Double-target small-molecule probe, radionuclide-labeled double-target small-molecule probe as well as preparation method and application of radionuclide-labeled double-target small-molecule probe

Abstract

The invention discloses a double-target small-molecule probe, a radionuclide-labeled double-target small-molecule probe as well as a preparation method and application of the radionuclide-labeled double-target small-molecule probe. The medicine < 68 > Ga-DOTA-PRE / RGD is obtained by labeling < 68 > Ga radionuclide, and is applied to PET / MR (positron emission tomography / magnetic resonance imaging) imaging of pancreatic cancer tumors. A radioactive product with high purity and high stability (greater than 93%) is obtained through a simple marking method; the obtained double-target radioactive small molecular probe 68Ga-DOTA-PRE / RGD has a huge potential for the accurate diagnosis of pancreatic cancer.

Description

Technical Field

[0001] This invention relates to the field of radiopharmaceutical technology, specifically to a dual-target small molecule probe, a radionuclide-labeled dual-target small molecule probe, its preparation method, and its application. Background Technology

[0002] Pancreatic cancer, as one of the most malignant tumors in clinical practice, has a five-year survival rate of less than 5%, and the vast majority of patients have already missed the best surgical opportunity by the time it is diagnosed. Therefore, early diagnosis and treatment intervention are essential to improve prognosis. With the exploration of the molecular mechanisms of pancreatic cancer development, a variety of monoclonal antibodies and small molecule inhibitors have been developed clinically to explore potential targets of pancreatic cancer, including epidermal growth factor receptor, mesothelin-urokinase plasminogen activator (uPA), insulin-like growth factor I receptor (IGF-1R), mucin 1, vascular endothelial growth factor receptor 2 (VEGFR2) zinc transporter 4 (ZIP4) (11), carbohydrate antigen CA199, etc. However, the heterogeneity among tumors and the heterogeneity of target ligand expression in tumors have increased the limitations of using single-target molecular drugs, thus giving rise to dual-target molecular probe drugs.

[0003] Targeted molecular probe imaging, as an emerging non-invasive imaging method for precise tumor diagnosis, has attracted significant clinical attention. Several molecular probes, such as PSMA and FAPI, have been developed and applied in clinical cancer diagnosis. A complete molecular probe typically consists of two parts: a highly selective ligand that binds to the tumor target and a radionuclide for visualization. Compared to single-target imaging agents, dual-target molecular probes can increase tumor affinity for drugs, enhancing tumor uptake. Furthermore, they reduce application limitations caused by heterogeneous expression of certain target ligands in tumors, expanding the range of drug applications. The presence of dual-target ligands may even achieve a synergistic imaging effect greater than the sum of its parts.

[0004] Plectin is a versatile cytoskeleton linker protein widely distributed in mammalian cells and many cell types. Plectin possesses binding sites for intermediate filaments (IFs), enabling it to interconnect IFs and anchor them to linker complexes, the nuclear membrane, and intracellular organelles. Beyond its physiological functions, plectin plays a crucial role in the migration, proliferation, and invasion of cancer cells. Aberrant expression of plectin is considered a biomarker and effective target for various cancers, including prostate and pancreatic cancer.

[0005] Integrins are a superfamily of cell adhesion receptors that can bind to extracellular matrix ligands, cell surface ligands, and soluble ligands, facilitating cell attachment to the extracellular matrix by recognizing RGD sequences. However, aberrant expression of integrins can contribute to tumor cell migration, proliferation, and survival. Interestingly, there is a close relationship between Plectin protein and integrins. Integrinβ4 plays an important role in pancreatic cancer metastasis, and aberrant expression of Integrinβ4 is an indispensable condition for the mislocalization of Plectin protein in pancreatic cancer. Integrinβ4 can directly interact with Plectin, interfering with the cell membrane localization of endogenous Plectin in pancreatic ductal cell carcinoma (PDAC).

[0006] Therefore, combining Plectin and Integrin as a dual target for radionuclide tracers may amplify the targeting imaging effect of the radionuclide tracers and improve the detection rate of PDAC. Based on the preceding discussion, this study designed a novel dual-target small molecule probe, which, through the use of… 68 The drug is labeled with a Ga radionuclide to obtain the drug. 68 Ga-DOTA-PLE / RGD was applied to PET / MR imaging of pancreatic cancer tumors. Summary of the Invention

[0007] The purpose of this invention is to provide a dual-target small molecule probe, a radionuclide-labeled dual-target small molecule probe, its preparation method, and its application. 68 The drug is labeled with a Ga radionuclide to obtain the drug. 68 Ga-DOTA-PLE / RGD was applied to PET / MR imaging of pancreatic cancer tumors.

[0008] To achieve the above objectives, the present invention provides a dual-target small molecule probe, the structural formula of which is as follows: .

[0009] This invention also provides a radionuclide-labeled dual-target small molecule probe, wherein the radionuclide-labeled dual-target small molecule probe is composed of the dual-target small molecule probe described in claim 1 and a radionuclide. 68 The radionuclide-labeled dual-target small molecule probe was prepared using Ga; the structural formula of the probe is as follows: .

[0010] This invention also provides a method for preparing a radionuclide-labeled dual-target small molecule probe as described above, the method comprising: dissolving the dual-target small molecule probe DOTA-PLE / RGD in sodium acetate, and using HCl to... 68 Ge- 68 Elution in Ga generator 68 GaCl3, and take 68 GaCl3 was added to the DOTA-PLE / RGD solution, heated at 95 °C for 10 min, cooled to room temperature, and then transferred to a Sep-Pak C18 column. After rinsing the Sep-Pak C18 silica column with ethanol, a radionuclide-labeled dual-target small molecule probe was obtained. 68 Ga-DOTA-PLE / RGD; .

[0011] This invention also provides a method for preparing the dual-target small molecule probe as described above, the method comprising: Step 1: Mix and react the amino acid Dde-Lys(Fmoc)-OH shown in Formula A, 2-chlorotriphenylmethyl chloro resin, N,N-diisopropylethylamine, and N,N-dimethylformamide to obtain the intermediate shown in Formula B; Step 2: The intermediate shown in Formula B is reacted with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group; then it is reacted with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetratert-butyl ester (DOTA(OtBu)3), N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and N,N-dimethylformamide to obtain the intermediate shown in Formula C; Step 3: The intermediate shown in Formula C is reacted with hydrazine hydrate / N,N-dimethylformamide solution to remove the Dde protecting group. Then it is reacted with amino acid Fmoc-Lys(Dde)-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide to obtain the intermediate shown in Formula D. Step 4: React the intermediate shown in Formula D with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group, and then react it with amino acid Fmoc-Pro-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide. Then, the above-mentioned amino acid Fmoc-Pro-OH was replaced in sequence with amino acid Fmoc-Thr(tBu)-OH, amino acid Fmoc-Pro-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Thr(tBu)-OH, and amino acid Fmoc-Lys(Fmoc)-OH, and the above reaction was repeated to obtain the intermediate shown in Formula E; Step 5: React the intermediate shown in Formula E with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group, and then react it with di-tert-butyl dicarbonate ((Boc)2O), N,N-diisopropylethylamine and N,N-dimethylformamide to obtain the intermediate shown in Formula F; Step 6: React the intermediate shown in Formula F with hydrazine hydrate / N,N-dimethylformamide solution to remove the Dde protecting group and obtain the intermediate shown in Formula G; Step 7: Mix and react the intermediate shown in Formula G, the amino acid Fmoc-Arg(Pbf)-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide. Then, the above-mentioned amino acid Fmoc-Pro-OH was replaced in sequence with amino acid Fmoc-Gly-OH, amino acid Fmoc-Asp(tBu)-OH, and amino acid Boc-Phe-OH, and the above reaction was repeated to obtain the intermediate shown in formula H. Step 8: Mix the intermediate shown in Formula H with trifluoroacetic acid (TFA), triisopropylsilane (Tri), 1,2-ethylenedithiol (EDT), and water to remove all protecting groups and obtain a dual-target small molecule probe.

[0012] This invention also provides a radionuclide-labeled dual-target small molecule probe for PET / MR imaging of pancreatic cancer tumors.

[0013] The technical solution of this invention designs a novel small molecule probe by combining Plectin with the Integrin target, and through... 68 Ga was used for labeling to obtain radioactive products. 68 Ga-DOTA-PLE / RGD. High-purity and highly stable (>93%) radioactive products were obtained using a simple labeling method. In Bxpc-3 cell experiments, results from the experimental group, single-blocking group, and double-blocking group indicate... 68Ga-DOTA-PLE / RGD exhibits high sensitivity and specificity, a result further validated in imaging experiments on Bxpc-3 mice, yielding excellent imaging results. At 30 min, the T / M value for the experimental group was 3.02±0.17, while the T / M value for the blocking group (PLE+RGD) was 1.75±0.32. At 1 h, the T / M value for the experimental group was 3.30±0.29, while the T / M value for the blocking group (PLE+RGD) was 1.72±0.03. At 2 h, the T / M value for the experimental group was 3.77±0.80, while the T / M value for the blocking group (PLE+RGD) was 1.88±0.47. All three time points showed significant statistical differences. Furthermore, the clearance half-life of this radioactive molecular probe indicates that the drug remains in the bloodstream for an appropriate period, providing a sufficient time window for target binding without increasing potential toxicity due to prolonged residence. Meanwhile, the biodistribution results indicate that the radiopharmaceutical is mainly distributed in the kidneys, with lower uptake in other organs, which reduces imaging interference from other organs during tumor imaging.

[0014] The novel dual-target radioactive small molecule probe provided by this invention 68 Ga-DOTA-PLE / RGD was used, and its imaging performance was evaluated using PET / MR. Based on the analysis and comprehensive consideration of the above experimental results, this dual-target radioactive small molecule probe... 68 Ga-DOTA-PLE / RGD has great potential for the precise diagnosis of pancreatic cancer.

[0015] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0016] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 yes 68 Schematic diagram of the synthesis and labeling process of Ga-DOTA-PLE / RGD; Figure 2 yes 68 Radiochemical purity (RCP) plot of Ga-DOTA-PLE / RGD; Figure 3 , 4 This is a graph showing the results of the in vitro stability test for Example 2; Figure 5 This is a graph showing the results of the cytotoxicity test for Example 3; Figure 6-11 This is a graph showing the results of the cell blocking experiment in Example 4; Figure 12This is a graph showing the results of the drug metabolism kinetics experiment in Example 5; Figure 13 This is a graph showing the results of the biodistribution experiment on normal mice in Example 6; Figure 14 This is a graph showing the results of the weight measurement experiment for mice in Example 7; Figure 15 , 16 This is a PET / MR imaging result of the mice in Example 8. Figure 17 This is a diagram showing the results of the biodistribution experiment in tumor-bearing mice (Example 9); Figure 18 This is a high-resolution mass spectrum of the DOTA-PLE / RGD obtained in Example 1. Detailed Implementation

[0017] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0018] All chemicals used in the experiment were commercially available and did not require purification before use. Sodium acetate and hydrochloric acid were purchased from Sigma-Aldrich, acetonitrile (ACN) from Chengdu Kelong Chemical Co., Ltd., and trifluoroacetic acid (TFA) from Aladdin. 68 Ge / 68 The Ga generator was purchased from [company name missing], and the Signa PET / MR system was purchased from General Electric Company, USA. Radioactivity counting was performed using a radiometric chromatography scanner (Hefei Zhongcheng Electromechanical Technology Development Co., Ltd.). The radiochemical purity of the product was analyzed by radiometric high-performance liquid chromatography (HPLC) (Shimadzu Instruments Suzhou Co., Ltd.) at a flow rate of 1 mL / min. Analytical methods: Solvent A, H₂O with 0.1% TFA; Solvent B, CH₃CN containing 0.1% TFA (gradient: 0–1 min 40% B, 1–9 min 40–90% B, 9–10 min 90–40% B).

[0019] Example 1 Dual-target small molecule probe 68 Preparation method of Ga-DOTA-PLE / RGD: Step 1: Place 2-CTC resin (2-chlorotriphenylmethyl chloride resin) into a reaction vessel, swell with dichloromethane for 1 hour, wash three times with anhydrous N,N-dimethylformamide, and dry under vacuum; add the first amino acid Dde-Lys(Fmoc)-OH to the reaction vessel, add 1.5 equivalents of N,N-diisopropylethylamine, and use N,N-dimethylformamide as a solvent to react with the resin for 2 hours; dry the solution under vacuum, wash three times with anhydrous N,N-dimethylformamide, dry under vacuum, add appropriate amounts of methanol and N,N-diisopropylethylamine, and react for half an hour to block the unreacted active sites on the resin, obtaining the resin compound, named compound B; Step 2: Weigh compound B and place it in a reaction vessel. Add 20% piperidine / N,N-dimethylformamide solution and react for 0.5 hours to remove the Fmoc protecting group. Then add 3.0 equivalents of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetratert-butyl ester (DOTA(OtBu)3), 3.0 equivalents of N,N-diisopropylethylamine, and 3.0 equivalents of benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU). Using N,N-dimethylformamide as a solvent, react with the resin for 2 hours to obtain the resin compound, named compound C. Step 3: Weigh compound C and place it in a reaction vessel. Add 2% hydrazine hydrate / N,N-dimethylformamide solution and react for 2 hours to remove the Dde protecting group. Then add 3.0 equivalents of amino acid Fmoc-Lys(Dde)-OH, 3.0 equivalents of N,N'-diisopropylcarbodiimide, and 3.0 equivalents of 1-hydroxybenzotriazole (HOBT). Using N,N-dimethylformamide as a solvent, react with the resin for 1.5 hours to obtain the resin compound, named compound D. Step 4: Weigh compound D and place it in a reaction vessel. Add 20% piperidine / N,N-dimethylformamide solution and react for 0.5 hours to remove the Fmoc protecting group. Then add 3.0 equivalents of amino acid Fmoc-Pro-OH, 3.0 equivalents of N,N'-diisopropylcarbodiimide, 3.0 equivalents of 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide as a solvent, and react with the resin for 1.5 hours to obtain the resin compound. Then, replace the above amino acid Fmoc-Pro-OH with amino acid Fmoc-Thr(tBu)-OH, amino acid Fmoc-Pro-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Thr(tBu)-OH, and amino acid Fmoc-Lys(Fmoc)-OH in sequence, and repeat the above reaction to finally obtain the resin compound, named compound E. Step 5: Weigh compound E into a reaction vessel, add 20% piperidine / N,N-dimethylformamide solution, and react for 0.5 hours to remove the Fmoc protecting group. Then add 3.0 equivalents of di-tert-butyl dicarbonate ((Boc)₂O), 3.0 equivalents of N,N-diisopropylethylamine, and N,N-dimethylformamide as solvent, and react with the resin for 2 hours to remove the Boc protecting groups on all terminal amino groups. The resulting resin compound is named compound F. Step Six: Weigh compound F and place it in a reaction vessel. Add 2% hydrazine hydrate / N,N-dimethylformamide solution and react for 2 hours to remove the Dde protecting group. The resulting resin compound is named compound G. Step 7: Weigh compound G into a reaction vessel, add 3.0 equivalents of amino acid Fmoc-Arg(Pbf)-OH, 3.0 equivalents of N,N'-diisopropylcarbodiimide, 3.0 equivalents of 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide as a solvent, and react with the resin for 1.5 hours to obtain the resin compound. Then, replace the above amino acid Fmoc-Pro-OH with amino acid Fmoc-Gly-OH, amino acid Fmoc-Asp(tBu)-OH, and amino acid Boc-Phe-OH in sequence, and repeat the above reaction to finally obtain the resin compound, named compound H; Step 8: Weigh compound H and place it in a reaction vessel. Add trifluoroacetic acid (TFA): triisopropylsilane (Tri): 1,2-ethylenedithiol (EDT): water in a volume ratio of 95%:2%:2%:1%. React for 2 hours to remove all protecting groups. The polypeptide compound is obtained by high performance liquid chromatography and named compound DOTA-PLE / RGD. Step 9: Dissolve 50 μg of DOTA-PLE / RGD in 125 μL of sodium acetate (1.25 M), and dilute with 5 mL of HCl (0.1 M). 68 Ge- 68 68GaCl3 was eluted in a Ga generator, and 1 mL of 68GaCl3 was added to the DOTA-PLE / RGD solution. The mixture was heated at 95°C for 10 min. After cooling to room temperature, the drug was transferred to a Sep-Pak C18 column, and then the Sep-Pak C18 silica column was washed with 0.8 mL of 80% ethanol to obtain the final radiopharmaceutical, which was named compound. 68 Ga-DOTA-PLE / RGD.

[0020] The compound DOTA-PLE / RGD prepared above was analyzed using high-resolution mass spectrometry, and the results are as follows: Figure 18 As shown, specifically, Figure 18This is a high-resolution mass spectrum of the pure compound DOTA-PLE / RGD, with the result being HRMS (ESI-TOF) m / z: [M+H]+calcd for C. 85 H 144 N 23 O 25 + 1887.07, found 1887.66. Detection Example 1 Figure 1 The synthesis and labeling process of 68Ga-DOTA-PLE / RGD is described. After radiopharmaceutical synthesis, rapid determination within one minute was performed using radioactive TLC. 68 Radiochemical purity (RCP) of Ga-DOTA-PLE / RGD, such as Figure 2 As shown in A, 68 The retention time of Ga-DOTA-PLE / RGD is 0.53, while 68 The retention time of GaCl3 is 0.22. For example... Figure 2 As shown in B, 68 The radiochemical purity of Ga-DOTA-PLE / RGD exceeded 99% after purification, indicating its high radiochemical purity. Furthermore, the octanol / water partition coefficient (Log P) of the final product was -1.96 ± 0.04, suggesting good hydrophilicity.

[0021] Detection Example 2 In vitro stability: In order to determine 68 To investigate the in vitro stability of Ga-DOTA-PLE / RGD, we mixed the radioactive product with PBS, mouse serum, and human serum, respectively, and incubated them. At 5 min, 30 min, 1 h, and 2 h after incubation, a certain volume of the drug was extracted using a micropipette for purity determination. The experimental results were as follows: Figure 3 Figure 4 .like Figure 4 As shown, the results of radioactive HPLC detection indicate that the drug did not exhibit any other significant peaks at the aforementioned time points. Furthermore, after incubation for 2 hours with PBS, mouse serum, and human serum, respectively, the radiochemical purity of the radioproduct remained greater than 93%. This demonstrates... 68 Ga-DOTA-PLE / RGD exhibits good stability in vitro.

[0022] The specific detection method includes: taking 10 µL 68 Ga-DOTA-PLE / RGD was mixed with 0.5 mL of n-octanol and 0.5 mL of water, and stirred thoroughly for 1 min using a vortex mixer. The organic and aqueous layers were separated by centrifugation. A 100 µL sample was taken from each layer and counted using a gamma counter. The experiment was repeated three times. A small amount was taken using a capillary tube.68 Ga-DOTA-PLE / RGD was spotted onto the bottom of a saturated filter paper for thin-layer chromatography, followed by the application of the saturated filter paper. 68 Saturated filter paper for Ga-DOTA-PLE / RGD thin-layer chromatography was placed in a developing solution containing methanol and ammonium acetate (1 M) (volume ratio 1:1). After a period of time, the developing solution was dried and placed on a radiometric thin-layer chromatography scanner for detection. 500 µL of each solution was taken... 68 Ga-DOTA-PLE / RGD (18.5 MBq) was mixed with 0.5 mL PBS, fresh mouse serum, and human serum and incubated at room temperature. Samples were taken at 5 min, 30 min, 1 h, and 2 h after mixing, and analyzed by Radio-HPLC. 68 Stability of Ga-DOTA-PLE / RGD.

[0023] Detection Example 3 Cytotoxicity assay: To determine the cytotoxicity of DOTA-PLE / RGD, we prepared DOTA-PLE / RGD solutions at different concentrations and used the viability of Bxpc-3 and Pacn-1 cells to reflect the drug's toxicity. The results of the cytotoxicity assay are as follows: Figure 5 As shown, at the highest concentration of 100 μM, the activity of Bxpc-3 cells was greater than 88%, and the activity of Pacn-1 cells was greater than 90%. However, the drug concentration required in animal experiments is much lower than 100 μM. The experimental results indicate that DOTA-PLE / RGD has no obvious cytotoxicity.

[0024] The specific experimental method included: uniformly spreading the two types of cells on a 96-well plate and culturing for 24 hours (4000 cells / well). On the second day, different concentrations of DOTA-PLE / RGD (1.56, 3.12, 6.25, 12.5, 25, 50, 100 µM) were mixed with the cells and incubated for 24 hours. After adding CCK8 reagent, the cells were incubated for 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader.

[0025] Detection Example 4 Cell blockade assay: This experiment used Bxpc-3 and Pacn-1 cells to perform the blockade assay to detect... 68 Ga-DOTA-PLE / RGD's binding ability to Plectin and Integrin targets.

[0026] In the Bxpc-3 cell blockade assay, compared with the PLE+RGD blockade group, the blockade rates at 5 min, 30 min, 1 h, and 2 h were 73.6%, 70.3%, 62.5%, and 58.8%, respectively (p < 0.05). In the PLE blockade group, the blockade rates at the same time points were 55.1%, 55.9%, 41.8%, and 52.2%, respectively; while the blockade rates after RGD blockade were 56.8%, 39.3%, 31.0%, and 30.3%, respectively (results shown in 6, 7, and 8).

[0027] In the Pacn-1 cell blockade experiment, compared with the PLE+RGD blockade group, the blockade rates of the experimental group at 5 min, 30 min, 1 h, and 2 h were 42.8%, 35.0%, 34.9%, and 33.0%, respectively (p < 0.05). However, the blockade effect was not significant in the PLE blockade group and the RGD blockade group (results are shown in the figure). Figure 8 , 10 (As shown in Figure 11).

[0028] The blocking results in the experimental group and the PLE+RGD group showed that both inhibitors had significant blocking effects on this dual-target drug. The blocking results in the experimental group, the PLE+RGD group, and the PLE group indicated that the single inhibitor PLE had some blocking effect on this dual-target drug; however, due to the presence of the RGD target, the dual-target drug could still specifically bind to the RGD target. Similarly, although the single RGD inhibitor had some blocking effect on this dual-target drug, its blocking effect was weaker than that in the PLE+RGD group due to the presence of the PLE target. The combined data from these four groups demonstrate the specific binding of this dual-target drug to both targets.

[0029] The specific experimental method included: seeding Bxpc-3 cells evenly in 24-well culture plates at a density of 2 × 10⁵ cells per well. The next day, the cells were then... 68 Ga-DOTA-PLE / RGD (20 µL, 10 µCi) was incubated at room temperature for 5 min, 30 min, 1 h, and 2 h. After each time point, the cells were washed three times with 0.5 mL PBS to remove unbound drug. Finally, 0.5 mL NaOH was added to each well, and the cells were repeatedly pipetted and the lysates were collected. Radioactivity was measured using a gamma counter. To verify the selectivity of the cells to the drug, three blocking groups were established: PLE blocking group, RGD blocking group, and PLE+RGD blocking group. An excess of the corresponding blocking agent was added to each group, and the cells were incubated at room temperature for 30 min. The cell uptake experiment was then repeated. Radioactivity is expressed as counts per minute (CPM).

[0030] Pacn-1 cells were seeded evenly in 24-well plates at a density of 2 × 10⁵ cells per well. The next day, the cells were... 68 Ga-DOTA-PLE / RGD (20 µL, 2 µCi) was incubated at room temperature for 5 min, 30 min, 1 h, and 2 h. After each time point, the cells were washed three times with 0.5 mL PBS to remove unbound drug. Finally, 0.5 mL NaOH was added to each well, and the cells were repeatedly pipetted and the lysates were collected. Radioactivity was measured using a gamma counter. To verify the selectivity of the cells to the drug, three blocking groups were established: PLE blocking group, RGD blocking group, and PLE+RGD blocking group. An excess of the corresponding blocking agent was added to each group, and the cells were incubated at room temperature for 30 min. The cell uptake experiment was then repeated. Radioactivity is expressed as counts per minute (CPM).

[0031] Case 5 Pharmacokinetics Experiment: 68 The distribution half-life of Ga-DOTA-PLE / RGD (Tα / 2 = 3.48 min) indicates its rapid distribution from the blood to target tissues, consistent with the early high uptake observed in experiments (liver, lung, kidney). The elimination half-life (Tβ / 2 = 11.90 min) indicates that the radiotracer has a moderate residence time in the blood, providing a sufficient time window for binding to the target without increasing potential toxicity due to prolonged residence (e.g., Figure 12 (As shown). These pharmacokinetic properties support... 68 Potential clinical applications of Ga-DOTA-PLE / RGD as a PET tracer.

[0032] The specific experimental methods included: taking 3 female BABL / C mice for testing. 68 Pharmacokinetics of Ga-DOTA-PLE / RGD in vivo. Mice were administered the drug via tail vein injection. 68 Ga-DOTA-PLE / RGD, dose 3.7 MBq / eye, volume approximately 150 µL. Blood samples were collected via capillary tube through the orbital rim at 3, 5, 10, 15, 30, 45, 60, 90, 120, 150, and 180 min post-injection. All samples were weighed, and their radioactivity was measured using a gamma counter. An additional dose was used as an internal control for calibration. Pharmacokinetic analysis was performed using Prism 9.5 (Graph-PAD software) with a two-phase attenuation least squares fitting method, and results are expressed as percentage of injected dose (%ID) / g.

[0033] Case 6 Biodistribution of normal mice: Figure 13The data presents biodistribution data for healthy mice at 5 min, 30 min, 1 h, and 2 h after drug injection. (See figure.) 68 The distribution ratio of Ga-DOTA-PLE / RGD in the kidneys is much higher than in other organs. This is related to the drug's good water solubility. The distribution ratio in other organs is lower, so when the drug is applied to imaging, it can avoid the result of absorption by other organs and interference with tumor imaging.

[0034] The specific experimental method included: taking 12 female BABL / C mice (approximately 6 weeks old) and injecting them via the tail vein. 68 Ga-DOTA-PLE / RGD (3.7 MBq, 150 µL per mouse) was administered via orbital blood sampling at 5 min, 30 min, 1 h, and 2 h post-injection. Mice were then immediately euthanized by cervical dislocation (n=3 per time point). Major organs (heart, liver, spleen, lung, kidney, stomach, small intestine, large intestine, muscle, bone, and brain) were dissected and collected. The gamma counters were used to measure and weigh the organs. The radioactivity absorption of each organ was expressed as a percentage of the injected radioactive dose per gram of tissue (%ID / g). An additional dose was used as an internal control for calibration.

[0035] Case 7 Mouse weight measurement: to verify 68 To determine the in vivo toxicity of Ga-DOTA-PLE / RGD in mice, we injected mice with an overdose of the drug and observed changes in their body weight. The experimental results are as follows: Figure 14 As shown, the mice in the experimental group (n=4) and the PBS blank control group (n=4) did not experience significant weight loss within one week after injection of the drug and PBS, respectively, demonstrating the biocompatibility of the drug in vivo.

[0036] The specific experimental method included: eight BABL / C mice were randomly selected and divided into an experimental group and a control group. The experimental group was injected with 500 times the human dose (1.85 MBq / Kg), while the control group was injected with the same volume of PBS. The changes in the weight of the mice were observed for one week.

[0037] Detection Example 8 PET / MR imaging of mice: respectively 68PET / MR imaging of tumor-bearing mice was performed at 30 min, 1 h, and 2 h after Ga-DOTA-PLE / RGD injection. In Pacn-1 tumor mice, significant radioactivity accumulation was observed in the tumors of the experimental group in the early stage, but the accumulation decreased rapidly over time, resulting in unsatisfactory imaging results. In Bxpc-3 tumor mice, significant radioactivity accumulation was observed in the tumors of the experimental group at 30 min, with a maximum SUV of 59.34±14.99. In contrast, the radioactivity accumulation in the tumors of the blockade group (PLE+RGD) was significantly reduced, with a maximum SUV of 21.80±4.21. The radioactivity of the tumors was also significantly reduced in both the PLE and RGD blockade groups, with maximum SUVs of 38.48±3.99 and 25.22±8.10, respectively, but both were greater than the maximum SUV of the blockade group (PLE+RGD). In the imaging results at 1h and 2h, the maximum SUV value of the tumor in the experimental group decreased from 29.52±12.30 to 13.86±6.75, while the maximum SUV value of the tumor in the blockade group (PLE+RGD) decreased from 13.47±6.37 to 5.20±2.70. The maximum SUV value of the tumor in the PLE blockade group decreased from 13.23±1.16 to 5.13±0.34, while the maximum SUV value of the tumor in the RGD blockade group decreased from 11.47±7.96 to 5.42±3.00. The experimental results indicate that when a single target is blocked, the presence of another target leads to partial uptake of the radioactive probe by the tumor. However, when both targets are blocked, the tumor uptake is lower than that of a single target. Furthermore, as shown in the figure, at 30 min, the T / M value of the experimental group was 3.02±0.17, while the T / M value of the blocking group (PLE+RGD) was 1.75±0.32. At 1 h, the T / M value of the experimental group was 3.30±0.29, while the T / M value of the blocking group (PLE+RGD) was 1.72±0.03. At 2 h, the T / M value of the experimental group was 3.77±0.80, while the T / M value of the blocking group (PLE+RGD) was 1.88±0.47. At all three time points, the T / M values ​​of the experimental group and the blocking group (PLE+RGD) were statistically significant (p<0.05*). (Results as follows) Figure 15 and 16 (As shown) This indicates that the tumor has a significant impact on... 68 The selective uptake of Ga-DOTA-PLE / RGD drugs also demonstrates the advantages of dual-target drugs over single-target drugs.

[0038] The specific experimental method included: dividing 12 nude mice bearing Bxpc-3 tumors into 4 groups of 3 mice each, and injecting the drug into each group via the tail vein. 68Ga-DOTA-PLE / RGD (2.96 MBq, 150 μL) was administered via PET / MR scanning at 30 min, 1 h, and 2 h post-injection. The blocking groups were divided into PLE blocking, RGD blocking, and PLE+RGD blocking groups. Mice in the blocking groups (n = 3) received an overdose of the corresponding blocking agent via tail vein injection 30 min before drug injection, and PET / MR scanning was performed 30 min later using the same method. During scanning, mice were anesthetized with 1.5% isoflurane gas in a 0.5 mL / min oxygen flow, and their body temperature was maintained by a heated airflow under the bed. For data analysis, regions of interest (ROIs) were plotted for the tumor and contralateral quadriceps muscle in the fused images. The tumor-to-muscle (T / M) ratio was calculated as the ratio of the mean SUVmax of the tumor to the mean SUVmax of the contralateral quadriceps muscle.

[0039] In addition, we administered the drug via tail vein to three nude mice carrying Pacn-1 tumors. 68 Ga-DOTA-PLE / RGD (2.96 MBq, 150 μL) was subjected to PET / MR scans at 30 min, 1 h, and 2 h after drug injection, and images were obtained.

[0040] Case 9 Biodistribution in tumor-bearing mice: A biodistribution experiment was conducted in tumor-bearing mice 30 minutes after drug injection. The results are as follows: Figure 17 As shown, the drug mainly accumulates in the kidneys, which is related to the drug's hydrophilicity. Furthermore, the radioactive uptake of the tumors in the experimental group (%ID / g: 0.40±0.07) was higher than that in the blocking group (PLE+RGD) (%ID / g: 0.14±0.01), indicating a statistically significant difference between the two groups (p<0.05*).

[0041] The specific experimental method included: 12 mice bearing Bxpc-3 tumors (6-8 weeks) were divided into 4 groups of 3 mice each, and all mice were injected via tail vein. 68 Following Ga-DOTA-PLE / RGD (2.96 MBq, 150 μL), biodistribution studies were conducted. Mice were euthanized 30 minutes after injection by cervical dislocation. Major organs (heart, liver, spleen, lungs, kidneys, stomach, small intestine, large intestine, muscles, bones, brain, tumors, and orbital blood) were dissected and collected. Radioactivity was measured using a gamma counter and the organs were weighed. The blocking groups were divided into PLE blocking group, RGD blocking group, and PLE+RGD blocking group. The blocking groups (n = 3) were injected with... 68 An overdose of the corresponding inhibitor was injected 30 minutes before Ga-DOTA-PLE / RGD administration. Radioactive absorption in each organ was expressed as a percentage of the injected radioactive dose per gram of tissue (%ID / g). A separate dose was used as an internal control for correction.

[0042] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0043] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0044] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A dual-target small molecule probe, characterized in that, The structural formula of the dual-target small molecule probe is as follows: 。 2. A radionuclide-labeled dual-target small molecule probe, characterized in that, The radionuclide-labeled dual-target small molecule probe is composed of the dual-target small molecule probe described in claim 1 and a radionuclide. 68 The radionuclide-labeled dual-target small molecule probe was prepared using Ga; the structural formula of the probe is as follows: 。 3. A method for preparing a radionuclide-labeled dual-target small molecule probe as described in claim 2, characterized in that, The preparation method includes: dissolving the dual-target small molecule probe DOTA-PLE / RGD in sodium acetate, and then using HCl to... 68 Ge- 68 Elution in Ga generator 68 GaCl3, and take 68 GaCl3 was added to the DOTA-PLE / RGD solution, heated at 95 °C for 10 min, cooled to room temperature, and then transferred to a Sep-Pak C18 column. After rinsing the Sep-Pak C18 silica column with ethanol, a radionuclide-labeled dual-target small molecule probe was obtained. 68 Ga-DOTA-PLE / RGD; 。 4. A method for preparing a dual-target small molecule probe as described in claim 1, characterized in that, The preparation method includes: Step 1: Mix and react the amino acid Dde-Lys(Fmoc)-OH shown in Formula A, 2-chlorotriphenylmethyl chloro resin, N,N-diisopropylethylamine, and N,N-dimethylformamide to obtain the intermediate shown in Formula B; Step 2: The intermediate shown in Formula B is reacted with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group; then it is reacted with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetratert-butyl ester (DOTA(OtBu)3), N,N-diisopropylethylamine, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), and N,N-dimethylformamide to obtain the intermediate shown in Formula C; Step 3: The intermediate shown in Formula C is reacted with hydrazine hydrate / N,N-dimethylformamide solution to remove the Dde protecting group. Then it is reacted with amino acid Fmoc-Lys(Dde)-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide to obtain the intermediate shown in Formula D. Step 4: React the intermediate shown in Formula D with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group, and then react it with amino acid Fmoc-Pro-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide. Then, the above-mentioned amino acid Fmoc-Pro-OH was replaced in sequence with amino acid Fmoc-Thr(tBu)-OH, amino acid Fmoc-Pro-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Leu-OH, amino acid Fmoc-Thr(tBu)-OH, and amino acid Fmoc-Lys(Fmoc)-OH, and the above reaction was repeated to obtain the intermediate shown in Formula E; Step 5: React the intermediate shown in Formula E with piperidine / N,N-dimethylformamide solution to remove the Fmoc protecting group, and then react it with di-tert-butyl dicarbonate ((Boc)2O), N,N-diisopropylethylamine and N,N-dimethylformamide to obtain the intermediate shown in Formula F; Step 6: React the intermediate shown in Formula F with hydrazine hydrate / N,N-dimethylformamide solution to remove the Dde protecting group and obtain the intermediate shown in Formula G; Step 7: Mix and react the intermediate shown in Formula G, the amino acid Fmoc-Arg(Pbf)-OH, N,N'-diisopropylcarbodiimide, 1-hydroxybenzotriazole (HOBT), and N,N-dimethylformamide. Then, the above-mentioned amino acid Fmoc-Pro-OH was replaced in sequence with amino acid Fmoc-Gly-OH, amino acid Fmoc-Asp(tBu)-OH, and amino acid Boc-Phe-OH, and the above reaction was repeated to obtain the intermediate shown in formula H. Step 8: Mix the intermediate shown in Formula H with trifluoroacetic acid (TFA), triisopropylsilane (Tri), 1,2-ethylenedithiol (EDT), and water to remove all protecting groups and obtain a dual-target small molecule probe.

5. The radionuclide-labeled dual-target small molecule probe according to claim 2 is applied to PET / MR imaging of pancreatic cancer tumors.

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