mL of trifluoroacetic acid and react at room temperature for 30 min, monitor the reaction using thin layer chromatography (TLC), after the reaction is complete, evaporate to dryness and then purify by column chromatography to obtain vandetanib-PEG4 intermediate 1.

[0150] (2) Vandetanib-PEG4 intermediate 1 (100 mg, 1.0 eq), S0456-Der-01 (158 mg, 1.1 eq), 2-(7-azabenzotriazole)- N,N,N',N' -Tetramethyluronium 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 for 1 hour in the dark. The reaction was monitored by liquid chromatography (HPLC). After the reaction was complete, it was purified by preparative liquid phase and freeze-dried to obtain green solid NY-VEGF-21.

[0151] The structural characterization was performed by H-NMR and C-NMR, and the results are as follows:

[0152] [M-2H] / 2- = 851.7;

[0153] 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.30 (d, J = 14.3 Hz, 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 = 52.9 Hz, 4H), 7.03 (s, 2H), 6.20 (s, 2H), 4.41 (d, J= 12.5 Hz, 1H), 4.21-4.03 (m,6H), 3.99(d, J = 4.9 Hz,4H), 3.93 (d, J = 13.0 Hz, 2H), 3.50-3.44 (m, 13H), 3.44-3.41 (m,2H), 3.37 (t, J = 5.9 Hz, 1H), 3.33 (t, J = 5.8 Hz, 2H), 3.15 (dq, J = 29.0, 5.8Hz, 3H), 3.00 (t, J = 12.4 Hz, 1H), 2.72 (s, 2H), 2.65 (s, 2H), 2.61 (p, J = 1.9Hz, 1H), 2.59-2.53 (m, 7H), 2.38 (p, J = 1.8 Hz, 0H), 2.28 (dt, J = 15.8, 7.1 Hz,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, 4.1 Hz, 1H);

[0154] 13C NMR (151 MHz, DMSO) δ 171.60, 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, 37.23, 34.97,32.93, 30.25, 28.84, 28.03, 27.15, 25.82, 23.75, 22.31, 20.74.

[0155] Example 2

[0156] This embodiment provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-22) has a structure as shown below:

[0157]

[0158] The synthetic route of the compound NY-VEGF-22 is as follows:

[0159]

[0160]

[0161] The preparation method of the compound NY-VEGF-22 is different from that of Example 1 only in that BOC-NH-PEG is replaced by (tert-butyloxycarbonyl)glycylglycylglycine 4 -COOH to react, and then prepared and freeze-dried to obtain green solid NY-VEGF-22.

[0162] The structural characterization was performed by H-NMR and C-NMR, and the results are as follows:

[0163] [M-2H] / 2- = 813.7;

[0164] 1H NMR (600 MHz, DMSO- d 6 ) δ 11.27 (s, 1H), 8.85 (s, 1H), 8.20 (t, J =5.8 Hz, 1H), 8.11 (t, J = 6.0 Hz, 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 = 38.7 Hz, 4H), 7.04 (s, 2H),6.21 (s, 2H), 4.35 (d, J = 12.4 Hz, 1H), 4.15-4.04 (m, 6H), 3.99 (s, 5H), 3.90(dd, J = 16.5, 5.2 Hz, 1H), 3.83 (d, J = 13.0 Hz, 1H), 3.70 (d, J = 5.9 Hz, 3H),3.64 (d, J = 5.6 Hz,3H), 3.02 (t, J = 12.7 Hz, 1H), 2.83-2.58 (m, 9H), 2.40-2.33(m, 3H), 2.12 (d, J = 11.9 Hz, 1H), 1.91-1.66 (m, 12H), 1.25 (s, 15H), 1.15(dd, J = 15.4, 9.1 Hz, 1H);

[0165] 13 C NMR (151 MHz, DMSO- d 6) δ 172.40, 172.06, 169.87, 169.31, 166.83,163.52, 159.52, 158.96, 158.71, 158.50, 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, 8, 27.58, 26.28, 24.18, 22.75, 21.19.

[0166] Example 3

[0167] This embodiment provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-23) has a structure as shown below:

[0168]

[0169] The synthetic route of the compound NY-VEGF-23 is as follows:

[0170]

[0171] The preparation method of the compound NY-VEGF-23 is as follows:

[0172] N -Demethylvandetanib (100 mg, 1.0 eq), S0456-Der-01 (252 mg, 1.1 eq), 2-(7-azabenzotriazole)- N,N,N',N' -Tetramethyluronium 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 the mixture was reacted at room temperature in the dark for 1 h. The reaction was monitored by liquid chromatography (HPLC). After the reaction was complete, the mixture was purified, separated and freeze-dried by preparative liquid phase to obtain green solid NY-VEGF-23.

[0173] The structural characterization was performed by H-NMR and C-NMR, and the results are as follows:

[0174] [M-2H] / 2- = 728.1;

[0175] 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.22 (s, 1H), 8.87 (s, 1H), 8.03 (s, 1H), 7.82 (d, J = 1.9 Hz, 1H), 7.81 (s, 2H), 7.69-7.46 (m,7H), 7.27 (d, J = 8.0 Hz,4H), 7.05 (d, J = 8.0 Hz, 2H), 6.21 (s, 2H), 4.31 (d, J = 13.0 Hz, 1H), 4.10 (s,4H), 3.97 (s,5H), 3.87 (dd, J = 10.2, 7.3 Hz,4H), 2.90 (t, J = 12.4 Hz, 1H), 2.82(s, 1H), 2.76-2.67 (m, 4H), 2.61 (t, J = 1.9 Hz, 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 = 6.2 Hz, 13H), 1.04 (dd, J = 12.3, 3.7 Hz, 1H), 0.95(dd, J = 12.0, 3.9 Hz, 1H);

[0176] 13 C NMR (151 MHz, DMSO- d 6) δ 172.01, 169.82, 163.54, 159.52, 158.49,158.11, 156.60, 156.44, 151.09, 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.

[0177] Example 4

[0178] This embodiment provides a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection. The near-infrared fluorescent probe (hereinafter referred to as compound NY-VEGF-24) has a structure as shown below:

[0179]

[0180] The synthetic route of the compound NY-VEGF-24 is as follows:

[0181]

[0182] The synthesis of the compound NY-VEGF-24 is referred to Example 2, which differs from Example 2 in that S0456-Der-02 is used to replace S0456-Der-01 for the reaction, and a green solid NY-VEGF-24 is obtained after preparation, purification and freeze-drying.

[0183] The structural characterization was performed by H-NMR and C-NMR, and the results are as follows:

[0184] [M-2H] / 2-=769.6;

[0185] 1 H NMR (600 MHz, DMSO- d 6 ) δ 11.24 (s, 1H), 8.86 (d, J = 7.3 Hz, 1H),8.71 (d, J= 14.0 Hz, 2H), 8.33 (t, J = 5.8 Hz, 1H), 8.21 (t, J = 6.0 Hz, 1H), 8.05(d, J = 4.5 Hz, 2H), 7.86-7.72 (m, 4H), 7.67-7.53 (m, 4H), 7.49 (s, 1H), 7.39(d, J = 8.4 Hz, 2H), 6.50 (d, J = 14.1 Hz, 2H), 4.33 (t, J = 8.0 Hz, 5H), 4.06 (td, J = 11.8, 9.8, 6.8 Hz, 3H), 3.99 (s, 4H), 3.94 (d, J = 5.3 Hz, 1H), 3.86 (dd, J =16.6, 5.3 Hz, 3H), 3.00 (q, J = 7.6 Hz, 3H), 2.71-2.57 (m, 9H), 2.48-2.36 (m,3H), 2.16-2.08 (m, 1H), 2.02 (t, J = 7.5 Hz, 4H), 1.93-1.72 (m, 5H), 1.68 (s,13H), 1.22 (d, J = 9.7 Hz, 2H), 1.10 (dd, J = 12.2, 4.0 Hz, 1H);

[0186] 13 C NMR (151 MHz, DMSO- d 6) δ 172.47, 170.79, 169.74, 169.26, 166.77,159.52, 158.97, 158.71, 158.10, 156.59, 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, 8, 27.85, 26.32, 23.74, 21.05.

[0187] Test Example 1

[0188] UV and fluorescence testing

[0189] Test samples: the four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0190] Test method:

[0191] (1) NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 were prepared into an approximately 10 nmol aqueous solution, and the absorption spectrum of each probe in the range of 500-1000 nm was measured using an ELISA reader (Thermo Fisher, Varioskan LUX).

[0192] (2) The fluorescence emission spectrum of each probe in the range of 8800-900 nm was measured using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10 B).

[0193] Test results:

[0194] like Figure 1 As shown, compounds NY-VEGF-21, NY-VEGF-22 and NY-VEGF-23 have maximum absorption wavelengths at about 770 nm, while compound NY-VEGF-24 has maximum absorption at 790 nm.

[0195] like Figure 2As shown, compounds NY-VEGF-21, NY-VEGF-22 and NY-VEGF-23 have maximum absorption at about 820 nm, and compound NY-VEGF-24 has a maximum emission wavelength at 840 nm.

[0196] Test Example 2

[0197] In vivo imaging test of HCC827 (human non-small cell lung cancer cell) subcutaneous tumor-bearing mouse model

[0198] Test samples: the four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0199] Test method:

[0200] In the HCC827 subcutaneous tumor-bearing mouse model, NY-VEGF-21~24 series probes (10 nmol / mouse, 100 μL of glucose injection) were administered via tail vein, and indocyanine green (ICG, 1.0 mg / kg, 100 μL of water for injection) was used as control. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B) after administration, at 0h (before administration), 2h, 4h, 6h, 9h, 12h, 24h, 48h, and 72h, respectively.

[0201] Test results:

[0202] like Figure 3 As shown in the figure, compared with ICG, the NY-VEGF-21~24 series probes all showed better tumor imaging effects, among which NY-VEGF-22 and NY-VEGF-23 were metabolized slowly and retained in the liver for a long time, while NY-VEGF-24 was metabolized rapidly in normal tissues and had almost no background signal 2 hours after administration. The duration of tumor fluorescence signal exceeded 72 hours, which means that the four fluorescent probes have good tumor imaging capabilities and have good development and application prospects for clinical intraoperative guided surgery.

[0203] Test Example 3

[0204] In vivo imaging of HepG2 (human hepatoma cell) subcutaneous tumor-bearing mouse model

[0205] Test samples: the four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0206] Test method:

[0207] In the HepG2 subcutaneous tumor-bearing mouse model, NY-VEGF-21~24 series probes (10 nmol / mouse, 100 μL of glucose injection) were administered via the tail vein, and 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, respectively.

[0208] Test results:

[0209] like Figure 4 As shown in the figure, in mice bearing subcutaneous HepG2 tumors, the NY-VEGF-21~24 series of probes all showed excellent tumor imaging capabilities. Among them, NY-VEGF-22 and NY-VEGF-23 have slower tissue metabolism than other probes, and their liver retention time is relatively long. NY-VEGF-24 has rapid tissue metabolism, and the tissue is basically metabolized 12 hours after administration. The four probes have good tumor imaging capabilities, with a display window of more than 72 hours, and have good prospects for clinical development and application.

[0210] Test Example 4

[0211] In vivo imaging of HCT116 (human colorectal cancer cell) subcutaneous tumor-bearing mouse model

[0212] Test samples: the four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0213] Test method:

[0214] In the HCT116 subcutaneous tumor-bearing mouse model, NY-VEGF-21 probe (10 nmol / mouse, 100 μL of glucose injection) was administered via tail vein, and indocyanine green (ICG, 1.0 mg / kg, 100 μL of water for injection) was used as 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, respectively.

[0215] Test results:

[0216] like Figure 5 As shown in the figure, compared with ICG, the probe NY-VEGF-21 showed better tumor imaging effect, with a tumor imaging time of more than 72 hours, and has good development and application prospects.

[0217] Test Example 5

[0218] In vivo imaging of PANC-1 (human pancreatic cancer cell) subcutaneous tumor-bearing mouse model

[0219] Test samples: the four compounds NY-VEGF-21, NY-VEGF-22, NY-VEGF-23 and NY-VEGF-24 provided in Examples 1 to 4.

[0220] Test method:

[0221] In the PANC-1 subcutaneous tumor-bearing mouse model, the NY-VEGF-21~24 series probes (10 nmol / mouse, 100 μL of glucose injection) were administered via the tail vein, and 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, respectively. Further, the probes NY-VEGF-22 and NY-VEGF-23 were used for imaging experiments in pancreatic cancer ASPC-1 tumor-bearing mice.

[0222] Test results:

[0223] like Figure 6 As shown, the metabolism of this series of probes is basically complete 24 hours after administration, the tumor morphology and edges are clearly visible, and the imaging duration can reach 72 hours. The four probes NY-VEGF-21~24 have good tumor imaging capabilities and have good clinical development and application prospects.

[0224] like Figure 7 As shown in the results, NY-VEGF-22 and NY-VEGF-23 also have good imaging ability and have potential for clinical application in intraoperative guided surgery.

[0225] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection, characterized in that: The near-infrared fluorescent probe is selected from the following compounds: 。 2. A method for preparing a near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection according to claim 1, characterized in that: The preparation method is selected from the following synthesis process: 。 3. Use of the near-infrared fluorescent probe for active targeted vascular epidermal growth factor receptor detection according to claim 1 in the preparation of a fluorescent contrast agent.

4. The use according to claim 3, characterized in that: The fluorescent contrast agent includes a fluorescent contrast agent for tumor diagnosis.

5. The use according to claim 4, characterized in that: The tumor includes any one of liver cancer, colorectal cancer, lung cancer, and pancreatic cancer.

Images