Application of a polypeptide TP-6 ​​in the preparation of tumor diagnostic and / or therapeutic reagents

Abstract

This invention relates to the fields of fluorescent contrast agents, radiopharmaceuticals, and nuclear medicine, specifically disclosing the application of a peptide TP-6 ​​in the preparation of tumor diagnostic and / or therapeutic reagents. The peptide TP-6 ​​is: D-Arg-L-Lys-L-Asp-L-Nva-D-Tyr, where D represents a non-natural D-type amino acid, L represents a natural L-type amino acid, and Nva is valine. The molecular probe constructed from the peptide TP-6 ​​of this invention can specifically target tumor sites and exhibits good uptake and retention capabilities at the tumor site, possessing a high target / non-target ratio. It is suitable for preparing intraoperative image-guided tumor diagnostic reagents and radiopharmaceuticals for tumor nuclear medicine diagnosis and precision radiotherapy.

Description

Technical Field

[0001] This invention is a divisional application of application No. 2021115423051, filed on December 16, 2021.

[0002] This invention relates to the fields of fluorescent contrast agents, radiopharmaceuticals, and nuclear medicine, specifically to the application of a polypeptide TP-6 ​​in the preparation of tumor diagnostic and / or therapeutic reagents. Background Technology

[0003] Malignant tumors have become the "number one killer" threatening human health and life. Undoubtedly, the diagnosis and effective treatment of malignant tumors are urgently needed. As is well known, tumor-targeting molecular imaging probes are powerful tools for tumor diagnosis, staging, and intraoperative navigation. Among these, tumor-specific ligands are key to tumor-targeting molecular probes. Current methods for designing and screening targeted ligands mainly include computer-aided drug design, lead compound modification, discovery from metabolites, discovery from drug synthesis intermediates, combinatorial chemistry and high-throughput screening, isolation and extraction from natural compounds, phage display library screening, and "drug repurposing." James Black, the 1988 Nobel laureate in Physiology or Medicine, proposed that the best path to new drug discovery begins with existing drugs. "Existing drugs" refer to marketed or clinically tested drugs with established pharmacokinetic and toxicological information, with high safety being their most prominent characteristic. The non-selective β-receptor antagonist propranolol and the first histamine H2 receptor antagonist cimetidine, developed by James Black, are typical examples of "drug repurposing." Propranolol is a classic drug for treating coronary heart disease and hypertension, and is now used to treat osteoporosis and melanoma; cimetidine is a revolutionary drug for treating peptic ulcers and has been proven to be suitable for treating chronic obstructive pulmonary disease, HIV infection, etc.; Nature pointed out that metformin, in combination with another "drug repurposing" drug, heme, can be used to treat triple-negative breast cancer; and arsenic trioxide, commonly known as "arsenic," is a highly toxic substance, but recent research has found that it can be used to treat acute promyelocytic leukemia. Therefore, the "drug repurposing" strategy has important guiding significance in drug development, and using this strategy to screen for targeted drugs for tumors is a rapid and effective method.

[0004] Thymopentin is an important bioactive substance in mammals, playing a crucial role in maintaining immune system balance and in anti-tumor and anti-microbial infection processes. Thymopentin is a fragment of amino acid residues 32-36 in thymopoietin II, retaining the biological activity of thymopoietin and possessing the same physiological functions and pharmacological effects as thymosin and thymopeptides extracted from the thymus. Based on the "drug repurposing" strategy, no further effects of thymopentin have been reported in current technology. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an application of peptide TP-6 ​​in the preparation of tumor diagnostic and / or therapeutic reagents, thus providing a new use for peptide TP-6.

[0006] This invention provides an application of peptide TP-6 ​​in the preparation of tumor diagnostic and / or therapeutic reagents. The amino acid sequence of peptide TP-6 ​​is D-Arg-L-Lys-L-Asp-L-Nva-D-Tyr, where D represents a non-natural D-type amino acid, L represents a natural L-type amino acid, and Nva is valine.

[0007] Furthermore, the tumor includes one or more of the following: lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer, and breast cancer.

[0008] Furthermore, the reagents include fluorescent imaging agents and / or radioactive reagents, wherein the fluorescent imaging agents include reagents for precise tumor boundary localization and / or intraoperative image-guided optical imaging.

[0009] Furthermore, the reagent has the following general formula: MLG;

[0010] M represents optical labeling, metal chelating agents and metal radionuclides complexes, and non-metal radionuclides. 18 F and 11 Any one of C;

[0011] L is a linking group;

[0012] G stands for polypeptide TP-6;

[0013] The optical labeling includes one or more of organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, and bioluminescent molecules;

[0014] The metal chelating agent is selected from hydrazine nicotinamide, 1,4,7-triazacyclononane-1,4,7-triacetic acid, 7-[(4-hydroxypropyl)methylene]-1,4,7-triazahybridizednonane-1,4-diacetic acid, 1,4,7,10-tetraazacyclotetraazacyclododecane-1,4,7,10-tetraacetic acid, mercaptoacetyltriglycine, diethyltriaminepentaacetic acid, or combinations thereof.

[0015] Furthermore, the optical labeling includes near-infrared I fluorescent dyes and / or near-infrared II fluorescent dyes, wherein the near-infrared I fluorescent dyes include one or more of MPA, IRDye800, Cy7.5, ICG, and Cy5.5.

[0016] Further, the linking group includes one or more of 6-aminohexanoic acid, NH2-PEG3-COOH, NH2-PEG4-COOH, NH2-PEG6-COOH and NH2-GGGGGG-COOH.

[0017] Furthermore, the reagent includes MPA-PEG4-TP-6.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. This invention provides an application of a polypeptide in the preparation of tumor diagnostic and / or therapeutic reagents. The molecular probe constructed from the polypeptide can specifically target the tumor site and has good uptake and retention capabilities at the tumor site, with a high target / non-target ratio. It is suitable for the preparation of intraoperative imaging navigation reagents for tumors and the preparation of radiopharmaceuticals for tumor nuclear medicine diagnosis and precision radiotherapy.

[0020] 2. Peptide-based in vivo molecular probes have unique advantages in terms of safety, and can significantly reduce drug development costs and risks;

[0021] Leveraging the tumor-specific targeting properties of thymopentin, fluorescent probes can be constructed by coupling peptides with fluorescent dyes. These probes can assist surgeons in precisely locating tumor boundaries during surgery, enabling accurate tumor resection. Furthermore, thymopentin and its derivatives can be coupled with radionuclides with diagnostic / therapeutic functions to construct corresponding radiopharmaceuticals, achieving the goals of tumor diagnosis and precise radiotherapy.

[0022] 3. The peptide-based molecular probes have been shown to have excellent targeting effects on various tumors, including liver cancer, lung cancer, colorectal cancer, breast cancer, pancreatic cancer, and gastric cancer, through in vivo optical and radionuclide imaging results. The probes' ability to specifically target tumor sites may enable nuclear medicine diagnosis, treatment, and optical surgical navigation for malignant tumors. Attached Figure Description

[0023] Figure 1 This is the mass spectrum of MPA-PEG3-TP-1-NH2.

[0024] Figure 2The images show 2-hour fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-1-NH2 in tumor-bearing mice; where A is fluorescence imaging in mice bearing lung cancer A549; B is fluorescence imaging in mice bearing pancreatic cancer AsPC-1; C is fluorescence imaging in mice bearing pancreatic cancer CFPAC-1; D is fluorescence imaging in mice bearing lung cancer H1299; E is fluorescence imaging in mice bearing colorectal cancer HCT116; and F is fluorescence imaging in mice bearing liver cancer. Fluorescence imaging in mice bearing HepG2 cancer; G represents fluorescence imaging in mice bearing HT29 colorectal cancer; H represents fluorescence imaging in mice bearing MAD-MB-231 breast cancer; I represents fluorescence imaging in mice bearing MGC-803 gastric cancer; J represents fluorescence imaging in mice bearing SGC-7901 gastric cancer; K represents fluorescence imaging in mice bearing MCF-7 breast cancer; L represents fluorescence imaging in mice bearing MiaPaPc-2 pancreatic cancer.

[0025] Figure 3 The images show the 2-hour fluorescence images of the prepared monomeric fluorescent compounds MPA-PEG4-TP-2, MPA-PEG4-TP-3, MPA-PEG4-TP-4, MPA-PEG4-TP-5, MPA-PEG4-TP-6, MPA-PEG4-TP-8, and MPA-PEG4-TP-9 in tumor-bearing mice; where A is the fluorescence image of MPA-PEG4-TP-2 in lung cancer A549 tumor-bearing mice; and B is the fluorescence image of MPA-PEG4-TP-3 in gastric cancer SGC-803 tumor-bearing mice. Fluorescence imaging: C shows the fluorescence imaging of MPA-PEG4-TP-4 in MDA-MB-468 breast cancer tumor-bearing mice; D shows the fluorescence imaging of MPA-PEG4-TP-5 in MCF-7 breast cancer tumor-bearing mice; E shows the fluorescence imaging of MPA-PEG4-TP-6 in AsPC-1 pancreatic cancer tumor-bearing mice; F shows the fluorescence imaging of MPA-PEG4-TP-8 in H1299 lung cancer tumor-bearing mice; G shows the fluorescence imaging of MPA-PEG4-TP-9 in HCT116 colon cancer tumor-bearing mice.

[0026] Figure 4 The images show the 2-hour fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-7 in tumor-bearing mice; where A is the fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice; B is the fluorescence imaging in colorectal cancer HCT116 tumor-bearing mice; C is the fluorescence imaging in breast cancer MDA-MB-231 tumor-bearing mice; D is the fluorescence imaging in pancreatic cancer MiaPaCa-2 tumor-bearing mice; and E is the fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice.

[0027] Figure 5The images show the 2-hour fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-10 in tumor-bearing mice; where A is the fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice; B is the fluorescence imaging in colorectal cancer HCT116 tumor-bearing mice; C is the fluorescence imaging in breast cancer MDA-MB-231 tumor-bearing mice; D is the fluorescence imaging in pancreatic cancer MiaPaCa-2 tumor-bearing mice; and E is the fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice.

[0028] Figure 6 Monomeric radioactive compounds prepared 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-1-NH2 in tumor-bearing mice at 1 hour; where A is SPECT-CT imaging of pancreatic cancer AsPC-1 tumor-bearing mice; B is SPECT-CT imaging of gastric cancer SGC-7901 tumor-bearing mice; C is SPECT-CT imaging of pancreatic cancer MiaPaPc-2 tumor-bearing mice; D is SPECT-CT imaging of breast cancer MCF-7 tumor-bearing mice; E is SPECT-CT imaging of colorectal cancer HT29 tumor-bearing mice.

[0029] Figure 7 Monomeric radioactive compounds prepared 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in tumor-bearing mice at 1 hour; where A is SPECT-CT imaging of MCF-7 breast cancer in mice; B is SPECT-CT imaging of AsPC-1 pancreatic cancer in mice; C is SPECT-CT imaging of MiaPaPc-2 pancreatic cancer in mice; D is SPECT-CT imaging of HT29 colorectal cancer in mice; E is SPECT-CT imaging of MGC-803 gastric cancer in mice; F is SPECT-CT imaging of SGC-7901 gastric cancer in mice.

[0030] Figure 8 For the preparation of dimeric radioactive compounds 99m SPECT-CT imaging of Tc-HYNIC-2Aca-(TP-1-NH2)2 in mice bearing pancreatic cancer AsPC-1 and colorectal cancer HT29. Detailed Implementation

[0031] This invention provides the use of thymopentin and its derivatives in the preparation of tumor diagnostic and / or therapeutic reagents, wherein the thymopentin is selected from one or more of the following polypeptides:

[0032] TP-1: L-Arg-L-Lys-L-Asp-L-Val-L-Tyr (Thymopentin);

[0033] TP-2: L-homo-Arg-L-Lys-L-Asp-L-Nva-L-Tyr;

[0034] TP-3: D-Arg-L-Lys-L-Asp-L-Val-L-Tyr;

[0035] TP-4: D-Arg-D-Lys-L-Asp-L-Val-L-Tyr;

[0036] TP-5: D-Arg-L-Lys-L-Asp-L-Val-D-Tyr;

[0037] Peptide TP-6: D-Arg-L-Lys-L-Asp-L-Nva-D-Tyr;

[0038] TP-7: L-Cys-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Cys, where Cys-Cys disulfide bonds form a ring;

[0039] TP-8: β-Ala-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp, wherein the N-terminal amino group and the C-terminal Asp side chain carboxyl group form an amide ring (novel cyclic peptide).

[0040] TP-9: D-Lys-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Glu, wherein the N-terminal main chain amino group and the C-terminal Glu side chain carboxyl group form an amide ring;

[0041] TP-10: D-Lys-L-Gly-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp, wherein the N-terminal main chain amino group and the C-terminal Asp main chain carboxyl group form an amide ring;

[0042] Wherein: D represents non-natural D-type amino acids, L represents natural L-type amino acids; homoArg is high arginine; Nva is positive valine.

[0043] In this invention, the tumor preferably includes one or more of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer, and breast cancer.

[0044] The present invention preferably involves coupling thymopentin and its derivatives with an imaging group to obtain a reagent. In the present invention, the reagent preferably includes a fluorescent imaging agent and / or a radioactive reagent, and the fluorescent imaging agent preferably includes a reagent for precise tumor boundary localization and / or intraoperative image-guided optical imaging.

[0045] In this invention, the reagent has the following general formula: MLG; where M represents optical labeling, a metal chelating agent and a metal radionuclide complex, or a non-metal radionuclide. 18 F and 11 Any of C; L is a linking group; G is thymopentin and its derivatives (polypeptides). In this invention, the photolabeling preferably includes one or more of organic chromophores, organic fluorophores, light-absorbing compounds, light-reflecting compounds, light-scattering compounds, and bioluminescent molecules. In this invention, the metal chelating agent is preferably selected from hydrazine nicotinamide, 1,4,7-triazacyclononane-1,4,7-triacetic acid, 7-[(4-hydroxypropyl)methylene]-1,4,7-triazahybridizednonane-1,4-diacetic acid, 1,4,7,10-tetraazacyclotetraazacyclododecane-1,4,7,10-tetraacetic acid, mercaptoacetyltriglycine, diethyltriaminepentaacetic acid, or combinations thereof. In this invention, the optical labeling preferably comprises a near-infrared I fluorescent dye and / or a near-infrared II fluorescent dye, wherein the near-infrared I fluorescent dye comprises one or more of MPA, IRDye800, Cy7.5, ICG, and Cy5.5. In this invention, the linking group preferably comprises one or more of 6-aminohexanoic acid, NH2-PEG3-COOH, NH2-PEG4-COOH, NH2-PEG6-COOH, and NH2-GGGGGG-COOH.

[0046] In this invention, the thymopentin and its derivatives, as well as the near-infrared fluorescent probe based thereon, are synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. using a solid-phase method, comprising:

[0047] 1) Synthesis of near-infrared fluorescent dye MPA

[0048] Glacial acetic acid, p-hydrazinobenzenesulfonic acid, methyl isopropyl ketone, and sodium acetate were mixed and reacted, and the resulting product, 2,2,3-trimethyl[3H]-indole-5-sulfonic acid, was purified. Then, o-dichlorobenzene was added to a mixture of 2,2,3-trimethyl[3H]-indole-5-sulfonic acid and 1,3-propanesulfonic acid lactone to prepare 2,2,3-trimethyl-5-sulfonic acid-1-(3-sulfonic acid-propyl)-[3H]-indole. This product was then reacted with... N The reaction of -[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]-anilinemonohydrochloride yields a green carbocyanine dye. Finally, the carbocyanine dye is reacted with mercaptopropionic acid and triethylamine to prepare a water-soluble near-infrared dye, MPA, through liquid-phase separation and purification.

[0049] 2) Synthesis of MPA-L-TP-X (X=1-10)

[0050] Ramage Amide AM resin with a loading of 0.45 mmol / g was selected, and after swelling, the Fmoc protecting group was removed. Following the peptide sequence, coupling was performed sequentially from the C-terminus to the N-terminus until the Fmoc-L-carboxyl group was reached. Small samples were cut, and the molecular weight of the peptide was determined by mass spectrometry. The side chains of Tyr, Asp, Lys, and Arg were protected with tBu, OtBu, Boc, and Arg, respectively. All amino acids used had the α-amino group protected by Fmoc. After confirming the correct mass spectrometry of the peptide Fmoc-L-TP-X-NH2, the Fmoc protecting group was removed, and a near-infrared dye MPA (1.2 times molar addition) was added for solid-phase reaction. The reaction was terminated after a negative ninhydrin test. MPA-L-TP-X-NH2, with all side-chain protecting groups removed, was obtained by reacting a lysis buffer (TFA: triisopropylsilane: water = 95:2.5:2.5) with a linear peptide resin. MPA-L-TP-X-NH2 was then dissolved in water and purified by semi-preparative chromatography. The liquid with acceptable purity was separated and collected by rotary evaporation and lyophilization to obtain the target product.

[0051] The preparation of radionuclide probes based on thymopentin and its derivatives is simpler than that based on their dimer form. The dimer structure contains thymopentin and its derivatives for targeting tumors and hydrazine nicotinamide (HYNIC), a bifunctional chelating agent for radiolabeling, as well as a linker L that increases the distance between thymopentin or its analogues and the radionuclide ligands N-tris(hydroxymethyl)methylglycine (Tricine) and triphenylphosphine tris(m-sulfonate) sodium salt (TPPTS) and regulates the pharmacokinetic properties in vivo. L is selected from any one or more of 6-aminohexanoic acid, NH2-PEG3-COOH, NH2-PEG4-COOH, NH2-PEG6-COOH or NH2-GGGGGG-COOH.

[0052] By modifying the bifunctional chelating agent, different radionuclides can be coupled. For example, replacing hydrazine nicotinamide with any one of the following bifunctional chelating agents: 1,4,7-triazacyclononane-1,4,7-triacetic acid, 7-[(4-hydroxypropyl)methylene]-1,4,7-triazahybridizednonane-1,4-diacetic acid, 1,4,7,10-tetraazacyclotetraazacyclododecane-1,4,7,10-tetraacetic acid, mercaptoacetyltriglycine, or diethyltriaminepentaacetic acid, can couple different radionuclides. 99m Tc can be replaced with 68 Ga、 64 Cu、 67 Ga、 90 Y、 111 In、 89 Zr or 177Lu. Or radioactive nuclides 124 I, 125 I, 131 I is directly labeled on Tyr in the structure of the free peptide TP-X to achieve the function of disease diagnosis / treatment.

[0053] In this invention, the method for preparing the single radionuclide probe includes:

[0054] 1) Synthesis of the bifunctional chelating agent HYNIC-L-NHS

[0055] 6-Chloronicotinic acid and 80% hydrazine hydrate were added to ethanol and heated under reflux. After the reaction was completed, the solvent was evaporated under reduced pressure. The resulting viscous substance was added to distilled water, the pH was adjusted to 5.5, and a solid precipitated. The solid was filtered and dried to obtain a yellow solid. The product was identified as 6-hydroxynicotinic acid by ESI-MS mass spectrometry and 1H NMR spectroscopy. The obtained 6-hydrazinoic acid and p-aminobenzaldehyde were added to dimethyl sulfoxide (DMSO) and heated for 5-6 hours. After the reaction was completed, water was added to precipitate the solid, which was then filtered to obtain a solid. This solid was dried and added to DMSO along with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) at room temperature. After the reaction was completed, water was added to precipitate the solid. This solid was purified by silica gel column chromatography and identified as intermediate HYNIC-NHS by ESI-MS mass spectrometry and 1H NMR spectroscopy. Then, this intermediate and the linker L were reacted under alkaline conditions. Finally, it was activated with EDCI and NHS and purified to obtain HYNIC-L-NHS solid for later use.

[0056] 2) Synthesis of HYNIC-L-TP-X (X=1-10)

[0057] The purified intermediate HYNIC-L-NHS was dissolved in DMSO, 1-1.5 moles of TP-X were added, followed by 2-3 moles of DIPEA. The reaction was carried out at room temperature for 1-2 hours. After the reaction was completed, the intermediate was separated and purified by preparative liquid chromatography and confirmed by mass spectrometry.

[0058] 3) 99m Synthesis of Tc-HYNIC-L-TP-X (X=1-10)

[0059] Prepare TPPTS (sodium triphenylphosphine trisulfonate) solutions with concentrations of 100.0-120 mg / mL, Tricine (trimethylglycine) solutions with concentrations of 130.0-150 mg / mL, and succinate-sodium succinate buffer solutions with concentrations of 102.4-110 mg / mL (77.0-88.8 mg succinate and 25.4-29.3 mg sodium succinate). Take 10.0 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer solution, and mix them with 10.0 μL (1.0 mg / mL) of the HYNIC-L-TP-X solution in a vial. Then add 10 mL of Na... 99m TcO4 was heated in a metal bath at 100 °C for 20-30 minutes. After the reaction was completed, the mixture was cooled to room temperature, and the product was identified by HPLC analysis.

[0060] In this invention, the method for preparing the dimer radionuclide probe preferably includes:

[0061] 1) Synthesis of the bifunctional chelating agent HYNIC-L-NHS

[0062] The method is the same as step 1 in the preparation of single radionuclide probes.

[0063] 2) Synthesis of the scaffold (2L-glutamate)

[0064] Dissolve an appropriate amount of Boc-glutamic acid in DMSO, add 2-3 molar amounts of EDCI and NHS, heat at 60°C for 0.5-1 hour, and HPLC analysis shows that the glutamic acid double-activated ester has been formed. Then add 2-3 molar amounts of linker L and 2-3 molar amounts of DIPEA to the solution, heat at 60°C for 0.5-1 hour, and HPLC analysis shows that 2 molecules of linker L have been linked to glutamic acid. Then add an equal volume of TFA and react at room temperature overnight to remove Boc protection. Finally, the crude product is freeze-dried after preparative HPLC separation for later use.

[0065] 3) Synthesis of intermediate 2L-E-HYNIC-NHS

[0066] The prepared scaffold 2L-glutamic acid was dissolved in DMSO, and then the same molar amount of HYNIC-L-NHS was added, followed by 2-3 times the molar amount of DIPEA. The reaction was carried out at room temperature for 1-2 hours. After the reaction was completed, the product was purified by preparative liquid chromatography and the target compound was confirmed by mass spectrometry. The purified product was activated with EDCI and NHS to obtain 2L-E-HYNIC-NHS for later use.

[0067] 4) Synthesis of (TP-X)2-2L-E-HYNIC (X=1-10)

[0068] The purified intermediate 2L-E-HYNIC-NHS was dissolved in DMSO, 1-1.5 moles of TP-X were added, followed by 2-3 moles of DIPEA. The reaction was carried out at room temperature for 1-2 hours. After the reaction was completed, the intermediate was separated and purified by preparative liquid chromatography and confirmed by mass spectrometry.

[0069] 5) Radioactive probe 99m Synthesis of Tc-HYNIC-2L-E-(TP-X)2 (X=1-10)

[0070] Prepare TPPTS (sodium triphenylphosphine trisulfonate) solutions with concentrations of 100.0-120 mg / mL, Tricine (trimethylglycine) with concentrations of 130.0-150 mg / mL, and succinate-sodium succinate buffer solutions with concentrations of 102.4-110 mg / mL (77.0-88.8 mg succinate and 25.4-29.3 mg sodium succinate). Take 10.0 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer solution, and mix them with 10.0 μL (1.0 mg / mL) of the HYNIC-2L-E-(TP-X)2 solution in a vial. Then add 10 mL of Na... 99m TcO4 was heated in a metal bath at 100℃ for 20-30 minutes. After the reaction was completed, it was cooled to room temperature, and the product was identified by HPLC analysis.

[0071] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0072] Example 1: Synthesis of MPA-PEG3-TP-1-NH2.

[0073] Ramage Amide AM resin with a loading of 0.45 mmol / g was selected, and after swelling, the Fmoc protecting group was removed. Following the thymopentin sequence: Arg-Lys-Asp-Val-Tyr, coupling was performed sequentially from the C-terminus to the N-terminus until Fmoc-PEG3-propionic acid was reached. The side chains of Tyr, Asp, Lys, and Arg were protected with tBu, OtBu, Boc, and Arg, respectively, with all amino acids using Fmoc-protected α-amino groups. After cutting a small sample, the molecular weight of the peptide was determined by mass spectrometry. After confirming the correct mass spectrometry of the peptide Fmoc-PEG3-TP-X-NH2, the Fmoc protecting group was removed, and a near-infrared dye MPA (1.2 times molar addition) was added for solid-phase reaction. The reaction was terminated after a negative ninhydrin test. MPA-PEG3-TP-1-NH2, with all side-chain protecting groups removed, was obtained by reacting a lysis buffer (TFA:triisopropylsilane:water = 95:2.5:2.5) with a linear peptide resin. MPA-PEG3-TP-1-NH2 was dissolved in water and purified using semi-preparative chromatography. The purified liquid was collected, lyophilized by rotary evaporation, and then confirmed as the target compound MPA-PEG3-TP-1-NH2 by ESI-MS mass spectrometry. ESI-MS: [M-2H] 2- =895.96 and [M-3H] 3- =597.26 ( Figure 1 ).

[0074] Example 2: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in A549 lung cancer tumor-bearing mice.

[0075] The prepared fluorescent compound MPA-PEG3-TP-1-NH2 was dissolved in physiological saline (100 nmol / mL). 0.1 mL (approximately 10 nmol) was injected intravenously into the tail vein of three A549 lung cancer-bearing nude mice (approximately 22 g in weight). Optical signals were acquired at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h post-administration. The distribution of the fluorescent drug in the model mice and its accumulation in the tumor region were observed. Results are shown below. Figure 2 The results showed that the fluorescent probe MPA-PEG3-TP-1-NH2 could specifically target the lung cancer (A549) site.

[0076] Example 3: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in pancreatic cancer AsPC-1 tumor-bearing mice.

[0077] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three nude mice bearing AsPC-1 pancreatic cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2 In B, it can be seen that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the pancreatic cancer (AsPC-1) site.

[0078] Example 4: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in pancreatic cancer CFPAC-1 tumor-bearing mice.

[0079] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three pancreatic cancer CFPAC-1-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 2 The C in the figure shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the pancreatic cancer (CFPAC-1) site.

[0080] Example 5: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in lung cancer H1299 tumor-bearing mice.

[0081] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three H1299 lung cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2 As shown in D, the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the lung cancer (H1299) site.

[0082] Example 6: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in HCT116 colorectal cancer-bearing mice.

[0083] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three nude mice bearing HCT116 colorectal cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h post-administration. The distribution of the fluorescent drug in the model mice and its enrichment in the tumor region were observed. Results are shown below. Figure 2 The E in the image shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the colorectal cancer (HCT116) site.

[0084] Example 7: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in HepG2 tumor-bearing mice.

[0085] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three HepG2 tumor-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2 The F in the figure shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the hepatocellular carcinoma (HepG2) site.

[0086] Example 8: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in HT29 colorectal cancer-bearing mice.

[0087] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three HT29 colorectal cancer-bearing nude mice. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2 The G in the figure shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the colorectal cancer (HT29) site.

[0088] Example 9: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in MAD-MB-231 breast cancer tumor-bearing mice.

[0089] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three MAD-MB-231 breast cancer-bearing nude mice, and fluorescence signals were collected at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after drug administration. Results are shown below. Figure 2 The presence of H indicates that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the breast cancer (MAD-MB-231) site.

[0090] Example 10: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in MGC-803 tumor-bearing mice with gastric cancer.

[0091] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three nude mice bearing MGC-803 gastric cancer tumors. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2The I in the figure shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the gastric cancer (MGC-803) site.

[0092] Example 11: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in SGC-7901 tumor-bearing mice with gastric cancer.

[0093] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three SGC-7901 tumor-bearing nude mice with gastric cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. The results are shown in […]. Figure 2 As shown in J, the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the gastric cancer (SGC-7901) site.

[0094] Example 12: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in breast cancer MCF-7 tumor-bearing mice.

[0095] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three MCF-7 breast cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 2 The K in the sample indicates that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the breast cancer (MCF-7) site.

[0096] Example 13: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-1-NH2 prepared in Example 1 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.

[0097] Following the same method as in Example 2, MPA-PEG3-TP-1-NH2 was injected into three pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 2 The L in the figure shows that the fluorescent probe MPA-PEG3-TP-1-NH2 can specifically target the pancreatic cancer (MiaPaPc-2) site.

[0098] Example 14: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-2 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-2) in lung cancer A549 tumor-bearing mice.

[0099] Following the same method as in Example 2, MPA-PEG4-TP-2 was injected into three A549 lung cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 3 As shown in A, the fluorescent probe MPA-PEG4-TP-2 can specifically target the lung cancer (A549) site.

[0100] Example 15: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-3 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-3) in SGC-803 tumor-bearing mice with gastric cancer.

[0101] Following the same method as in Example 2, MPA-PEG4-TP-3 was injected into three SGC-803 tumor-bearing nude mice with gastric cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 3 In B, it can be seen that the fluorescent probe MPA-PEG4-TP-3 can specifically target the gastric cancer (SGC-803) site.

[0102] Example 16: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-4 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-4) in breast cancer MDA-MB-468 tumor-bearing mice.

[0103] Following the same method as in Example 2, MPA-PEG4-TP-4 was injected into three MDA-MB-468 breast cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 3 The C in the figure shows that the fluorescent probe MPA-PEG4-TP-4 can specifically target the breast cancer (MDA-MB-468) site.

[0104] Example 17: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-5 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-5) in breast cancer MCF-7 tumor-bearing mice.

[0105] Following the same method as in Example 2, MPA-PEG4-TP-5 was injected into three MCF-7 breast cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 3 As shown in D, the fluorescent probe MPA-PEG4-TP-5 can specifically target the breast cancer (MCF-7) site.

[0106] Example 18: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-6 (prepared in the same way as in Example 1, except that TP-1-NH2 is replaced with TP-6) in pancreatic cancer AsPC-1 tumor-bearing mice.

[0107] Following the same method as in Example 2, MPA-PEG4-TP-6 was injected into three nude mice bearing pancreatic cancer AsPC-1 tumors. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 3 The E in the image shows that the fluorescent probe MPA-PEG4-TP-6 can specifically target the pancreatic cancer (AsPC-1) site.

[0108] Example 19: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-8 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-8) in lung cancer H1299 tumor-bearing mice.

[0109] Following the same method as in Example 2, MPA-PEG4-TP-8 was injected into three H1299 lung cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 3 The F in the figure shows that the fluorescent probe MPA-PEG4-TP-8 can specifically target the lung cancer (H1299) site.

[0110] Example 20: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG4-TP-9 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-9) in HCT116 tumor-bearing mice with colon cancer.

[0111] Following the same method as in Example 2, MPA-PEG4-TP-8 was injected into three nude mice bearing HCT116 colon cancer tumors. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 3 The G in the figure shows that the fluorescent probe MPA-PEG4-TP-9 can specifically target the colon cancer (HCT116) site.

[0112] Example 21: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-7 (preparation method is the same as in Example 1, except that TP-1-NH2 is replaced with TP-7) in pancreatic cancer AsPC-1 tumor-bearing mice.

[0113] Following the same method as in Example 2, MPA-PEG3-TP-7 was injected into three nude mice bearing pancreatic cancer AsPC-1 tumors. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 4 As shown in A, the fluorescent probe MPA-PEG3-TP-7 can specifically target the pancreatic cancer (AsPC-1) site.

[0114] Example 22: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-7 prepared in Example 21 in HCT116 colorectal cancer-bearing mice.

[0115] Following the same method as in Example 2, MPA-PEG3-TP-7 was injected into three nude mice bearing HCT116 colorectal cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 4 In B, it can be seen that the fluorescent probe MPA-PEG3-TP-7 can specifically target the colorectal cancer (HCT116) site.

[0116] Example 23: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-7 prepared in Example 21 in MAD-MB-231 breast cancer tumor-bearing mice.

[0117] Following the same method as in Example 2, MPA-PEG3-TP-7 was injected into three MAD-MB-231 breast cancer-bearing nude mice, and fluorescence signals were collected at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. Results are shown below. Figure 4 The C in the figure shows that the fluorescent probe MPA-PEG3-TP-7 can specifically target the breast cancer (MAD-MB-231) site.

[0118] Example 24: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-7 prepared in Example 21 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.

[0119] Following the same method as in Example 2, MPA-PEG3-TP-7 was injected into three pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 4 In the D region, it can be seen that the fluorescent probe MPA-PEG3-TP-7 can specifically target the pancreatic cancer (MiaPaPc-2) site.

[0120] Example 25: Fluorescence imaging of the monomeric fluorescent compound MPA-PEG3-TP-7 prepared in Example 21 in SGC-7901 tumor-bearing mice with gastric cancer.

[0121] Following the same method as in Example 2, MPA-PEG3-TP-7 was injected into three SGC-7901 tumor-bearing nude mice with gastric cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 4 E in the image shows that the fluorescent probe MPA-PEG3-TP-7 can specifically target the gastric cancer (SGC-7901) site.

[0122] Example 26: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-10 (prepared in the same way as in Example 1, except that TP-1-NH2 is replaced with TP-10) in pancreatic cancer AsPC-1 tumor-bearing mice.

[0123] Following the same method as in Example 2, MPA-PEG3-TP-10 was injected into three nude mice bearing pancreatic cancer AsPC-1 tumors. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 5 In Figure A, it can be seen that the fluorescent probe MPA-PEG3-TP-10 can specifically target the pancreatic cancer (AsPC-1) site.

[0124] Example 27: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-10 in HCT116 colorectal cancer-bearing mice.

[0125] Following the same method as in Example 2, MPA-PEG3-TP-10 was injected into three nude mice bearing HCT116 colorectal cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 5 In B, it can be seen that the fluorescent probe MPA-PEG3-TP-10 can specifically target the colorectal cancer (HCT116) site.

[0126] Example 28: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-10 in MAD-MB-231 breast cancer-bearing mice.

[0127] Following the same method as in Example 2, MPA-PEG3-TP-10 was injected into three MAD-MB-231 breast cancer-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after administration. Results are shown below. Figure 5 In the C, it can be seen that the fluorescent probe MPA-PEG3-TP-10 can specifically target the breast cancer (MAD-MB-231) site.

[0128] Example 29: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-10 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.

[0129] Following the same method as in Example 2, MPA-PEG3-TP-10 was injected into three pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, and fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 5 In the D region, it can be seen that the fluorescent probe MPA-PEG3-TP-10 can specifically target the pancreatic cancer (MiaPaPc-2) site.

[0130] Example 30: Fluorescence imaging of the prepared monomeric fluorescent compound MPA-PEG3-TP-10 in SGC-7901 tumor-bearing mice with gastric cancer.

[0131] Following the same method as in Example 2, MPA-PEG3-TP-10 was injected into three SGC-7901 tumor-bearing nude mice with gastric cancer. Fluorescence signals were collected at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h after drug administration. Results are shown below. Figure 5 E in the image shows that the fluorescent probe MPA-PEG3-TP-10 can specifically target the gastric cancer (SGC-7901) site.

[0132] Example 31: Monomeric Radioactive Compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-1-NH2 in AsPC-1 pancreatic cancer-bearing mice.

[0133] 1) Synthesis of the bifunctional chelating agent HYNIC-PEG4-NHS

[0134] 1 g of 6-chloronicotinic acid and 2.0 mL of 80% hydrazine hydrate were added to 10 mL of ethanol, and the mixture was heated under reflux for 4 hours. After the reaction was completed, the solvent was rotary evaporated under reduced pressure. The resulting viscous substance was added to distilled water, and the pH was adjusted to approximately 5.5. A solid precipitated, which was then filtered and dried to obtain 0.86 g of a yellow solid. The product was identified as 6-chloronicotinic acid by ESI-MS mass spectrometry and 1H NMR spectroscopy. The obtained 0.86 g of 6-chloronicotinic acid and 0.61 g of p-aminobenzaldehyde were added to 3.0 mL of dimethyl sulfoxide (DMSO), and the mixture was heated for 5-6 hours. After the reaction was completed, the solid precipitated in water, was filtered, and dried to obtain 1.2 g of solid. The dried 1.2 g solid was added to DMSO along with 2.5 g EDCI and 1.5 g NHS and reacted at room temperature. After the reaction was complete, water was added to precipitate a solid. This solid was purified by silica gel column chromatography, dried, and weighed 1.3 g. ESI-MS mass spectrometry and 1H NMR spectroscopy identified it as HYNIC-NHS, ESI-MS: [M+H] = 382.1508. This purified product was then added to PEG4 containing DIPEA and reacted at room temperature for 2 hours. After the reaction was complete, two molar amounts of EDCI and NHS were added to the solution. After the reaction was complete, the product was purified by preparative liquid chromatography, freeze-dried, and confirmed by mass spectrometry as the target product HYNIC-PEG4-NHS, ESI-MS: [M+H] = 630.3 and [M+Na] = 630.3. + =652.3.

[0135] 2) 5 mg of the purified intermediate HYNIC-PEG4-NHS was dissolved in 0.3 mL of DMSO. 3 mg of TP-1 and 5.6 mg of DIPEA were added to the mixture, and the reaction was carried out at room temperature for 2 hours. After the reaction was complete, the product was separated and purified by preparative liquid chromatography, yielding 2.8 mg of a yellow solid. Mass spectrometry confirmed this to be the target product HYNIC-PEG4-TP-1-NH2. ESI-MS: [M+2H] 2+ =596.81 and [M+3H] 3+ =398.18.

[0136] 0) Radioactive compounds 99m Synthesis of Tc-HYNIC-PEG4-TP-1-NH2

[0137] Prepare solutions of 100.0 mg / mL TPPTS (sodium triphenylphosphine trisulfonate), 130.0 mg / mL Tricine (trimethylglycine), and 102.4 mg / mL succinate-sodium succinate buffer (77.0 mg succinate and 25.4 mg sodium succinate). Take 10.0 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer, and mix them with 10.0 μL (1.0 mg / mL) of HYNIC-PEG4-TP-1-NH2 in a vial. Then add 10 mL of Na... 99m TcO4 was heated in a metal bath at 100 °C for 20 minutes, and after the reaction was completed, it was cooled to room temperature to obtain the radiopharmaceutical. 99m The product, Tc-HYNIC-PEG4-TP-1-NH2, was identified by HPLC analysis.

[0138] radioactive compounds 999m Tc-HYNIC-PEG4-TP-1-NH2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into the tail vein of three nude mice bearing AsPC-1 pancreatic cancer tumors. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h post-administration. The distribution of the radionuclide probe in the mice and its enrichment in the tumor region were observed. Results are shown below. Figure 6 In Figure A, the nuclide probe can be seen. 99m Tc-HYNIC-PEG4-TP-1-NH2 can specifically target the pancreatic cancer (AsPC-1) site.

[0139] Example 32: Monomeric radioactive compounds 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-1-NH2 in SGC-7901 tumor-bearing mice with gastric cancer.

[0140] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-1-NH2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three SGC-7901 tumor-bearing nude mice with gastric cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h after drug administration. Results are shown below. Figure 6 B in the figure shows the nuclide probe. 99m Tc-HYNIC-PEG4-TP-1-NH2 can specifically target the gastric cancer (SGC-7901) site.

[0141] Example 33: Monomeric Radioactive Compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-1-NH2 in MiaPaPc-2 tumor-bearing mice with pancreatic cancer.

[0142] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-1-NH2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing MiaPaPc-2 pancreatic cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 6 C in the figure, as can be seen from the nuclide probe 99m Tc-HYNIC-PEG4-TP-1-NH2 can specifically target pancreatic cancer (MiaPaPc-2).

[0143] Example 34: Monomeric Radioactive Compound 99m SPECT-CT imaging in MCF-7 mice bearing Tc-HYNIC-PEG4-TP-1-NH2 breast cancer.

[0144] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-1-NH2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three MCF-7 breast cancer-bearing nude mice. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 6 D in the figure represents the nuclide probe. 99m Tc-HYNIC-PEG4-TP-1-NH2 can specifically target the breast cancer (MCF-7) site.

[0145] Example 35: Monomeric Radioactive Compound 99m SPECT-CT imaging of HT29 tumor-bearing mice with Tc-HYNIC-PEG4-TP-1-NH2 colorectal cancer.

[0146] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-1-NH2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing HT29 colorectal cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h after drug administration. Results are shown below. Figure 6 E in the figure represents the nuclide probe.99m Tc-HYNIC-PEG4-TP-1-NH2 can specifically target the colorectal cancer (HT29) site.

[0147] Example 36: Monomeric Radioactive Compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 (prepared in the same way as in Example 31, except that TP-1-NH2 is replaced with TP-7) in breast cancer MCF-7 tumor-bearing mice.

[0148] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three MCF-7 breast cancer-bearing nude mice. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 In Figure A, the nuclide probe can be seen. 99m Tc-HYNIC-PEG4-TP-7 can specifically target the breast cancer (MCF-7) site.

[0149] Example 37: Preparation of a monomeric radioactive compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in AsPC-1 tumor-bearing mice with pancreatic cancer.

[0150] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing AsPC-1 pancreatic cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 B in the figure shows the nuclide probe. 99m Tc-HYNIC-PEG4-TP-7 can specifically target pancreatic cancer (AsPC-1).

[0151] Example 38: Preparation of a monomeric radioactive compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in mice bearing pancreatic cancer MiaPaPc-2 tumors.

[0152] The radioactive compound was prepared using the same method as in Example 31. 99mTc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing MiaPaPc-2 pancreatic cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 C in the figure, as can be seen from the nuclide probe 99m Tc-HYNIC-PEG4-TP-7 can specifically target pancreatic cancer (MiaPaPc-2).

[0153] Example 39: Monomeric Radioactive Compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in HT29 colorectal cancer-bearing mice.

[0154] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing HT29 colorectal cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 D in the figure represents the nuclide probe. 99m Tc-HYNIC-PEG4-TP-7 can specifically target the colorectal cancer (HT29) site.

[0155] Example 40: Preparation of a monomeric radioactive compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in MGC-803 tumor-bearing mice with gastric cancer.

[0156] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing MGC-803 gastric cancer tumors. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 E in the figure represents the nuclide probe. 99m Tc-HYNIC-PEG4-TP-7 can specifically target the gastric cancer (MGC-803) site.

[0157] Example 41: Preparation of a monomeric radioactive compound 99m SPECT-CT imaging of Tc-HYNIC-PEG4-TP-7 in SGC-7901 tumor-bearing mice with gastric cancer.

[0158] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-PEG4-TP-7 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing MGC-803 gastric cancer tumors. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h post-administration. Results are shown below. Figure 7 F in the figure represents the nuclide probe. 99m Tc-HYNIC-PEG4-TP-7 can specifically target the gastric cancer (SGC-7901) site.

[0159] Example 42: Dimer 99m Radiosynthesis of Tc-HYNIC-2Aca-E-(TP-1-NH2)2.

[0160] 1) Synthesis of the bifunctional chelating agent HYNIC-NHS

[0161] 1 g of 6-chloronicotinic acid and 2.0 mL of 80% hydrazine hydrate were added to 10 mL of ethanol, and the mixture was heated under reflux for 4 hours. After the reaction was completed, the solvent was rotary evaporated under reduced pressure. The resulting viscous substance was added to distilled water, and the pH was adjusted to approximately 5.5. A solid precipitated out, which was then filtered and dried to obtain 0.86 g of a yellow solid. The product was identified as 6-chloronicotinic acid by ESI-MS mass spectrometry and 1H NMR spectroscopy. The obtained 0.86 g of 6-chloronicotinic acid and 0.61 g of p-aminobenzaldehyde were added to 3.0 mL of dimethyl sulfoxide (DMSO), and the mixture was heated for 5-6 hours. After the reaction was completed, the solid precipitated out by adding water, filtering, and drying to obtain 1.2 g of solid. The 1.2 g dried solid was then added to DMSO along with 2.5 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) and 1.5 g of N-hydroxysuccinimide (NHS) and reacted at room temperature. After the reaction was complete, water was added to precipitate the solid. This solid was purified by silica gel column chromatography, dried, and weighed 1.3 g. It was identified as the target product HYNIC-NHS by ESI-MS mass spectrometry and 1H NMR spectroscopy. ESI-MS: [M+H] = 382.15.

[0162] 2) Synthesis of stent (Aca)2-E

[0163] 5.0 g of t-Butyloxycarbony-protected glutamic acid (E), 8.3 g of dicyclohexylcarbodiimide (DCC), and 4.6 g of NHS were added to 100 mL of tetrahydrofuran (THF) organic solvent. The mixture was stirred overnight at room temperature to activate the dicarboxyl group. After the reaction was complete, the mixture was filtered, and the filtrate was washed with THF. After washing, without further purification, the filtrate was directly dissolved in 50 mL of dimethyl sulfoxide (DMSO), followed by the addition of 10 g of aminocaproic acid (Aca). Finally, 14.6 g of DIPEA was added, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed, 3.0 mL of trifluoroacetic acid (TEA) was added to the reaction to remove the Boc protecting group. After the reaction was completed, the mixture was separated and purified by preparative liquid chromatography. Finally, 7.8 g of viscous solid was obtained by drying, and the solid was verified by mass spectrometry to be the expected target compound (Aca)2-E.

[0164] 3) Synthesis of the intermediate HYNIC-E-(Aca)2-2NHS

[0165] The prepared 0.5 g scaffold (Aca)2-E was dissolved in DMSO, then 0.28 g HYNIC-NHS was added, followed by 0.32 g DIPEA. The mixture was reacted at room temperature for 2 hours, and then EDCI and NHS were added for activation. After the reaction was completed, the mixture was purified by preparative liquid chromatography and freeze-dried to obtain 0.34 g of yellow solid, which was verified by mass spectrometry to be the expected target compound HYNIC-E-(Aca)2-2NHS.

[0166] 4) Synthesis of HYNIC-2Aca-E-(TP-1-NH2)2

[0167] The purified intermediate HYNIC-E-(Aca)2-2NHS was dissolved in 0.3 mL DMSO. After the reaction was complete, 5 mg TP-1 was added, followed by 5.6 mg DIPEA. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the product was separated and purified by preparative liquid chromatography. Finally, 3.5 mg of yellow solid was obtained, which was confirmed by mass spectrometry to be the target product.

[0168] 5) 99m Preparation of Tc-HYNIC-2Aca-E-(TP-1-NH2)2

[0169] Prepare TPPTS (sodium triphenylphosphine tris(m-sulfonate)) solution at a concentration of 100.0 mg / mL, Tricine (trimethylglycine) at a concentration of 130.0 mg / mL, and succinate-sodium succinate buffer at a concentration of 102.4 mg / mL (77.0 mg succinate and 25.4 mg sodium succinate). Take 10 μL of each TPPTS solution, Tricine solution, and succinate-sodium succinate buffer solution, and mix them with 10 μL (1.0 mg / mL) of HYNIC-2Aca-E-(TP-1-NH2)2 in a vial. Then add 10 mL of Na... 99m TcO4 was heated in a metal bath at 100 °C for 20 minutes, and after the reaction was completed, it was cooled to room temperature to obtain the radiopharmaceutical. 99m Tc-HYNIC-2Aca-E-(TP-1-NH2)2, the product was identified by HPLC analysis.

[0170] Example 43: Dimeric radioactive compound prepared in Example 42 99m SPECT-CT imaging of Tc-HYNIC-2Aca-E-(TP-1-NH2)2 in AsPC-1 tumor-bearing mice with pancreatic cancer.

[0171] The radioactive compound was prepared using the same method as in Example 31. 99m Tc-HYNIC-2Aca-E-(TP-1-NH2)2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing AsPC-1 pancreatic cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h after drug administration. Results are shown below. Figure 8 In Figure A, the nuclide probe can be seen. 99m Tc-HYNIC-2Aca-E-(TP-1-NH2)2 can specifically target the pancreatic cancer (AsPC-1) site.

[0172] Example 44: Preparation of a dimeric radioactive compound 99m SPECT-CT imaging of Tc-HYNIC-2Aca-E-(TP-1-NH2)2 in HT29 colorectal cancer-bearing mice.

[0173] The radioactive compound was prepared using the same method as in Example 31. 99mTc-HYNIC-2Aca-E-(TP-1-NH2)2 was prepared as a physiological saline solution (3 mCi / mL). 0.1 mL (approximately 300 μCi) was injected into three nude mice bearing HT29 colorectal cancer. SPECT-CT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 4 h after drug administration. Results are shown below. Figure 8 B in the figure shows the nuclide probe. 99m Tc-HYNIC-2Aca-E-(TP-1-NH2)2 can specifically target the colorectal cancer (HT29) site.

Claims

1. The application of a polypeptide TP-6 ​​in the preparation of tumor diagnostic reagents, characterized in that, The amino acid sequence of the polypeptide TP-6 ​​is D-Arg-L-Lys-L-Asp-L-Nva-D-Tyr, where D represents a non-natural D-type amino acid, L represents a natural L-type amino acid, and Nva is valine. The reagent is MPA-PEG4-TP-6; The tumor is pancreatic cancer; The synthesis steps of the near-infrared fluorescent dye MPA are as follows: Glacial acetic acid, p-hydrazinobenzenesulfonic acid, methyl isopropyl ketone, and sodium acetate are mixed and reacted, and purified to obtain the product 2,2,3-trimethyl[3H]-indole-5-sulfonic acid; then, o-dichlorobenzene is added to a mixture of 2,2,3-trimethyl[3H]-indole-5-sulfonic acid and 1,3-propanesulfonic acid lactone to obtain 2,2,3-trimethyl-5-sulfonic acid-1-(3-sulfonic acid-propyl)-[3H]-indole; then, this product is combined with... N The green carbocyanine dye was obtained by reacting -[(3-(anilinomethylene)-2-chloro-1-cyclohexen-1-yl)methylene]-aniline monohydrochloride. Finally, the carbocyanine dye was reacted with mercaptopropionic acid and triethylamine to prepare the water-soluble near-infrared dye MPA by liquid-phase separation and purification.

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