A small molecule near-infrared fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a small-molecule near-infrared fluorescent probe and a preparation method and application thereof, and belongs to the technical field of organic fluorescent probe molecules. The small-molecule near-infrared fluorescent probe comprises a compound or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers, optical isomers, deuterium compounds or prodrugs of the compound or mixtures thereof, wherein the compound has a structural formula selected from formula (I), wherein X is a linking group, Y is a near-infrared fluorescent dye molecule group; the X is selected from any one of none, -NH(CH2CH2O) a CH2CH2CO- or -NH(CH2) b CO-, wherein a is 1-6, and b is 2-6. The small-molecule near-infrared fluorescent probe has tumor-targeting recognition ability and near-infrared fluorescent imaging performance, and can be used as a fluorescent contrast agent and a tumor diagnosis reagent, and has a wide application prospect in the field of clinical surgery, especially in tumor resection surgery under fluorescent guidance.

Description

Technical Field

[0001] This invention belongs to the field of organic fluorescent probe molecular technology, and more specifically, relates to a small molecule near-infrared fluorescent probe, its preparation method and application. Background Technology

[0002] Malignant tumors have become a major health threat to human life. With the increasing aging of society and changes in lifestyle, the prevention and control of this disease is becoming increasingly complex. In medical practice, the main treatments for malignant tumors include surgical resection, chemotherapy, and radiotherapy. Among these, surgical resection is widely recognized as the core treatment for solid tumors; precise tumor resection can significantly improve treatment outcomes and prolong patient life. To achieve accurate tumor localization and surgical guidance, the medical community has widely applied various imaging technologies, such as computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and ultrasound, to achieve precise imaging of cancerous tissue. Due to inherent limitations in traditional imaging technologies, such as insufficient spatial resolution and sensitivity, it is difficult to accurately define the boundaries of lesions. However, emerging optical molecular imaging navigation systems, with their real-time, non-invasive, and ultra-high resolution characteristics, have opened up entirely new image-assisted approaches for precise tumor surgery. Near-infrared fluorescence imaging technology demonstrates significant advantages throughout the entire tumor diagnosis and treatment cycle, from early detection to mid-stage diagnosis, late-stage treatment, and postoperative follow-up. This technology uses near-infrared fluorescent probes in the 650–1000 nm wavelength range for specific detection, which not only acquires high-resolution images of tissues and organs, but also has unique advantages such as good biocompatibility and low background fluorescence interference.

[0003] As a near-infrared fluorescent marker approved by the U.S. Food and Drug Administration (FDA), indocyanine green (ICG) rapidly binds to plasma proteins after intravenous injection, is subsequently metabolized by the liver and enters the biliary system, and can accumulate at tumor sites through enhanced permeability and retention (EPR). However, this dye suffers from insufficient specificity, making it difficult to effectively distinguish malignant lesions from surrounding inflammatory tissue or benign hyperplasia, thus increasing the risk of misdiagnosis; moreover, the dye's retention time at tumor sites is relatively short, limiting its clinical application.

[0004] Therefore, how to develop a near-infrared fluorescent probe that combines specificity, targeting, and long retention time at the tumor site is an urgent technical problem to be solved in the development of tumor-specific fluorescent probes in this field. Summary of the Invention

[0005] 1. The problem to be solved During the research and development of this invention, it was noted that many solid tumors exhibit high or abnormal expression of EGFR (epidermal growth factor receptor). EGFR is a member of the epidermal growth factor receptor family, a glycoprotein located on the cell membrane surface, and belongs to the tyrosine kinase type receptor, which is activated by binding to a ligand. EGFR is closely related to tumor cell proliferation, angiogenesis, and tumor invasion and metastasis.

[0006] Osimertinib, approved by the FDA in November 2015, is a third-generation, oral, irreversible, selective EGFR mutation inhibitor, first marketed in the United States. Clinically, it is used to treat locally advanced or metastatic non-small cell lung cancer with EGFR T790M mutations. As a targeted anti-tumor drug, osimertinib can precisely act on tumor tissue, inducing programmed cell death in cancer cells by activating relevant signaling pathways, thereby exerting its therapeutic efficacy. Therefore, this invention aims to develop a near-infrared fluorescent probe based on osimertinib that possesses specificity, targeting, and a long retention time at the tumor site.

[0007] Based on this, the first objective of this invention is to develop a small molecule compound based on osimertinib, which, when used as a near-infrared fluorescent probe, can specifically target solid tumors and has a long retention time at the tumor site, with an imaging time of up to 72 hours. Based on the first objective, the second objective of this invention is to provide a small molecule near-infrared fluorescent probe that targets tumors and can remain at the tumor site for a long time, which can form a relatively clear fluorescence contrast between tumor tissue and surrounding normal tissue during fluorescence imaging. Based on the first and second objectives, the third objective of this invention is to use osimertinib-based small molecule compounds as small molecule near-infrared fluorescent probes for fluorescence imaging of tumors, which can provide a longer imaging time window for tumor localization, tumor boundary identification, and fluorescence-guided tumor resection.

[0008] 2. Technical Solution Based on the above objectives, the technical solution adopted by the present invention is as follows: [1. Compound] The first aspect of this invention provides a class of compounds or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers, optical isomers, deuterated derivatives or prodrugs or mixtures thereof, wherein the compounds have a structural formula selected from formula (I):

[0009] (I) In formula (Ⅰ), X is a linking group and Y is a near-infrared fluorescent dye molecule group; X is selected from none, -NH (CH2CH2O)a CH2CH2CO- or -NH(CH2) b Any one of CO-, wherein a is 1 to 6, preferably 3 to 5, most preferably 4, and b is 2 to 6, preferably 2 to 5.

[0010] The compounds provided by this invention, or their pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers, optical isomers, deuterated derivatives, or prodrugs or mixtures thereof, wherein the osimertinib-derived group is coupled to a near-infrared fluorescent dye via a linker group, can serve as small molecule fluorescent probes possessing both tumor-targeting recognition capabilities and near-infrared fluorescence imaging performance. Specifically, the osimertinib-derived group can act as an EGFR-targeting recognition unit, while the near-infrared fluorescent dye provides the fluorescence signal, which is beneficial for improving the probe's targeted enrichment ability in tumor tissues and overcoming the problem of insufficient specificity in traditional non-targeting fluorescent dyes.

[0011] As a preferred embodiment of any of the first aspects of the present invention, Y has a structural formula selected from formula (II):

[0012] (II) In equation (II), R1 is -(CH2). m SO3H, where m is 3 or 4; R2 is -(CH2) n SO3H, where n is 3 or 4; Z is or ; in, The linking site of the representative group.

[0013] As a preferred embodiment of any of the first aspects of the present invention, X is selected from -NH(CH2CH2O). a CH2CH2CO- or -NH(CH2) b When CO-, X and Y are connected by an amide bond, and the N bonded to X is also connected to X by an amide bond.

[0014] As a preferred embodiment of any of the first aspects of the present invention, the compound is selected from any of the following structures:

[0015] NY-EGFR-T-111;

[0016] NY-EGFR-T-112;

[0017] NY-EGFR-T-113;

[0018] NY-EGFR-T-114;

[0019] NY-EGFR-T-115;

[0020] NY-EGFR-T-116.

[0021] As a preferred embodiment of any of the first aspects of the present invention, the pharmaceutically acceptable salt is a sodium salt or an ammonium salt.

[0022] [2. Pharmaceutical Composition] A second aspect of the present invention provides a pharmaceutical composition comprising a compound provided in the first aspect of the present invention or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or a prodrug or mixture thereof, and a pharmaceutically acceptable carrier.

[0023] As a preferred embodiment of any of the second aspects of the present invention, a pharmaceutically acceptable carrier includes physiological saline.

[0024] [3. Near-infrared fluorescent probe] A third aspect of the present invention provides a near-infrared fluorescent probe comprising a compound provided in the first aspect of the present invention or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or a prodrug or a mixture thereof, or comprising a pharmaceutical composition provided in the second aspect of the present invention.

[0025] [4. Applications of Fluorescent Contrast Agents] The fourth aspect of the present invention provides the use of the near-infrared fluorescent probe provided in the third aspect of the present invention in the preparation of fluorescent contrast agents.

[0026] As a preferred embodiment of any of the fourth aspects of the present invention, the fluorescent contrast agent is a tumor fluorescent contrast agent.

[0027] [5. Applications in the preparation of tumor diagnostic drugs] The fifth aspect of the present invention provides the use of the near-infrared fluorescent probe provided in the third aspect of the present invention in the preparation of tumor diagnostic drugs.

[0028] As a preferred embodiment of any fifth aspect of the present invention, the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma, colorectal cancer, ovarian cancer, and pancreatic cancer.

[0029] Furthermore, the near-infrared fluorescent probe comprises NY-EGFR-T-111 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or its prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, colorectal cancer and ovarian cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-112 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof; the tumor is selected from one or more of hepatocellular carcinoma, non-small cell lung cancer, lung adenocarcinoma and colorectal cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-113 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or its prodrug or mixture thereof; the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma, ovarian cancer and pancreatic cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-114 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of hepatocellular carcinoma, non-small cell lung cancer and lung adenocarcinoma; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-115 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or both of non-small cell lung cancer and ovarian cancer; and / or The near-infrared fluorescent probe contains NY-EGFR-T-116 or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or its prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma, ovarian cancer, colorectal cancer and pancreatic cancer.

[0030] [6. Use as a reagent for image guidance] The sixth aspect of the present invention provides the use of the compound provided in the first aspect of the present invention or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated product or a prodrug or a mixture thereof, or the pharmaceutical composition provided in the second aspect of the present invention, in the preparation of an image-guided agent for administering surgery to a subject suffering from a disease, wherein the agent is used to visualize the designated surgical site by excitation light irradiation, and the disease is cancer.

[0031] [7. Reagent Kit] The seventh aspect of the present invention provides a kit comprising a compound provided in the first aspect of the present invention or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated compound or a prodrug or a mixture thereof or a pharmaceutical composition provided in the second aspect of the present invention, and instructions for use in diagnosing a disease.

[0032] [8. Preparation method] The eighth aspect of the present invention provides a method for preparing compounds provided in the first aspect of the present invention, or pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers, optical isomers, deuterated derivatives, or prodrugs thereof, or mixtures thereof, comprising the following steps: The target molecule undergoes a condensation reaction with an arbitrarily selected linker molecule to obtain an intermediate; The intermediate or target molecule undergoes a condensation reaction with the dye molecule to obtain the target compound; The structure of the targeting group in the targeting molecule is shown below: ; The linking group in the linker molecule is selected from -NH(CH2CH2O). a CH2CH2CO- and -NH(CH2) b Any one of CO-, where a is 1~6 and b is 2~6; The structure of the linkage group in the dye molecule is shown in formula (II):

[0033] (II) In equation (II), R1 is -(CH2). m SO3H, where m is 3 or 4; R2 is -(CH2) n SO3H, where n is 3 or 4; Z is or , in, The linking site of the representative group.

[0034] It should be noted that "any choice" means that you can choose to choose or not to choose.

[0035] 3. Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The present invention provides a near-infrared region fluorescence emission spectrum of a compound based on osimertinib or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated compound or a prodrug or a mixture thereof, which makes such compound or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated compound or a prodrug or a mixture thereof available as a near-infrared fluorescent probe.

[0036] Specifically, the fluorescence emission spectra of NY-EGFR-T-111 to NY-EGFR-T-116 show that the maximum emission wavelength of NY-EGFR-T-111 to NY-EGFR-T-116 is located in the near-infrared region of 800nm ​​to 820nm, and can be used as near-infrared fluorescent probes.

[0037] (2) The compounds provided by this invention, or their pharmaceutically acceptable salts, esters, hydrates, solvates, tautomers, optical isomers, deuterated derivatives, or prodrugs or mixtures thereof, wherein the osimertinib derivative group is coupled to a near-infrared fluorescent dye via a linker group, can serve as small molecule fluorescent probes possessing both tumor-targeting recognition capabilities and near-infrared fluorescence imaging performance. The osimertinib derivative group can act as an EGFR-targeting recognition unit, which is beneficial for improving the probe's targeting enrichment ability for tumor tissues and its long-term imaging capability, thus addressing the problem of insufficient specificity in traditional non-targeting fluorescent dyes.

[0038] Moreover, small molecule fluorescent probes have significant advantages over large molecule fluorescent probes such as antibodies and proteins in terms of synthesis, quality control and immunogenicity, especially in terms of strong controllability of synthesis and lack of immunogenicity.

[0039] (3) The small-molecule near-infrared fluorescent probes provided by this invention have good tumor targeting enrichment and long-term imaging capabilities. Animal in vivo imaging results show that the small-molecule near-infrared fluorescent probes provided by this invention can generate strong fluorescence signals in the tumor area; 24 h after administration, the fluorescence signal in normal tissue has significantly weakened, while the tumor area still maintains a significant fluorescence signal, and the imaging time can reach 72 h. Some small-molecule near-infrared fluorescent probes, such as NY-EGFR-T-116, can achieve the above effects after 12 h of administration. Thus, a relatively clear fluorescence contrast between tumor tissue and surrounding normal tissue can be formed, providing a longer imaging time window for tumor localization, tumor boundary identification, and fluorescence-guided tumor resection.

[0040] (4) The near-infrared fluorescent probe provided by the present invention can not only be used as a fluorescent contrast agent and tumor diagnostic reagent, but also shows broad application prospects in the field of clinical surgery, especially in fluorescence-guided tumor resection surgery.

[0041] Specifically, on the one hand, in existing technologies, osimertinib's mechanism of action involves specifically binding to exon 19-deleted EGFR and L858R / T790M mutant EGFR, inhibiting related signaling pathways, and thereby inducing programmed cell death in tumor cells. This invention, based on the targeted anti-tumor drug osimertinib, can actively target epidermal growth factor receptors that are highly or abnormally expressed in solid tumors, possessing specific targeting capabilities. On the other hand, near-infrared fluorescent probes cleared by the kidneys are rapidly eliminated from normal tissues, while exhibiting high affinity for tumor tissues and remaining at the tumor site for an extended period. This enhances the contrast between the tumor and surrounding tissues, reduces interference from non-tumor tissue fluorescence signals on imaging results, significantly lowers the misjudgment rate, and helps surgeons more accurately define the tumor extent during surgery, ensuring complete removal of lesions, thereby optimizing patient treatment prognosis and quality of life. On the other hand, the novel near-infrared fluorescent probe constructed by this invention, which is originally insoluble in water, has good aqueous dispersion or solubility properties. It can use physiological saline as the drug delivery solvent, making drug delivery convenient. It improves the formulation and drug delivery limitations caused by the poor water solubility of osimertinib, making it easier to prepare injectable formulations and administer intravenously. This is beneficial for its application in fields such as fluorescence imaging, tumor diagnosis, and intraoperative fluorescence navigation.

[0042] In summary, the innovative near-infrared fluorescent probe obtained by the molecular design of this invention not only expands the functional boundaries of drugs, but also provides a precise real-time navigation tool for tumor surgery, significantly improving the accuracy of tumor resection and treatment efficacy, and is of great significance for the development of drugs with precise anti-cancer properties.

[0043] (5) In the preparation method of the near-infrared fluorescent probe provided by the present invention, chemical coupling technology is used to carry out a condensation reaction under the action of condensation reagent, so that the amino group in the osimertinib derivative reacts with the carboxyl group in the dye molecule or the linking group to form a structurally stable amide bond, and a novel small molecule near-infrared fluorescent probe is successfully constructed.

[0044] This preparation method can prepare different types of probes by changing the structure of the linking group and the near-infrared fluorescent dye, providing a structural basis for regulating the probe's optical properties, targeting enrichment properties and in vivo metabolic properties, and has good designability and scalability. Attached Figure Description

[0045] Figure 1 The ultraviolet absorption spectra of NY-EGFR-T-111 to NY-EGFR-T-116 in Test Example 1; Figure 2 The fluorescence spectra of NY-EGFR-T-111 to NY-EGFR-T-116 in Test Example 1; Figure 3The images show in vivo images of NY-EGFR-T-111~NY-EGFR-T-116 and ICG in the HCC827 subcutaneous tumor-bearing mouse model in test example 2. Figure 4 This is an in vivo imaging image of NY-EGFR-T-111~NY-EGFR-T-116 in the H1975 subcutaneous tumor-bearing mouse model from test example 3; Figure 5 The images show in vivo images of NY-EGFR-T-111~NY-EGFR-T-116 and ICG in the subcutaneous tumor SKOV3 mouse model, as shown in Test Example 4. Figure 6 The images show in vivo images of NY-EGFR-T-112~NY-EGFR-T-114 and NY-EGFR-T-116 in the subcutaneous tumor HepG2 mouse model, as shown in Test Example 5. Figure 7 In vivo imaging of NY-EGFR-T-112, NY-EGFR-T-113 and NY-EGFR-T-116 in the subcutaneous tumor AsPC-1 tumor-bearing mouse model in test example 6; Figure 8 In vivo imaging of NY-EGFR-T-111, NY-EGFR-T-112 and NY-EGFR-T-116 in the HuH-7 subcutaneous tumor-bearing mouse model in test example 7; Figure 9 In vivo imaging of NY-EGFR-T-111, NY-EGFR-T-112 and NY-EGFR-T-116 in the subcutaneous tumor HCT116-bearing mouse model in Test Example 8. Detailed Implementation

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0047] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0048] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0049] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0050] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0051] It should be noted that the criteria for confirming the completion of a reaction using thin-layer chromatography (TLC) are as follows: The reaction is considered complete if the compound with the smallest molar amount in the reaction has been completely consumed by the reaction. If the TLC test shows that the compound with the smallest molar amount in the reaction is absent, the reaction is considered complete. Similarly, the criteria for confirming the completion of a reaction using high-performance liquid chromatography (HPLC) are as follows: The reaction is considered complete if the peak of the compound with the smallest molar amount in the reaction has disappeared by the HPLC test.

[0052] It should be noted that the subcutaneous tumor HCC827 tumor-bearing mouse model was developed by inoculating human non-small cell lung cancer cells (HCC827) into the axillae of Balb / c nude mice, and waiting for the tumor volume to grow to 100 mm. 3 Testing was conducted at the appropriate time. Similarly, subcutaneous tumor-bearing mouse models H1975, SKOV3, HepG2, AsPC-1, HuH-7, and HCT116 were obtained by inoculating and culturing human non-small cell lung cancer (H1975), human ovarian cancer cells (SKOV3), human liver cancer cells (HepG2), human pancreatic cancer cell line (AsPC-1), human liver cancer cell line (HuH-7), and colorectal cancer cells (HCT116), respectively. Balb / cnude mice weighing 20±2g were selected for inoculation.

[0053] The present invention will be further described below with reference to specific embodiments.

[0054] Preparation materials N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1-methylindazole-3-yl)pyrimidin-2-amine (AZD9291 intermediate 2, CAS No.: 1421372-94-2), Boc-6-aminohexanoic acid were purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd., and N-Boc-N,N'-dimethylethylenediamine, 2-(7-azabenzotriazole)-N,N,N', N'-Tetramethylurea hexafluorophosphate (HATU) and Boc-β-alanine were purchased from Jiangsu Aikon Biomedical R&D Co., Ltd.; N,N-diisopropylethylamine (DIPEA) was purchased from Shanghai Mairui Chemical Technology Co., Ltd.; dimethyl sulfoxide (DMSO) was purchased from Tianjin Xins Biochemical Technology Co., Ltd.; reduced iron powder (item number: K100014) and ammonium chloride were purchased from Shanghai Dipo Chemical Technology Co., Ltd.; anhydrous ethanol was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; tetrahydrofuran (THF) was purchased from Sinopharm Chemical Reagent Co., Ltd.; 3-chloropropionyl chloride was purchased from Beijing Inokai Technology Co., Ltd.; sodium hydroxide was purchased from Shanghai Titan Technology Co., Ltd.; trifluoroacetic acid (TFA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and Boc-NH-PEG4-CH2CH2COOH was purchased from Shanghai Adamas Reagent Co., Ltd.

[0055] Methanol, diatomaceous earth, ethyl acetate, and dichloromethane (DCM) are all commercially available.

[0056] It should be noted that the preparation method of S0456-Der-01 includes the following steps: S0456 (CAS No.: 1252007-83-2, 200 mg, 1 eq), potassium 4-hydroxyphenylpropionate (66 mg, 1.2 eq), N,N-diisopropylethylamine (59 mg, 2 eq), and DMSO (2 mL) are mixed and stirred, reacted at room temperature for 1 h, and the reaction is monitored by liquid chromatography (HPLC). After the reaction is complete, the mixture is purified by liquid chromatography to obtain the target fraction solution, which is then lyophilized to obtain a green solid (205 mg), denoted as S0456-Der-01.

[0057] The preparation method of S0456-Der-02 is basically the same as that of S0456-Der-01, the only difference being that 3-mercaptopropionic acid is used instead of potassium 4-hydroxyphenylpropionate. Its structural formula is as follows: .

[0058] Preparation Example 1 This preparation example provides a near-infrared fluorescent probe, the preparation method of which includes the following steps: 1. 2 g of compound 1 (N-(4-fluoro-2-methoxy-5-nitrophenyl)-4-(1-methylindazole-3-yl)pyrimidin-2-amine, AZD9291 intermediate 2) (1.0 eq), 1.052 g of compound 2 (N-Boc-N,N'-dimethylethylenediamine) (1.1 eq), 1.33 mL of N,N-diisopropylethylamine (DIPEA) (1.5 eq), and 4 mL of dimethyl sulfoxide (DMSO) (11 eq) were mixed and stirred and reacted at 85 °C for 2 hours. The reaction was confirmed to be complete by thin-layer chromatography (TLC). The reaction solution was cooled to room temperature, 20 mL of ice water was added, and the mixture was filtered, washed with water, and dried to obtain a red solid, which was designated as intermediate a.

[0059] 2. Weigh 500 mg of intermediate a (1.0 eq), 497.73 mg of reduced iron powder (10 eq), and 524.5 mg of ammonium chloride (NH4Cl, 11 eq). Measure 4 mL of anhydrous ethanol and 1 mL of water. Mix and reflux at 85 °C for 5 hours under nitrogen protection. Confirm the reaction is complete using thin-layer chromatography (TLC). Add 5 mL of methanol to the reaction solution for dilution. Filter with diatomaceous earth and evaporate the filtrate by rotary evaporation to obtain a light brown compound, denoted as intermediate b.

[0060] 3. Dissolve 300 mg of intermediate b (1.0 eq) in a mixed solvent of 2.5 mL tetrahydrofuran (THF) and 0.25 mL water. Add 85.4 mg of compound 3 (3-chloropropionyl chloride, 1.2 eq) dropwise in an ice bath (0 °C). After the addition is complete, gradually raise the temperature to room temperature and react for 1 hour. Then add 67.8 mg of sodium hydroxide (3 eq). After the addition is complete, raise the temperature to 60 °C and react for 3 hours. Confirm the reaction is complete using thin-layer chromatography (TLC) to obtain a reaction solution containing intermediate c. Add the reaction solution dropwise to 10 mL of water to form a suspension. Add 10 mL of ethyl acetate for extraction. After concentrating the organic layer, add 1 mL of trifluoroacetic acid (TFA) and react at room temperature for 30 min. Monitor and confirm the reaction is complete using thin-layer chromatography (TLC). Evaporate the reaction solution to dryness and then purify it by column chromatography to obtain intermediate d.

[0061] 4. Mix 50 mg of intermediate d (1.0 eq), 125.69 mg of SO456-Der-01 (1.2 eq), 58.76 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.5 eq), 23.94 mg of N,N-diisopropylethylamine (1.8 eq), and 1 mL of DMSO (150 eq) and stir. React at room temperature in the dark for 30 min. Monitor the reaction using high-performance liquid chromatography (HPLC) and confirm its completion. Purify the target fraction solution by preparative liquid chromatography. After lyophilization, obtain a near-infrared fluorescent probe that is a green solid, denoted as NY-EGFR-T-111.

[0062] The specific synthesis route is as follows:

[0063] 5. The structure of NY-EGFR-T-111 was characterized by mass spectrometry, hydrogen NMR, and carbon NMR. The specific characterization results are as follows: [M-2H] 2- / 2=740.75; 1 H NMR (600 MHz, DMSO- d 6) δ 8.80 (s, 1H), 8.25–7.94 (m, 3H), 7.75 (dd, J =25.6, 13.8 Hz, 2H), 7.64–7.54 (m, 5H), 7.44 (dd, J = 15.4, 6.4 Hz, 1H), 7.37–7.08 (m, 8H), 7.06–6.84 (m, 3H), 6.63 (ddd, J = 17.2, 10.4, 6.6 Hz, 1H), 6.25–6.13 (m, 3H), 5.73 (dd, J = 17.7, 10.5 Hz, 2H), 4.11 (s, 4H), 3.94–3.80 (m,5H), 3.64 (s, 1H), 3.41–3.15 (m, 5H), 2.92 (s, 2H), 2.78–2.64 (m, 9H), 2.60(q, J = 7.5,7.0 Hz, 4H), 2.46–2.37 (m, 2H), 2.00–1.61 (m, 11H), 1.29-1.11 (m,13H); 13 C NMR (151 MHz, DMSO-d6) δ 171.92, 171.61, 163.10, 158.84, 158.59,158.33,158.08,144.85, 142.22, 140.96, 140.37, 138.29, 135.53, 130.54, 126.23,125.40, 123.54,122.85,122.15, 119.62, 117.97, 116.06, 114.41, 114.15,111.62,111.33,110.51,100.69,56.44,56.17,50.71, 50.69, 48.48, 48.44, 43.62, 35.28,34.75, 33.76, 33.71, 29.44, 27.14, 27.08,25.90,23.81, 22.31, 20.81.

[0064] Preparation Example 2 This preparation example provides a near-infrared fluorescent probe, the preparation method of which includes the following steps: 1. Mix 2 g of compound 1 (AZD9291 intermediate 2) (1.0 eq), 1.052 g of compound 2 (1.1 eq), 1.33 mL of DIPEA (1.5 eq), and 4 mL of DMSO (11 eq) and stir. React at 85 °C for 2 hours. After confirming the completion of the reaction by thin-layer chromatography (TLC), cool the reaction solution to room temperature, add 20 mL of ice water, filter, wash with water, and dry to obtain a red solid.

[0065] 2. Weigh 500 mg of the red solid from step 1 above and add 5 mL of dichloromethane (DCM). Then add 200 mg of TFA for Boc deprotection. React at 25-30℃ for 30 min. After confirming the reaction is complete by thin-layer chromatography (TLC), add sodium hydroxide solution to adjust the pH to 7-8. Then extract with DCM, combine the organic phases, dry with anhydrous sodium sulfate and concentrate to obtain intermediate e.

[0066] 3. Mix 700 mg of intermediate e (1.0 eq), 665 mg of Boc-NH-PEG4-CH2CH2COOH (1.2 eq), 1.154 g of HATU (2 eq), 528.9 μL of DIPEA (2 eq), and 2 mL of DMSO (19 eq) and stir. React at room temperature for 1 hour. Monitor and confirm the completion of the reaction using thin-layer chromatography (TLC). Add the reaction solution dropwise to 15 mL of water to form a suspension. Extract with ethyl acetate, concentrate the organic layer, and purify by column chromatography to obtain intermediate f.

[0067] 4. Weigh 500 mg of intermediate f (1.0 eq), 346.53 mg of reduced iron powder (10 eq), and 364.17 mg of ammonium chloride (11 eq), and measure 4 mL of anhydrous ethanol and 1 mL of water. Mix and reflux at 85 °C for 5 hours under nitrogen protection. Confirm the reaction is complete by thin-layer chromatography (TLC). Add 10 mL of methanol to the reaction solution to dilute it. Filter with diatomaceous earth and evaporate the filtrate to dryness to obtain a light brown compound, which is denoted as intermediate h.

[0068] 5. Dissolve 600 mg of intermediate h (1.0 eq) in a mixed solvent of 5 mL THF and 0.5 mL water, and add 116.6 mg of compound 3 (3-chloropropionyl chloride, 1.2 eq) dropwise in an ice bath (0 °C). After the addition is complete, gradually raise the temperature to room temperature and react for 1 hour. Then add 92.5 mg of sodium hydroxide (3 eq). After the addition is complete, raise the temperature to 60 °C and react for 3 hours. Use thin-layer chromatography (TLC) to confirm that the reaction is complete and obtain a reaction solution containing intermediate i. Add the reaction solution dropwise to 10 mL of water to form a suspension, then extract with DCM. After concentrating the organic layer, add 1 mL of TFA to remove the Boc protecting group. React at 25-30 °C for 30 min. Use thin-layer chromatography (TLC) to monitor and confirm that the reaction is complete. Evaporate the reaction solution to dryness and then purify it by column chromatography to obtain intermediate j.

[0069] 6. Mix 80 mg of intermediate j (1.0 eq), 133.24 mg of SO456-Der-01 (1.2 eq), 62.3 mg of HATU (1.5 eq), 22.33 mg of DIPEA (1.8 eq), and 1.16 mL of DMSO and stir. React at room temperature in the dark for 30 min. Monitor the reaction using high-performance liquid chromatography (HPLC) and confirm the completion of the reaction. Purify the target fraction solution by preparative high-performance liquid chromatography. After lyophilization, obtain a near-infrared fluorescent probe in the form of a green solid, denoted as NY-EGFR-T-112.

[0070] The specific synthesis route is as follows:

[0071] 7. The structure of NY-EGFR-T-112 was characterized by mass spectrometry, hydrogen NMR, and carbon NMR. The specific characterization results are as follows: [M-2H] 2- / 2=864; 1 H NMR (600 MHz, DMSO- d 6) δ 8.80 (d, J = 8.6 Hz, 1H), 8.16 (s, 4H), 7.87–7.82 (m, 1H), 7.75 (d, J = 13.9 Hz, 3H), 7.62–7.51 (m, 5H), 7.41 (d, J = 6.9 Hz, 1H), 7.28 (t, J = 8.6 Hz, 3H), 7.20–7.09 (m, 4H), 6.99 (d, J = 8.2 Hz, 2H), 6.67–6.59 (m, 2H), 6.21–6.13 (m, 4H), 5.70 (t, J = 10.9 Hz, 2H), 4.07 (t, J = 7.1 Hz,7H), 3.57–3.51 (m, 4H), 3.48–3.35 (m, 14H), 3.31–3.23 (m, 4H), 3.08 (dq, J =6.0, 3.8 Hz, 2H), 2.87 (s, 5H), 2.78–2.60 (m, 8H), 2.60–2.49 (m, 5H), 2.22(t, J = 7.9 Hz, 2H), 1.87 (dt, J = 13.2, 6.5 Hz, 3H), 1.76–1.64 (m, 8H), 1.25–1.16 (m, 12H); 13C NMR (151 MHz, DMSO-d6) δ 171.57, 171.34, 163.10, 158.55, 158.29,158.05, 145.01, 142.14, 140.91, 140.31, 138.27, 135.41, 130.13, 126.23,125.44, 123.51, 122.74, 122.11, 119.66, 114.32 (d, J = 38.6 Hz), 111.62,110.47, 100.65, 69.78 (d, J = 9.4 Hz), 69.54 (d, J = 4.2 Hz), 69.07, 66.93,66.66, 50.69, 48.52, 43.62, 38.53, 37.26, 35.38, 33.73, 33.18, 32.41, 30.26,27.15, 25.91, 23.78, 22.38, 20.79.

[0072] Preparation Example 3 This preparation example provides a near-infrared fluorescent probe, which is prepared in basically the same way as Preparation Example 2, except that Boc-β-alanine is used instead of Boc-NH-PEG4-CH2CH2COOH in Preparation Example 2 to obtain the near-infrared fluorescent probe, denoted as NY-EGFR-T-113, with the following specific structural formula:

[0073] The structure of NY-EGFR-T-113 was characterized by mass spectrometry and proton NMR and carbon NMR spectrometry. The specific characterization results are as follows: [M-2H] 2- / 2 = 776.3; 1H NMR (600 MHz, DMSO-d6) δ 8.82 (d, J = 9.1 Hz, 1H), 8.34–7.99 (m,3H), 7.93–7.70 (m, 4H), 7.66–7.53 (m, 5H), 7.45 (dd, J = 17.5, 6.9 Hz, 2H),7.37–7.07 (m, 7H), 7.01 (t, J = 9.0 Hz, 2H), 6.63-6.57 (m, 2H), 6.26-6.15 (m,3H), 5.74 (dd, J = 23.7, 10.5 Hz, 2H), 4.10 (d, J = 7.3 Hz, 7H), 3.49–3.09(m, 8H), 3.00 (s, 2H), 2.78 (d, J = 16.5 Hz, 4H), 2.73–2.64 (m, 5H), 2.63–2.52 (m, 5H), 2.38 (tt, J = 6.2, 3.4 Hz, 1H), 2.29–2.18 (m, 4H), 2.05–1.55(m, 11H), 1.27-1.17 (m, 12H); 13 C NMR (151 MHz, DMSO-d6) δ 171.44, 171.12, 157.91, 144.71, 142.10,140.78, 140.22, 139.00, 138.13, 130.07, 122.71, 122.00, 119.54, 114.31,100.53, 50.55, 48.37, 43.48, 33.61 (d, J = 4.5 Hz), 33.01, 32.65, 30.12,27.01 (d, J = 5.0 Hz), 25.81, 22.23 (d, J = 7.6 Hz).

[0074] Preparation Example 4 This preparation example provides a near-infrared fluorescent probe, which is prepared in basically the same way as Preparation Example 2, except that Boc-6-aminohexanoic acid is used instead of Boc-NH-PEG4-CH2CH2COOH in Preparation Example 2 to obtain the near-infrared fluorescent probe, denoted as NY-EGFR-T-114, with the following specific structural formula:

[0075] The structure of NY-EGFR-T-114 was characterized by mass spectrometry and proton NMR and carbon NMR spectrometry. The specific characterization results are as follows: [M-2H] 2- / 2 = 797.3; 1 H NMR (600 MHz, DMSO- d 6) δ 8.82 (d, J = 8.4 Hz, 1H), 8.20 (s, 3H), 7.84–7.70 (m, 3H), 7.67–7.56 (m, 4H), 7.44 (t, J = 6.4 Hz, 1H), 7.37–7.11 (m, 6H), 7.01 (dd, J = 8.5, 4.5 Hz, 2H), 6.67-6.61 (m, 1H), 6.21 (dd, J = 15.4, 10.2 Hz, 3H), 5.73 (t, J = 12.2 Hz, 1H), 4.10 (t, J = 7.0 Hz, 5H), 3.92 (d, J = 2.5 Hz, 8H), 3.84 (m, 13H), 3.45 (q, J = 9.9 Hz, 3H), 3.00–2.82 (m, 6H), 2.79–2.65 (m, 6H), 2.62–2.53 (m, 4H), 2.27–2.09 (m, 4H), 1.95–1.86 (m, 2H), 1.76–1.67 (m, J =14.6, 5.5 Hz, 6H), 1.41–1.30 (m, 2H), 1.24 (d, J = 7.8 Hz, 10H), 1.18–1.05 (m,2H); 13C NMR (151 MHz, DMSO-d6) δ 171.27 (d, J = 86.1 Hz), 158.16 (d, J =39.2 Hz), 144.99, 142.14, 140.31, 138.26, 130.17, 126.22, 122.13, 119.65,114.42, 110.46, 100.65, 50.68, 48.51, 33.74, 27.16 (d, J = 2.2 Hz), 23.79,22.37.

[0076] Preparation Example 5 This preparation example provides a near-infrared fluorescent probe, which is prepared in basically the same way as Preparation Example 1, except that S0456-Der-01 in Preparation Example 1 is replaced with S0456-Der-02 in the reaction to obtain the near-infrared fluorescent probe, denoted as NY-EGFR-T-115, with the following specific structural formula:

[0077] The structure of NY-EGFR-T-115 was characterized by mass spectrometry and proton NMR and carbon NMR spectroscopy. The specific characterization results are as follows: [M-2H] 2- / 2 = 696.7; 1 H NMR (600 MHz, DMSO- d 6) δ 8.81 (d, J = 15.7 Hz, 1H), 8.65 (d, J = 12.3 Hz, 3H), 8.56 (d, J = 11.8 Hz, 3H), 8.13 (s, 4H), 7.75 (dd, J = 10.9, 1.5 Hz, 2H),7.70–7.54 (m, 3H), 7.51–7.36 (m, 3H), 7.34–7.09 (m, 3H), 6.62 (dd, J = 17.1, 10.4 Hz, 1H), 6.47 (t, J = 14.0 Hz, 2H), 6.30–6.09 (m, 2H), 5.74 (d, J = 10.8 Hz, 2H), 5.67 (d, J= 7.2 Hz, 2H), 4.00–3.73 (m, 8H), 3.60 (pd, J = 6.6, 3.9 Hz, 2H),3.45–3.22 (m, 4H), 3.16–3.09 (m, 1H), 3.08–2.83 (m, 5H), 2.83–2.58 (m, 9H),2.12–1.88 (m, 4H), 1.62 (d, J = 3.6 Hz, 12H), 1.24 (dd, J = 9.4, 6.7 Hz, 6H). 13 C NMR (151 MHz, DMSO-d6) δ 171.95, 170.26, 158.31, 145.11, 144.97,144.87, 142.35, 142.30, 140.43, 140.36, 138.26, 134.10, 126.24, 125.34,123.51, 122.90, 119.82, 111.57, 110.31, 102.09, 53.64, 48.73, 48.70, 47.88,42.73, 41.91, 35.14, 33.76, 33.52, 33.43, 27.38, 27.33, 25.83, 23.28, 20.63, 18.11, 16.76, 12.56.

[0078] Preparation Example 6 This preparation example provides a near-infrared fluorescent probe, which is prepared in basically the same way as Preparation Example 2, except that S0456-Der-01 in Preparation Example 2 is replaced with S0456-Der-02 in the reaction to obtain the near-infrared fluorescent probe, denoted as NY-EGFR-T-116, with the following specific structural formula:

[0079] The structure of NY-EGFR-T-116 was characterized by mass spectrometry and proton NMR and carbon NMR spectroscopy. The specific characterization results are as follows: [M-2H] 2- / 2 = 820.3; 1 H NMR (600 MHz, DMSO- d 6) δ 8.82 (d, J = 7.6 Hz, 1H), 8.67 (d, J= 13.9 Hz,2H), 8.22 (s, 2H), 8.06–7.96 (m, 2H), 7.76 (d, J = 1.6 Hz, 2H), 7.67–7.55 (m,3H), 7.41 (d, J = 7.6 Hz, 3H), 7.35–7.12 (m, 4H), 6.63 (ddd, J = 17.0, 10.0, 6.6Hz, 1H), 6.48 (d, J = 14.1 Hz, 2H), 6.23 (dd, J = 17.2, 8.8 Hz, 1H), 5.75 (t, J =11.7 Hz, 1H), 3.95–3.80 (m, 7H), 3.65–3.51 (m, 3H), 3.48–3.36 (m, 14H), 3.34(q, J = 6.1 Hz, 3H), 3.19–3.08 (m, 3H), 3.05–2.84 (m, 7H), 2.76 (s, 1H), 2.65(q, J = 6.3 Hz, 8H), 2.36 (q, J = 7.7 Hz, 3H), 2.02 (p, J = 6.9 Hz, 4H), 1.85–1.53(m, 15H), 1.24 (dd, J = 9.5, 6.7 Hz, 4H); 13C NMR (151 MHz, DMSO-d6) δ 172.01, 169.71, 158.58, 158.32, 155.34,144.95, 142.37, 140.45, 139.15, 138.28, 133.94, 126.23, 125.41, 123.55,122.84, 119.83, 116.05, 114.14, 111.62, 111.34, 110.35, 102.03, 69.81, 69.79,69.77, 69.75, 69.71, 69.60, 69.57, 69.04, 66.91, 66.60, 56.42, 53.65, 48.80, 47.89, 42.83, 41.92, 35.52, 35.43, 33.76, 33.23, 33.15, 32.84, 32.39, 27.43, 25.90, 23.31, 20.66, 18.12, 16.76, 12.56.

[0080] Test Example 1 The NY-EGFR-T-111 to NY-EGFR-T-116 series probes were prepared into 1 μmol / L aqueous solutions. The absorption spectra of each probe in the 500-900 nm range were first detected using a UV spectrophotometer (HITACHI, 3J1-0015). The results are as follows: Figure 1 As shown. From Figure 1 As can be seen, the maximum absorption wavelength of NY-EGFR-T-111, NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-114 is approximately 780 nm, while the maximum absorption wavelength of NY-EGFR-T-115 and NY-EGFR-T-116 is approximately 792 nm. This indicates that the NY-EGFR-T-111 to NY-EGFR-T-116 series probes exhibit the strongest fluorescence at their corresponding maximum absorption wavelengths and can be used for fluorescence imaging.

[0081] Then, using the maximum absorption wavelength of each probe as the excitation wavelength, a fluorescence spectrophotometer was used to detect the fluorescence emission spectrum of each probe in the range of 720-880 nm. The results are as follows: Figure 2 As shown. From Figure 2As can be seen, the maximum emission spectra of NY-EGFR-T-111, NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-114 are approximately 805 nm, while the maximum emission spectra of NY-EGFR-T-115 and NY-EGFR-T-116 are approximately 815 nm. This indicates that the NY-EGFR-T-111~NY-EGFR-T-116 series probes are near-infrared fluorescent probes with deep tissue penetration capability, extremely high detection signal-to-noise ratio, and low biological damage, making them suitable for in vivo imaging.

[0082] Test Example 2 Human non-small cell lung cancer subcutaneous tumor HCC827 tumor-bearing mice imaging This test case is used to evaluate the in vivo imaging performance of the NY-EGFR-T-111~NY-EGFR-T-116 series probes and ICG in the HCC827 subcutaneous tumor-bearing mouse model.

[0083] Six subcutaneous tumor-bearing mice (HCC827) were selected and administered NY-EGFR-T-111~116 series probes via tail vein at a dose of 10 nmol / mouse, with physiological saline as the solvent and an injection volume of 100 μL. One additional HCC827 subcutaneous tumor-bearing mouse was used as a control group and administered indocyanine green (ICG) via tail vein at a dose of 25 nmol / mouse, with physiological saline as the solvent and an injection volume of 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h. The fluorescence imaging results are shown below. Figure 3 As shown.

[0084] from Figure 3 It was found that in the HCC827 subcutaneous tumor-bearing mouse model, NY-EGFR-T-111~116 exhibited stronger tumor fluorescence signals in the axillary tumor region compared to ICG; they also demonstrated good tumor targeting ability, with fluorescence signals remaining almost exclusively in the tumor region 24 hours after administration, and imaging time exceeding 72 hours; NY-EGFR-T-114 showed a longer retention time in the liver after administration; NY-EGFR-T-111, NY-EGFR-T-112, NY-EGFR-T-113, NY-EGFR-T-115, and NY-EGFR-T-116 exhibited good in vivo metabolic characteristics (renal excretion), clearing rapidly from normal tissues and enabling clear contrast in fluorescence imaging between tumor and normal tissues. Therefore, the NY-EGFR-T-111~116 probes possess potential for lung cancer tumor imaging and hold promise for further development and application in clinical fluorescence-guided tumor surgical resection.

[0085] Test Example 3 Human lung adenocarcinoma subcutaneous tumor H1975 was visualized in tumor-bearing mice. This test case was used to evaluate the in vivo imaging performance of the NY-EGFR-T-111 to NY-EGFR-T-116 series probes in the H1975 subcutaneous tumor-bearing mouse model.

[0086] Six subcutaneous tumor-bearing mice (H1975 model) were selected and administered NY-EGFR-T-111~116 series probes via tail vein at a dose of 10 nmol / mouse. The solvent was physiological saline, and the injection volume was 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h. The fluorescence imaging results are shown below. Figure 4 As shown.

[0087] from Figure 4 It was found that in the H1975 subcutaneous tumor-bearing mouse model, NY-EGFR-T-111 and NY-EGFR-T-115 showed low tumor uptake, with very weak fluorescence signals in the tumor area. NY-EGFR-T-112 and NY-EGFR-T-113 showed the most significant tumor fluorescence signals, followed by NY-EGFR-T-114 and NY-EGFR-T-116. NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-114 showed significant uptake in the liver, while NY-EGFR-T-116 was cleared most rapidly in normal tissues. Approximately 12 hours after administration, only the tumor and bladder showed significant fluorescence signals, and the liver essentially metabolized it completely after 24 hours. Therefore, NY-EGFR-T-112~114 and NY-EGFR-T-116 probes have promising prospects for clinical development and application in lung cancer.

[0088] Test Example 4 Human ovarian cancer subcutaneous tumor SKOV3 tumor-bearing mice imaging This test case is used to evaluate the in vivo imaging performance of the NY-EGFR-T-111~NY-EGFR-T-116 series probes and ICG in a subcutaneous tumor-bearing mouse model of SKOV3.

[0089] Six SKOV3 subcutaneous tumor-bearing mice were selected, and NY-EGFR-T-111~116 series probes were administered via tail vein at a dose of 10 nmol / mouse, with physiological saline as the solvent and an injection volume of 100 μL. One additional SKOV3 subcutaneous tumor-bearing mouse was selected as the control group and administered indocyanine green (ICG) via tail vein at a dose of 25 nmol / mouse, with physiological saline as the solvent and an injection volume of 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h. The fluorescence imaging results are shown below. Figure 5 As shown.

[0090] from Figure 5 It was found that in the SKOV3 subcutaneous tumor-bearing mouse model, NY-EGFR-T-111~116 showed significant tumor fluorescence signals compared to ICG, exhibiting systemic fluorescence after administration. Over time, only the tumor site showed fluorescence. Therefore, the probes NY-EGFR-T-111~116 have potential for clinical development and application in ovarian cancer. Among them, NY-EGFR-T-111, NY-EGFR-T-113, NY-EGFR-T-115, and NY-EGFR-T-116 showed superior in vivo metabolic characteristics, while NY-EGFR-T-112 and NY-EGFR-T-114 showed some hepatic uptake and required a longer metabolic clearance time. Therefore, NY-EGFR-T-111, NY-EGFR-T-113, NY-EGFR-T-115, and NY-EGFR-T-116 have potential for further clinical development in the diagnosis and treatment of ovarian cancer.

[0091] Test Example 5 HepG2 subcutaneous tumor of human liver cancer in mice was visualized. This test case was used to evaluate the in vivo imaging performance of the NY-EGFR-T-112~114 and NY-EGFR-T-116 series probes in a subcutaneous tumor-bearing mouse model of HepG2.

[0092] Four subcutaneous tumor-bearing HepG2 mice were selected and administered NY-EGFR-T-112, NY-EGFR-T-113, NY-EGFR-T-114, and NY-EGFR-T-116 via tail vein, respectively. The dosage was 10 nmol / mouse, the solvent was physiological saline, and the injection volume was 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. The fluorescence imaging results are shown below. Figure 6 As shown.

[0093] from Figure 6 In the HepG2 subcutaneous tumor-bearing mouse model, probes NY-EGFR-T-112, NY-EGFR-T-113, NY-EGFR-T-114, and NY-EGFR-T-116 exhibited good tumor targeting ability, showing significant tumor uptake within 72 hours of administration, indicating potential for clinical development and application in liver cancer. Among them, NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-114 showed some hepatic uptake and a long metabolic time of up to 72 hours, while NY-EGFR-T-116 exhibited good in vivo metabolic characteristics, with fluorescence signals primarily remaining in the tumor after 48 hours. Therefore, probe NY-EGFR-T-116 shows promise for further clinical development in the diagnosis and treatment of liver cancer.

[0094] Test Example 6 AsPC-1 subcutaneous tumor of human pancreatic cancer was visualized in mice. This test case was used to evaluate the in vivo imaging performance of the NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-116 series probes in the AsPC-1 subcutaneous tumor-bearing mouse model.

[0095] Three subcutaneous tumor-bearing AsPC-1 mice were selected and administered NY-EGFR-T-112, NY-EGFR-T-113, and NY-EGFR-T-116 via tail vein, respectively. The dosage was 10 nmol / mouse, the solvent was physiological saline, and the injection volume was 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. The fluorescence imaging results are shown below. Figure 7 As shown.

[0096] from Figure 7It was found that in the AsPC-1 subcutaneous tumor-bearing mouse model, all three fluorescent probes exhibited strong tumor fluorescence signals, demonstrating potential pancreatic cancer tumor imaging capabilities. Among them, NY-EGFR-T-113 and NY-EGFR-T-116 showed good in vivo metabolic characteristics (renal excretion), rapid clearance from normal tissues, and clear contrast between tumor and normal tissue fluorescence imaging. They also demonstrated good tumor targeting ability; 24 hours after administration, only the tumor area showed fluorescence signals, and imaging time exceeded 72 hours. Therefore, probes NY-EGFR-T-113 and NY-EGFR-T-116 show promising clinical development and application prospects in the diagnosis and treatment of pancreatic cancer.

[0097] Test Example 7 Human liver cancer subcutaneous tumor HuH-7 tumor-bearing mice imaging This test case was used to evaluate the in vivo imaging performance of the NY-EGFR-T-111, NY-EGFR-T-112, and NY-EGFR-T-116 series probes in the HuH-7 subcutaneous tumor-bearing mouse model.

[0098] Three subcutaneous tumor-bearing HuH-7 mice were selected and administered NY-EGFR-T-111, NY-EGFR-T-112, and NY-EGFR-T-116 via tail vein, respectively. The dosage was 10 nmol / mouse, the solvent was physiological saline, and the injection volume was 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h. The fluorescence imaging results are shown below. Figure 8 As shown.

[0099] from Figure 8 It was found that in the HuH-7 subcutaneous tumor-bearing mouse model, all three fluorescent probes exhibited strong tumor fluorescence signals, demonstrating potential hepatocellular carcinoma imaging capabilities. Among them, NY-EGFR-T-111 and NY-EGFR-T-112 showed some hepatic uptake, while NY-EGFR-T-116 exhibited good in vivo metabolic characteristics (renal excretion), clearing rapidly from normal tissues and providing clear contrast in fluorescence imaging between tumor areas and normal tissues. It also demonstrated good tumor targeting ability; 24 hours after administration, virtually only the tumor showed fluorescence, with imaging time exceeding 72 hours. Therefore, the NY-EGFR-T-116 probe shows promising clinical development and application prospects in the diagnosis and treatment of hepatocellular carcinoma.

[0100] Test Example 8 Human colorectal cancer subcutaneous tumor HCT116 tumor-bearing mice imaging This test case is used to evaluate the in vivo imaging performance of the NY-EGFR-T-111, NY-EGFR-T-112, and NY-EGFR-T-116 series probes in the HCT116 subcutaneous tumor-bearing mouse model.

[0101] Three subcutaneous tumor-bearing mice (HCT116) were selected, and probes NY-EGFR-T-111, NY-EGFR-T-112, and NY-EGFR-T-116 were administered via tail vein at a dose of 10 nmol / mouse, using physiological saline as the solvent, with an injection volume of 100 μL. Fluorescence imaging was performed using a surgical fluorescence imaging system (Nanjing Nuoyuan Medical Instrument Co., Ltd., 10B, excitation wavelength 785 nm) after administration. The fluorescence imaging time points were 0 h (before administration), 6 h, 12 h, 24 h, 48 h, and 72 h, respectively. The fluorescence imaging results are shown below. Figure 9 As shown.

[0102] from Figure 9 It was found that in the HCT116 subcutaneous tumor-bearing mouse model, probes NY-EGFR-T-111, NY-EGFR-T-112, and NY-EGFR-T-116 exhibited good tumor-targeting capabilities. Among them, NY-EGFR-T-111 and NY-EGFR-T-112 showed some hepatic uptake, while NY-EGFR-T-116 demonstrated good in vivo metabolic characteristics (renal excretion), rapid clearance from normal tissues, and improved fluorescence imaging contrast between tumor and normal tissues. It also exhibited good tumor-targeting capabilities; 24 hours after administration, virtually only the tumor showed fluorescence, and imaging time exceeded 72 hours. Therefore, probe NY-EGFR-T-116 shows promise for further clinical application in fluorescence-guided tumor resection during colorectal cancer surgery.

[0103] The above description provides an illustrative overview of the present invention and its embodiments. This description is not restrictive, and the embodiments shown are merely one example of the invention's implementation. Actual implementations are not limited to these examples. Therefore, if those skilled in the art are inspired by this description and design similar implementations and examples without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

Claims

1. A compound or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or a prodrug or mixture thereof, said compound having a structural formula selected from formula (I): （Ⅰ） In formula (Ⅰ), X is a linking group and Y is a near-infrared fluorescent dye molecule group; The X is selected from none, -NH(CH2CH2O). a CH2CH2CO- or -NH(CH2) b Any of the following CO- groups, where a is 1 to 6 and b is 2 to 6.

2. The compound according to claim 1, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug or mixture thereof, characterized in that, The Y has a structural formula selected from formula (II): （Ⅱ） In equation (II), R1 is -(CH2). m SO3H, where m is 3 or 4; R2 is -(CH2) n SO3H, where n is 3 or 4; Z is or , in, The linking site of the representative group.

3. The compound according to claim 2, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug or mixture thereof, characterized in that, X is selected from -NH(CH2CH2O). a CH2CH2CO- or -NH(CH2) b When CO-, the connection between X and Y is an amide bond, and the connection between N and X is an amide bond.

4. The compound according to claim 2, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug or mixture thereof, characterized in that, The compound is selected from any of the following structures: NY-EGFR-T-111; NY-EGFR-T-112; NY-EGFR-T-113; NY-EGFR-T-114; NY-EGFR-T-115; NY-EGFR-T-116.

5. The compound according to any one of claims 1 to 4, or its pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug or mixture thereof, characterized in that, The pharmaceutically acceptable salt is a sodium salt or an ammonium salt.

6. A pharmaceutical composition comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, Pharmaceutically acceptable carriers include physiological saline.

8. A near-infrared fluorescent probe comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, or comprising the pharmaceutical composition of claim 6 or 7.

9. The use of the near-infrared fluorescent probe according to claim 8 in the preparation of fluorescent contrast agents.

10. The use of the near-infrared fluorescent probe according to claim 9 in the preparation of fluorescent contrast agents, characterized in that, The fluorescent contrast agent is a tumor fluorescent contrast agent.

11. Use of the near-infrared fluorescent probe of claim 8 in the preparation of tumor diagnostic drugs.

12. The use of the near-infrared fluorescent probe according to claim 11 in the preparation of tumor diagnostic drugs, characterized in that, The tumor is selected from one or more of the following: liver cancer, non-small cell lung cancer, lung adenocarcinoma, colorectal cancer, ovarian cancer, and pancreatic cancer.

13. The use of the near-infrared fluorescent probe according to claim 12 in the preparation of tumor diagnostic drugs, characterized in that, The near-infrared fluorescent probe comprises NY-EGFR-T-111 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, colorectal cancer and ovarian cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-112 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma and colorectal cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-113 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma, ovarian cancer and pancreatic cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-114 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of hepatocellular carcinoma, non-small cell lung cancer and lung adenocarcinoma; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-115 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or both of non-small cell lung cancer and ovarian cancer; and / or The near-infrared fluorescent probe comprises NY-EGFR-T-116 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative or prodrug or mixture thereof, and the tumor is selected from one or more of liver cancer, non-small cell lung cancer, lung adenocarcinoma, ovarian cancer, colorectal cancer and pancreatic cancer.

14. The use of the compound of any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug thereof, or a mixture thereof, or the pharmaceutical composition of claim 6 or 7, in the preparation of an image-guided agent for surgical administration to a subject suffering from a disease, wherein, The reagent is exposed to excitation light at a designated surgical site to induce visualization; the disease is cancer.

15. A kit comprising the compound of any one of claims 1 to 5 or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated compound or a prodrug or a mixture thereof or a pharmaceutical composition of claim 6 or 7, and instructions for use in diagnosing a disease.

16. A method for preparing the compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt, ester, hydrate, solvate, tautomer, optical isomer, deuterated derivative, or prodrug thereof, or a mixture thereof, characterized in that, Includes the following steps: The target molecule undergoes a condensation reaction with an arbitrarily selected linker molecule to obtain an intermediate; The intermediate or the target molecule undergoes a condensation reaction with the dye molecule to obtain the target compound; The structure of the targeting group in the targeting molecule is shown below: ； The linking group in the linker molecule is selected from -NH(CH2CH2O). a CH2CH2CO- and -NH(CH2) b Any one of CO-, where a is 1~6 and b is 2~6; The structure of the linkage group in the dye molecule is shown in formula (II): （Ⅱ） In equation (II), R1 is -(CH2). m SO3H, where m is 3 or 4; R2 is -(CH2) n SO3H, where n is 3 or 4; Z is or , in, The linking site of the representative group.

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