A fibroblast activation protein FAP and α v Dual-targeting radiopharmaceuticals of β3 integrin, their preparation methods and applications

Abstract

This invention discloses a series of dual-targeting radiopharmaceuticals targeting FAPI-2286 and cyclic RGDfK peptides, and their optimization through linker engineering, belonging to the fields of nuclear medicine, molecular imaging, and tumor targeted therapy. This invention relates to dual-targeting radiopharmaceutical precursors, wherein the dual-targeting radiopharmaceutical precursors are FR, FPR, and FPNR, and the precursors consist of an FAPI-2286 derivative targeting fibroblast activation proteins and an integrin α-targeting protein. v The invention utilizes the following structural strategies: (1) Directly linking functional groups simplifies the molecular structure, reduces molecular weight, and facilitates probe metabolism; (2) Using PEG4 as a linker reduces the steric hindrance between FAPI and the RGD functional module, increases probe flexibility, and enhances the synergistic recognition ability of dual-target probes on the target site; (3) Introducing a naphthyl structure on the basis of the PEG4 linker can further regulate the hydrophobicity of the probe and prolong the plasma half-life. This invention achieves synergistic targeting of the tumor microenvironment and tumor cells through linker optimization, wherein the PEG4 linker structure... 177 Lu-FPR exhibits higher tumor uptake, longer tumor retention time, and superior radionuclide therapy efficacy, and can be used for tumor imaging, efficacy evaluation, and targeted radiotherapy.

Description

Technical Field

[0001] This invention relates to the fields of nuclear medicine, molecular imaging, and targeted tumor therapy, specifically to a fibroblast activation protein FAP and α. v Dual-targeting radiopharmaceuticals of β3 integrin, their preparation methods and applications. Background Technology

[0002] Targeted radiopharmaceuticals, by combining radionuclides with targeted ligands that have specific recognition capabilities, enable precise imaging and treatment of diseased tissues, and have received widespread attention and rapid development in the field of tumor diagnosis and treatment in recent years. Especially... 177 Therapeutic radionuclides, represented by Lu, have shown promising clinical application prospects in the treatment of various solid tumors due to their suitable beta-ray energy and long half-life.

[0003] However, most approved or research-stage radiopharmaceuticals currently rely primarily on single molecular targets, such as PSMA, SSTR, or FAP. This single-target strategy is susceptible to the heterogeneity of tumor molecular expression and temporal-spatial dynamics in practical applications, leading to insufficient uptake in some tumor lesions, heterogeneous imaging, and limited therapeutic efficacy, thus affecting overall treatment outcomes.

[0004] The complexity of the tumor microenvironment further exacerbates these problems. Tumor tissue is composed not only of tumor cells but also of various components such as cancer-associated fibroblasts (CAFs), tumor blood vessels, and immune cells. Among these, CAFs are highly enriched in various solid tumors and restrict drug penetration and distribution within tumor tissue by forming a dense matrix barrier, thereby reducing treatment efficiency. FAP, a transmembrane serine protease highly expressed on the surface of CAFs, has become an important molecular target for tumor microenvironment targeting in recent years.

[0005] On the other hand, α v β3 integrins are highly expressed in various tumor cells and neovascular endothelial cells, playing a crucial role in tumor cell adhesion, invasion, and angiogenesis. Their classic ligand, RGD, has been widely used in tumor-targeting molecular design. Therefore, simultaneously targeting FAP and α... v β3 integrins are expected to synergistically enhance the tumor recognition capabilities of radiopharmaceuticals at both the tumor microenvironment and tumor cell levels.

[0006] Although existing research has reported FAP / α v Dual-targeting molecular probes such as β3 are used, but existing technologies still have the following shortcomings: (1) The complex structure of dual-target molecules usually leads to an increase in molecular weight, which affects the pharmacokinetic behavior in vivo; (2) Spatial conformational mismatch between different target groups may reduce receptor binding efficiency; (3) The lack of systematic optimization in the design of the connecting arm makes it difficult to balance tumor uptake and in vivo clearance; (4) Existing dual-target probes still have room for improvement in tumor retention time and therapeutic effect.

[0007] Therefore, how to achieve dual-target radiopharmaceuticals with high tumor uptake, long retention time and excellent pharmacokinetic properties through rational molecular structure design, especially through Linker engineering to optimize the spatial configuration and physicochemical properties of dual-target molecules, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention proposes a fibroblast activation protein FAP and α... v Dual-targeting radiopharmaceuticals of β3 integrin, their preparation methods and applications.

[0009] The objective of this invention can be achieved through the following technical solutions: A first aspect of the present invention relates to a dual-targeting radiopharmaceutical precursor, the dual-targeting radiopharmaceutical precursor being an FPR, the FPR comprising an FAPI-2286 derivative, a cyclic RGDfK peptide, a DOTA, and a PEG4 linker arm; the FAPI-2286 derivative being linked to the cyclic RGDfK peptide via the PEG4 linker arm, and the DOTA being linked to a dual-targeting intermediate formed by the FAPI-2286 derivative and the cyclic RGDfK peptide.

[0010] Optionally, the FAPI-2286 derivative is a maleimide-modified FAPI-2286 derivative, the cyclic RGDfK peptide is a thiol-modified cyclic RGDfK peptide, and a thiosuccinimide linkage structure is formed between the FAPI-2286 derivative and the cyclic RGDfK peptide.

[0011] Optionally, the PEG4 linker includes four ethylene glycol repeating units.

[0012] A second aspect of the present invention relates to a dual-targeting radiopharmaceutical, the dual-targeting radiopharmaceutical comprising the above-described dual-targeting radiopharmaceutical precursor and a radionuclide chelated with the DOTA.

[0013] Optionally, the radionuclide is 177 Lu.

[0014] A third aspect of the present invention relates to a method for preparing the above-mentioned dual-targeted radiopharmaceutical precursor, comprising the following steps: Synthesize maleimide-modified FAPI-2286 derivatives; Synthesize cyclic RGDfK derivatives containing PEG4 linker arms and modified with thiol groups; The maleimide-modified FAPI-2286 derivative was subjected to a thiol-maleimide coupling reaction with the cyclic RGDfK derivative containing a PEG4 linker and modified with thiol to obtain a dual-targeting intermediate. The dual-targeting intermediate is reacted with NHS-DOTA to obtain the dual-targeting radiopharmaceutical precursor.

[0015] Optionally, the cyclic RGDfK derivative containing a PEG4 linker and modified with thiol groups is prepared by Fmoc solid-phase peptide synthesis and then purified by Alloc deprotection, DPPA-mediated coupling, acid cleavage deprotection, and preparative high-performance liquid chromatography.

[0016] Optionally, the maleimide-modified FAPI-2286 derivative is prepared by the following steps: preparing FAPI-2286 related intermediates using 2-chlorotriphenylmethyl chloride resin and Fmoc solid-phase peptide synthesis method, reacting them with 1,3,5-tris(bromomethyl)benzene after acid cleavage and deprotection, and then reacting them with 3-(maleimide)propionic acid-N-hydroxysuccinimide ester.

[0017] A fourth aspect of the invention relates to a 177 The preparation method of Lu-labeled dual-targeting radiopharmaceutical includes the following steps: The above-mentioned dual-targeting radiopharmaceutical precursor was dissolved in dimethyl sulfoxide to obtain a precursor stock solution; Will 177 LuCl3 was added to sodium acetate buffer, and the precursor stock solution was also added. The resulting reaction mixture was reacted at 90°C for 30 minutes to obtain a 177Lu-labeled dual-targeting radiopharmaceutical; The sodium acetate buffer solution has a pH of 4.5.

[0018] The fifth aspect of the present invention relates to a 177 Lu-FPR in the preparation of FAP and α v The application of β3 integrin-related solid tumor diagnostic reagents, efficacy assessment reagents, or radionuclide therapy drugs, wherein... 177 Lu-FPR is a precursor composed of FAPI-2286 derivative, PEG4 linker, cyclic RGDfK peptide, and DOTA. 177 Complexes formed by Lu.

[0019] More specifically, the connecting arm module is selected from: (a) Direct connection structure; (b) PEG4 linkage structure; (c) PEG4-naphthalene ring linkage structure.

[0020] Its structural formula is: Optionally, the dual-targeting radiopharmaceutical is FR, whose structure includes: FAPI-2286-RGD-DOTA.

[0021] Optionally, the dual-targeting radiopharmaceutical is FPR, whose structure includes: FAPI-2286-PEG4-RGD-DOTA.

[0022] Optionally, the dual-targeting radiopharmaceutical is FPNR, whose structure includes: FAPI-2286-PEG4-Naph-RGD-DOTA.

[0023] Optionally, the radionuclide is selected from: 177 Lu、 68 Ga、 64 Cu、 90 Y、 111 In and 225 One or more of Ac.

[0024] The beneficial effects of this invention are: (1) By constructing FAP / α v The β3 dual-targeting system enables the synergistic recognition of tumor microenvironment (CAFs) and tumor cells / angiogenesis, thereby improving the overall tumor targeting coverage. (2) By introducing linker arms with different structures, the spatial conformation and flexibility of the dual-target molecules can be precisely controlled, thereby optimizing the receptor binding efficiency and in vivo behavior; (3) The PEG4 linker significantly improves molecular conformational matching, enabling FPR to exhibit optimal cellular uptake and receptor binding capacity. (4) FPR showed higher FAP binding affinity and cell uptake capacity in in vitro experiments, and higher tumor uptake and longer retention time in vivo. (5) The above 177 Lu-labeled FPR showed significant tumor growth inhibition in tumor treatment models and had good biocompatibility. (6) This invention achieves a systematic improvement in the performance of dual-target radiopharmaceuticals through “connector arm engineering optimization”, providing a new structural optimization strategy for the design of multi-target radiopharmaceuticals. Attached Figure Description

[0025] The invention will now be further described with reference to the accompanying drawings.

[0026] Figure 1 This is a diagram showing the radiochemical characterization results of a dual-targeted radioactive probe; where A represents free radioactive material. 177 Lu、 177 Lu-FR, 177 Lu-FPR and 177 Radio-TLC analysis plot of Lu-FPNR, B is... 177 The radio-HPLC chromatogram of Lu-FR, C is... 177 Radio-HPLC chromatogram of Lu-FPR, D is... 177 The radio-HPLC chromatogram of Lu-FPNR, E is... 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR lipid-water partition coefficient logD 7.4 Measurement results graph, F is 177 Figure showing the in vitro stability of Lu-FPR in PBS and serum.

[0027] Figure 2 The figure shows the in vitro cell experiments and in vivo pharmacokinetics results of the dual-targeted radioactive probe; where A is... 177 Lu-FR, 177 Lu-FPR and 177 A) shows the time-dependent uptake results of Lu-FPNR in U87-MG-FAP cells; B) shows the cellular uptake results of each probe after treatment with different blocking agents; C) shows the competitive inhibition curves of each probe binding to FAP; D) shows the cytotoxicity evaluation results of the non-radioactive precursors FR, FPR, and FPNR; E) shows... 177 The cellular radiotoxicity evaluation results of Lu-FPR are shown in the figure, where F to I represent... 177 Lu-FAPI-2286 177 Lu-FR, 177 Lu-FPR and 177 The blood clearance curve and half-life fitting results of Lu-FPNR in mice.

[0028] Figure 3 The images show the Micro-SPECT / CT imaging and tissue distribution results of various radioactive probes in tumor-bearing mice; where A represents... 177 Lu-FAPI-2286 177 Lu-FR, 177 Lu-FPR and 177Micro-SPECT / CT images of Lu-FPNR at 1 h, 4 h, 8 h, 24 h and 48 h after administration. B is the time-uptake curve of each probe in the tumor tissue. C is the radioactivity distribution of major tissues and organs at 48 h after administration. D is the tumor / normal tissue uptake ratio analysis of each probe at 48 h after administration.

[0029] Figure 4 for 177 The in vivo competitive blockade experiment results of Lu-FPR are shown in the figure; the figure shows the Micro-SPECT / CT imaging and tumor uptake quantitative analysis results of the unblocked group, FAPI-2286 blockade group, c(RGDfK) blockade group, and the combined FAPI-2286 and c(RGDfK) blockade group, which were used to verify the results. 177 Lu-FPR on FAP and α v Dual-target specificity of β3 integrin.

[0030] Figure 5 for 177 The results of the in vivo radionuclide therapy efficacy evaluation of Lu-FPR are shown in the figure. Among them, A is a schematic diagram of the drug administration and treatment observation process in tumor-bearing mice, B is a photograph of the tumors in each group after treatment, C is a curve of tumor volume change in each group of mice during treatment, D is a statistical result of tumor growth inhibition rate in each group, E is a statistical result of tumor weight in each group, and F is a result of H&E staining and FAP immunohistochemical staining of tumor tissues in each group.

[0031] Figure 6 for 177 The results of the Lu-FPR treatment safety evaluation in mice's peripheral blood are shown in the figure; the figure shows the saline group, 177 Lu-FAPI-2286 group and 177 The white blood cell count, red blood cell count, hemoglobin content, and platelet count of mice in the Lu-FPR group were used to evaluate whether significant bone marrow suppression or hematological toxicity occurred after treatment.

[0032] Figure 7 for 177 The results of serum biochemical safety evaluation of mice after Lu-FPR treatment are shown in the figure; the figure shows the saline group, 177 Lu-FAPI-2286 group and 177 The levels of alanine aminotransferase, aspartate aminotransferase, creatinine, and urea in Lu-FPR mice were used to evaluate changes in liver and kidney function after treatment.

[0033] Figure 8 for 177The figure shows the pathological evaluation results of major organ tissues in mice after Lu-FPR treatment; the figure also shows the saline group, 177 Lu-FAPI-2286 group and 177 The H&E staining results of the heart, liver, spleen, lung and kidney tissues of Lu-FPR group mice were used to evaluate whether there was significant tissue damage in the major organs after treatment.

[0034] Figure 9 This is a schematic diagram illustrating the experimental principle. Figure 10 The reaction route for solid-phase synthesis of polypeptide compounds 5, 6, and 7; Figure 11 The reaction route diagrams for compounds 11, 12, and 13 are shown. Figure 12 Reaction route diagram for maleimide functionalization of FAPI-2286 intermediate; Figure 13 This is a reaction route diagram for compound FR; Figure 14 This is a reaction route diagram for compound FPR; Figure 15 For compound FPNR or 177 Lu-labeled reaction route map. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Example 1: Dual-targeting precursors FR, FPR, FPNR and their 177 Preparation of Lu-labeled compounds I. General SPPS Preparation Example like Figure 10 As shown, 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis (Fmoc-SPPS) is a well-established chemical synthesis method. Compounds 5, 6, and 7 were synthesized using the following method; the synthetic route is shown in [link to synthetic route]. Figure 10 The specific steps are as follows: 1. Resin swelling: Weigh 2.146 g of 2-chlorotriphenylmethyl resin with an active group loading of 1.398 mmol / g (1% DVB, 3 mmol effective loading, 1 eq) into a dry solid-phase reaction tube, add 15 mL of anhydrous DMF to soak the resin, shake on a shaker for 15 min to allow the resin to fully disperse and activate the reactive groups of the resin, and then remove the DMF; 2. Weigh 2221.83 mg (5.4 mmol, 1.8 eq) of the first amino acid Fmoc-Asp(OtBu)-OH into a test tube, add 15 mL of anhydrous DMF to dissolve it completely, then add 1305 μL (7.5 mmol, 2.5 eq) of DIPEA, mix well, and then add the mixture into a solid-phase reaction tube to fully immerse the resin. Seal the tube and react it on a shaker at room temperature for 6 h. 3. End-capping: After the first amino acid reaction, remove the reaction liquid, add DMF to wash the resin three times, 10 mL each time, shaking for 3 min each time to remove unreacted amino acids and organic bases. Then add 10 mL of a 5% volume mixed DMF solution of methanol and DIPEA (i.e., MeOH:DIPEA:DMF = 1:1:20, v:v:v), shake for 30 min to seal the unreacted active groups on the resin. Then wash the resin three times with DMF to remove MeOH. 4. Deprotection of Fmoc protecting groups: Prepare a 20% piperidine DMF solution (piperidine:DMF = 1:4, v:v), add 10 mL of the mixture to fully soak the resin, shake and react for 8 minutes. After each reaction, remove the piperidine solution and add fresh piperidine solution. Repeat the reaction 3 times. After the reaction, wash the resin three times with DMF to remove the piperidine. Then, take a small amount of resin and use ninhydrin solution to test whether the protecting group has been removed. 5. Adding the second amino acid Fmoc-D-Phe-OH: Weigh Fmoc-D-Phe-OH (1743.5 mg, 4.5 mmol, 1.5 eq), HBTU (2047.95 mg, 5.4 mmol, 1.8 eq), and HOBT (729.65 mg, 5.4 mmol, 1.8 eq) into a test tube, add 15 mL of DMF, and sonicate to dissolve completely. Then add 1305 μL of DIPEA (7.5 mmol, 2.5 eq) to the solution and mix well. Add the mixture to a solid-phase reaction tube to immerse the resin and shake to react for 4 h. 6. Repeat steps 4 and 5 to react with Alloc-Lys-(Fmoc)-OH to obtain compound 1 [(Alloc-Lys(Fmoc)-D-Phe-Asp(OtBu)-Resin]. After removing the Fmoc from the Lys side chain, amino acids are sequentially linked according to different needs to obtain compounds 2, 3, and 4. Figure 10 Among: (a) Boc-Cys(Trt)-OH; (b) Fmoc-NH-PEG4-CH2CH2-COOH, Boc-Cys(Trt)-OH; (c) Fmoc-NH-PEG4-CH2CH2-COOH, Fmoc-3-(2-Naphthyl)-L-OH, Boc-Cys(Trt)-OH. 7. Removal of Alloc Protecting Group: After washing the resin three times with anhydrous DCM, 1043.6 mg (0.9 mmol, 0.3 eq) of tetra(triphenylphosphine)palladium and 7.838 mL (90 mmol, 30 eq) of morpholine were dissolved in anhydrous DCM to prepare a mixed solution with a total volume of 15 mL. After thorough sonication, the solution was added to a solid-phase reaction tube and the reaction was shaken for 2 h. Then the reaction solution was removed, and the resin was washed with 1% DIPEA / DMF solution and 15 mg / mL sodium diethyldithiocarbamate trihydrate solution for 5 minutes each time, until the removed solution was no longer yellow. The target catalyst was removed, and finally, excess sodium diethyldithiocarbamate was washed away with DMF.

[0037] 8. Then repeat steps 4 and 5 to link the remaining amino acids in sequence. Figure 10 In the middle: (d) Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, and finally the Fmoc protecting group of Fmoc-Gly-OH is removed; 9. Resin preparation: Wash the resin three times each with DMF, MeOH, petroleum ether, and DCM in sequence. Then, immerse the resin in 15 mL of 1% TFA / DCM solution and shake for 15 min. Collect the reaction solution in a flask and repeat the reaction until the resin turns black. Collect the reaction solution and concentrate the filtrate using a rotary evaporator. Add the concentrate dropwise to an ice-cold ether solution, centrifuge at low temperature, remove the ether solution, and collect the precipitate to obtain compounds 5, 6, and 7 as yellow solids with yields of 85%, 84%, and 65%, respectively.

[0038] like Figure 11 As shown, compounds 5, 6, and 7 obtained can be directly proceeded to the next reaction without further purification.

[0039] 1. Molecular self-cyclization: 0.7 mmol (1 eq) of compounds 5, 6, and 7 were weighed and placed in three different drying flasks. 3.5 mmol (294.04 mg, 5 eq) of NaHCO3 powder and 15 mL of DMF solution were added to each flask. After thorough sonication to dissolve, 453 μL (2.1 mmol, 3 eq) of DPPA solution was added, and the mixture was stirred at room temperature for 8 h. After the reaction was complete, the DMF was removed by vacuum evaporation and concentration. An appropriate amount of deionized water was added, and the mixture was freeze-dried in a lyophilizer to obtain crude products compounds 8, 9, and 10. 2. Removal of protecting groups by trifluoroacetic acid: Crude products 8, 9, and 10 were placed in different round-bottom flasks, and 10 mL of a mixed acid solution of TFA / TIS / H2O / EDT (94:2.5:2.5:1, v:v:v:v) was added to each flask. The mixture was stirred at room temperature for 2 h. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The concentrate was added dropwise to 40 mL of ice-cold diethyl ether, centrifuged at low temperature, and the supernatant diethyl ether solution was removed. The precipitate was collected, dried in a vacuum drying oven to remove the diethyl ether, and the products were separated and purified by preparative high-performance liquid chromatography to obtain compounds 11, 12, and 13, all of which were white solid compounds. The yields of the three compounds after the two-step reaction were 27%, 22%, and 36%, respectively.

[0040] 3. Preparation of high performance liquid chromatography separation and purification method: Compound 11: Compound 12: Compound 13: 4. Characterization of compounds 11, 12, and 13 Compound 11: 1 H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.24 (dq, J = 14.4, 7.5Hz, 5H), 4.78 (s, 1H), 4.44 (t, J = 7.7 Hz, 1H), 4.32-4.23 (m, 1H), 4.08-4.01(m, 1H), 3.95 (d, J = 5.5 Hz, 1H), 3.94-3.78 (m, 2H), 3.23 (t, J = 6.4 Hz, 2H),3.16-2.85 (m, 6H), 2.65 (ddd,J = 99.6, 16.8, 6.8 Hz, 2H), 1.86-1.66 (m, 4H),1.55-1.47 (m, 2H), 1.30 (s, 2H), 1.22 (d, J = 8.3 Hz, 2H). 13 C-NMR ( d 4 -MeOD, 100MHz, ppm) δ = 173.68, 172.79, 172.59, 172.25, 172.07, 170.70, 166.97, 157.25,136.66, 128.84, 128.20, 126.50, 56.18, 55.99, 54.78, 52.26, 50.05, 42.09,40.36, 39.17, 35.59, 34.59, 31.87, 28.29, 27.47, 25.24, 24.97, 22.81. MS-ESI: Calcd. For C 30 H 46 N 10 O8S [M+H] + 707.3, found : 707.4. For [M+2H] 2+ 354.2, found 354.2. 。

[0041] Compound 12: 1 H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.25 (dd, J = 16.8, 7.4Hz, 5H), 4.75 (d, J = 6.1 Hz, 1H), 4.41 (d, J = 7.4 Hz, 1H), 4.34-4.29 (m, 1H),4.07-4.00 (m, 1H), 3.96-3.89 (m, 1H), 3.73 (t, J = 6.1 Hz, 2H), 3.67-3.54 (m,12H), 3.43 (dd, J = 23.1, 5.0 Hz, 2H), 3.28-3.08 (m, 6H), 3.06-2.89 (m, 4H),2.77 (dd,J = 16.7, 6.1 Hz, 1H), 2.57-2.49 (m, 1H), 2.44 (t, J = 6.1 Hz, 2H),1.82 (d, J = 9.1 Hz, 2H), 1.70 (d, J = 7.4 Hz, 2H), 1.52 (t, J = 14.5 Hz, 2H), 1.30(d, J = 9.9 Hz, 6H). 13 C-NMR ( d 6 -DMSO, 100 MHz, ppm) δ = 172.39, 172.31, 172.06,171.82, 170.98, 170.44, 169.88, 167.23, 157.41, 138.03, 129.50, 128.58,126.78, 124.54, 110.16, 70.20, 70.17, 70.12, 70.03, 69.93, 69.15, 67.30,63.22, 56.02, 55.43, 54.37, 52.15, 49.67, 42.90, 38.90, 36.55, 36.09, 35.69,34.73, 34.32, 32.57, 29.05, 28.14, 25.74, 25.59, 23.06. MS-ESI: Calcd. For C 41 H 67 N 11 O 13 S [M+H] + 954.5, found : 954.5. For [M+2H] 2+ 477.9, found 477.9. 。

[0042] Compound 13: 1 H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.81 (t, J= 7.6 Hz,3H), 7.72 (s, 1H), 7.48-7.40 (m, 3H), 7.29-7.19 (m, 5H), 4.78-4.74 (m, 2H),4.41 (t, J = 7.7 Hz, 1H), 4.31 (dd, J = 8.9, 5.6 Hz, 1H), 4.10-4.04 (m, 1H), 3.99(t, J = 7.4 Hz, 1H), 3.94-3.83 (m, 2H), 3.69 (t, J = 6.1 Hz, 2H), 3.61-3.41 (m,12H), 3.40-3.35 (m, 2H), 3.22 (ddd, J = 24.0, 13.8, 7.4 Hz, 6H), 3.10 (t, J = 6.9Hz, 2H), 3.06-3.03 (m, 2H), 2.89 (dd, J = 13.6, 7.5 Hz, 2H), 2.78-2.50 (m, 2H),2.41 (t, J = 6.1 Hz, 2H), 1.79-1.65 (m, 4H), 1.64-1.40 (m, 4H), 1.30 (d, J = 9.4Hz, 2H), 1.27-1.15 (m, 2H). 13 C-NMR ( d 4-CD3OD, 100 MHz, ppm) δ = 173.55,172.81, 172.58, 172.47, 172.17, 172.05, 171.55, 170.60, 166.95, 157.24,136.67, 134.20, 133.56, 132.59, 128.93, 128.83, 128.19, 127.83, 127.64,127.28, 127.07, 126.50, 125.84, 125.43, 70.06, 70.03, 70.01, 69.86, 69.73, 68.92, 66.88, 56.12, 55.91, 55.25, 54.37, 52.20, 50.02, 47.90, 42.07, 40.34, 39.01, 38.82, 37.81, 36.22, 35.63, 34.50, 31.75, 28.43, 27.48, 25.19, 25.13, 22.80. MS-ESI: Calcd. For C 54 H 78 N 12 O 14 S [M+H] + 1151.6, found 1151.6. For [M+2H] 2+ 576.4, found 576.4.

[0043] II. Synthesis of maleimide-functionalized FAPI-2286 1. For example Figure 11As shown, amino acids Fmoc-Cys(Trt)-OH, Fmoc-Phe-OH, Fmoc-Gln-OH, Fmoc-Thr-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH were sequentially linked to the resin. After the reaction was completed, the resin was cut and dried to obtain polypeptide chain compound 14. Then, a mixed solution of TFA / TIS / H2O / EDT (94:2.5:2.5:1, v:v:v:v) was added and stirred for 2 h to remove the protecting group. The reaction solution was concentrated by vacuum evaporation and then added dropwise to ice-cold diethyl ether. After centrifugation to remove the supernatant, the diethyl ether was removed in a vacuum drying oven to obtain a yellow solid. The crude product was then separated and purified by preparative HPLC to obtain a white solid compound 15: Hex-Cys-Pro-Pro-Thr Gln-Phe-Cys-OH, with a yield of 58%. 528 mg of compound 15 (0.588 mmol, 1 eq) was dissolved in 15 mL of a mixed solution of ethanol and acetonitrile (EtOH:ACN = 1:1, v:v). 410 μL of DIPEA (2.352 mmol, 4 eq) was added and mixed thoroughly. Then, TBMB was added dropwise to the stirred reaction mixture in solution, for a total of 272.8 mg (0.764 mmol, 1.3 eq) of TBMB. The mixture was stirred at room temperature for 1 h. Then, 308 mg of β-mercaptoethylamine (6.468 mmol, 11 eq) was added and the mixture was stirred for another 2 h. After the reaction was complete, the ethanol and acetonitrile were evaporated under reduced pressure. The mixture was purified by preparative HPLC to obtain a white solid compound 16 in 65% yield. 2. Compound 16, 200 mg (0.184 mmol, 1 eq), and BMPS, 49.1 mg (0.221 mmol, 1.2 eq) were dissolved in DMF solvent. 65 μL of DIPEA (0.369 mmol, 2 eq) was added, and the mixture was stirred overnight. The product was concentrated under vacuum, lyophilized with water, and purified by preparative HPLC to obtain a white solid compound 17, with a yield of 37%. 3. Preparation of high performance liquid chromatography separation and purification method: Compound 15: Compound 17: 3. Characterization of Compound 17 1 H-NMR ( d 4-CD3OD, 400 MHz, ppm) δ = 7.33-7.22 (m, 8H), 6.82 (s, 2H),4.75-4.65 (m, 4H), 4.58 (t, J = 6.0 Hz, 1H), 4.34 (d, J = 8.3 Hz, 2H), 4.20 (dd, J = 7.9, 5.3 Hz, 1H), 3.94-3.71 (m, 10H), 3.65 (d, J = 9.8 Hz, 2H), 3.31 (d, J =7.0 Hz, 2H), 3.00-2.78 (m, 4H), 2.52 (dd, J = 24.3, 6.9 Hz, 4H), 2.37-1.83 (m,16H), 1.68-1.62 (m, 2H), 1.37 (q, J = 3.7 Hz, 4H), 1.20 (d, J = 6.1 Hz, 3H), 0.95(t, J = 6.9 Hz, 3H). 13 C-NMR ( d 4 -CD3OD, 100 MHz, ppm) δ = 176.43, 174.80, 172.82,171.96, 171.92, 171.89, 171.60, 171.48, 171.26, 170.81, 169.84, 139.85,138.92, 138.74, 134.11, 129.91, 128.34, 128.15, 126.45, 67.18, 60.38, 58.63,58.38, 54.62, 54.28, 51.97, 50.75, 38.32, 36.96, 35.56, 35.26, 35.11, 34.90,34.42, 34.10, 32.77, 31.28, 31.12, 30.91, 30.18, 28.31, 27.88, 26.16, 25.25,24.97, 24.51, 22.05, 18.81, 12.93. MS-ESI: Calcd. For C 58 H 78 N 10 O14 S [M+H] + 1235.5, found 1235.5. For [M+2H] 2+ 618.4, found 1235.5.

[0044] III. Preparation of FR / FPR / FPNR precursors 1. For example Figure 13 As shown, the synthesis and characterization of FR were as follows: 20 mg of compound 11 (0.028 mmol, 1.2 eq) and 29 mg of compound 17 (0.024 mmol, 1 eq) were weighed into a dry round-bottom flask, mixed thoroughly with 3 mL of DMF solution, and stirred overnight. No further purification was required. Then, 14.1 mg of DOTA-NHS Ester (0.028 mmol, 1.2 eq) and 17 μL of DIPEA were added to the reaction solution, and the reaction was continued at room temperature for 8 h. After the reaction, DMF was removed, and the crude product was lyophilized with water. The product was purified by preparative HPLC to obtain a white solid FR with a yield of 41%.

[0045] The preparation method is as follows: The characteristics are as follows: 1 H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.37-7.15 (m, 13H), 4.81 (t, J = 6.7Hz, 2H), 4.68 (d, J = 13.6 Hz, 5H), 4.57 (s, 1H), 4.45 (s, 1H), 4.34 (d, J = 12.3Hz, 3H), 4.21-4.15 (m, 1H), 4.09-3.61 (m, 23H), 3.35 (s, 10H), 3.30-3.07 (m,12H), 3.04-2.45 (m, 16H), 2.35-2.03 (m, 10H), 2.01-1.80 (m, 6H), 1.77-1.71(m, 2H), 1.69-1.46 (m, 6H), 1.34 (d, J = 19.6 Hz, 6H), 1.21 (d, J = 5.4 Hz, 3H),0.97-0.90 (m, 3H). HR-MS (ESI) m / z: Calculated. For C 104 H 150 N 24 O 29 S4, [M+H] + , 2328.9996, [M+2H] 2+ ,1165.0032, found 1165.0027.

[0046] 2. Synthesis and Characterization of FPR: The synthesis of FPR was similar to that of FR: 18 mg of compound 12 (0.019 mmol, 1.2 eq) and 20 mg of compound 17 (0.016 mmol, 1 eq) were weighed into a dry round-bottom flask, mixed thoroughly with 3 mL of DMF solution, and stirred overnight. No further purification was required. Then, 10.2 mg of DOTA-NHS Ester (0.019 mmol, 1.2 eq) and 16 μL of DIPEA were added to the reaction solution, and the reaction was continued at room temperature for 8 h. After the reaction, DMF was removed, water was added, and the crude product was lyophilized. The product was purified by preparative HPLC to obtain a white solid FPR with a yield of 36%.

[0047] The preparation method is as follows: The characteristics are as follows: 1H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.25 (dd, J = 16.8, 7.4 Hz, 5H), 4.75 (d, J = 6.1 Hz, 1H), 4.41 (d, J = 7.4 Hz, 1H), 4.34-4.29 (m, 1H), 4.07-4.00(m, 1H), 3.96-3.89 (m, 1H), 3.73 (t, J = 6.1 Hz, 2H), 3.67-3.54 (m, 12H), 3.43(dd, J = 23.1, 5.0 Hz, 2H), 3.28-3.08 (m, 6H), 3.06-2.89 (m, 4H), 2.77 (dd, J =16.7, 6.1 Hz, 1H), 2.57-2.49 (m, 1H), 2.44 (t, J= 6.1 Hz, 2H), 1.82 (d, J = 9.1Hz, 2H), 1.70 (d, J = 7.4 Hz, 2H), 1.52 (t, J = 14.5 Hz, 2H), 1.30 (d, J = 9.9 Hz, 6H). HR-MS (ESI) m / z: Calculated. For C 115 H 171 N 25 O 34 S4, [M+H] + , 2576.1416, [M+2H] 2+ 1288.5706 found 1288.5734.

[0048] 3. For example Figure 14 As shown, the synthesis and characterization of FPNR were as follows: The synthesis of FPNR was similar to that of FR: 31.3 mg of compound 13 (0.032 mmol, 1.4 eq) and 28 mg of compound 17 (0.023 mmol, 1 eq) were weighed into a dry round-bottom flask, mixed thoroughly with 3 mL of DMF solution, and stirred overnight. No further purification was required. Then, 20 mg of DOTA-NHS Ester (0.039 mmol, 1.7 eq) and 20 μL of DIPEA were added to the reaction solution, and the reaction was continued at room temperature for 8 h. After the reaction, DMF was removed, water was added, and the crude product was lyophilized. The product was purified by preparative HPLC to obtain white solid FPNR with a yield of 40%.

[0049] The preparation method is as follows: The characteristics are as follows: 1 H-NMR ( d 4 -CD3OD, 400 MHz, ppm) δ = 7.81 (d, J = 9.6 Hz, 3H), 7.72 (s,1H), 7.50-7.39 (m, 3H), 7.32-7.19 (m, 13H), 4.82-4.62 (m, 8H), 4.56 (d, J = 2.2Hz, 1H), 4.40 (s, 1H), 4.35 (d, J= 7.7 Hz, 3H), 4.26-3.69 (m, 24H), 3.60 (s,12H), 3.39 (d, J = 15.7 Hz, 10H), 3.31-2.97 (m, 23H), 2.95-2.41 (m, 17H), 2.31-2.02 (m, 10H), 1.89 (dd, J = 52.0, 7.1 Hz, 6H), 1.72 (s, 2H), 1.65 (s, 6H), 1.40-1.28 (m, 6H), 1.20 (d, J = 6.0 Hz, 3H), 0.94 (t, J = 6.9 Hz, 3H). HR-MS (ESI) m / z: Calculated. For C 128 H 180 N 26 O 35 S4, [M+H] + , 2773.2256, [M+2H] 2+ ,1387.1162, found 1387.1156.

[0050] IV. Example of 177Lu labeling preparation Labeling experiments such as Figure 14 As shown, the precursor probes FR, FPR, and FPNR were dissolved in biological-grade DMSO to prepare a 1 mM stock solution for later use. A carrier-free solution was also prepared. 177 37.0 MBq of LuCl3 solution (dissolved in 0.04 M HCl solution) was added to a centrifuge tube, diluted with 50 μL of NaOAc-HOAc buffer (0.4 M, pH = 4.5), followed by 5 μL (5 nmol) of the precursor stock solution. The mixture was then thoroughly vortexed and incubated at 90 °C and 800 rpm for 30 min with shaking. After the reaction, the sample was allowed to return to room temperature to obtain the final product. 177 Lu-labeled radioactive probe 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR.

[0051] Example 2 Characterization of dual-targeted radioactive probes (1) Determination of radioactive labeling efficiency like Figure 1 As shown in Figure A, thin-layer chromatography (radio-TLC) was used for evaluation. 177 Lu-FR,177 Lu-FPR, 177 The radiolabeling efficiency of Lu-FPNR was determined. The labeled reaction system was sampled and spotted onto a silica gel TLC plate, and developed using 50 mmol / L EDTA solution as the developing solvent. After development, the free radiolabeled components were detected using a radiometric TLC scanner to calculate the efficiency. 177 The ratio of Lu to labeled products was determined. Results showed that the radiolabeling yields of all three probes were greater than 98%.

[0052] (2) Radiochemical purity analysis like Figure 1 As shown in B-1D, the labeled product was further analyzed using high performance liquid chromatography (radio-HPLC).

[0053] The chromatographic conditions are as follows: Column: C18 reversed-phase column Mobile phase: Methanol / water (containing 0.1% TFA) gradient elution Method: 0-2 min, 5% (phase B); 2-23 min, 5%-100% (phase B). Detection method: Combined ultraviolet detection and radioactivity detection The results show that: 177 Lu-FR showed a single radioactive peak at 15.0 min. 177 Lu-FPR showed a single radioactive peak at 14.8 min. 177 A single radioactive peak appeared in Lu-FPNR at 15.9 min. All probes had a radiochemical purity greater than 95%, and no obvious impurity peaks or free nuclide peaks were detected.

[0054] (3) Determination of octanol / water partition coefficient (logD7.4) like Figure 1 As shown in Figure E, the lipid-water partition coefficient of the probe at pH 7.4 was determined using the shake-flask method. The specific method is as follows: Add approximately 1 MBq of radioactive probe to a centrifuge tube containing 1 mL of PBS (pH 7.4) and 1 mL of n-octanol. Vortex vigorously for 1 min, then centrifuge at 6000 × g for 5 min to separate the two phases. Take 100 μL of each phase and use a gamma counter to determine the radioactivity count.

[0055] Calculate the value of logD7.4 using the following formula: logD = log10 (C octanol / C aqueous) Each experiment was repeated 3 times, and the results are expressed as mean ± standard deviation.

[0056] The results are as follows: 177 Lu-FR: -2.90 ± 0.09 177 Lu-FPR: -2.83 ± 0.20 177 Lu-FPNR: -1.35 ± 0.06 (4) Evaluation of in vitro stability like Figure 1 As shown in Figure F, the three radioactive probes were placed in PBS buffer (pH 7.4) and 50% fetal bovine serum (FBS) and incubated at 37 °C. Samples were taken at 0, 1, 4, 24, 48, and 96 h, and radiochemical purity was determined by radio-TLC.

[0057] The results showed that all three probes maintained good stability within 96 h, and their radiochemical purity was all above 92%. No obvious dechelation or degradation products were observed.

[0058] Example 3: In vitro cell experiments and in vivo pharmacokinetic experiments Unless otherwise specified, all experiments in this example used U87-MG cells transfected with FAP. In this example, the parental U87-MG cells were α-cells. v β3 high expression models have been widely used in existing technologies for integrin α v In vitro experiments related to β3-targeting molecules (e.g.: [1]Shaw SK, Schreiber CL, Roland FM, et al. High expression ofintegrin α v β3enables uptake of targeted fluorescent probes into ovarian cancer cells and tumors[J]. Bioorganic&Medicinal Chemistry, 2018, 26(8): 2085-2091. [2]Perrins RD, McCarthy LA, Robinson A, et al. Targeting ultrasmallgold nanoparticles with cRGD peptide increases the uptake and efficacy ofcytotoxic payload[J]. Nanomaterials, 2022, 12(22): 4013.).

[0059] (1) Cell uptake experiment like Figure 2 As shown in Figure A, U87-MG-FAP cells stably expressing FAP were seeded in 24-well plates at a seeding density of 1 × 10⁻⁶. 5 Cells / wells were cultured overnight at 37 °C and 5% CO2. [The following appears to be a separate, unrelated sentence:] Add [to / to / the cells / wells]. 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR (0.037 MBq / well) was incubated at 37 °C for 1 h, 2 h, and 4 h, respectively. After incubation, the culture medium was discarded, and the cells were washed three times with PBS buffer to remove unbound radioactive probes. Cells were then lysed with 0.3 mM NaOH, and the lysates were collected and the cell radioactivity was measured using a gamma counter. Cell uptake results were expressed as per 10⁻⁶ cells / well. 6 Percentage of cells injected (%ID / 10) 6 (cells) is the unit of measurement.

[0060] The results showed that all three probes exhibited a time-dependent increase in uptake, among which 177 Lu-FPR reached 8.32 ± 0.37 %ID / 10 at 4 h. 6 cells, 1.34 times higher than 177 Lu-FR and 1.27 times higher 177 Lu-FPNR.

[0061] (2) Receptor blocking experiment like Figure 2 As shown in Figure B, to verify the dual-target specificity, U87-MG-FAP cells stably expressing FAP were seeded in 24-well plates at a seeding density of 1×10⁻⁶. 5 Cells / well were cultured overnight at 37 °C and 5% CO2. The following inhibitors were added 30 min before the cell uptake experiment: FAPI-2286 (FAP blocker, 2.5 nmol / well) c(RGDfK)(α v β3 integrin blocker, 2.5 nmol / well FAPI-2286 combined with c(RGDfK) blockade (FAP blocker + α) v β3 integrin blocker, 2.5 nmol / well Then joined 177 Lu-FR, 177 Lu-FPR or 177 Lu-FPNR was co-incubated for 2 hours.

[0062] The results show that: exist 177 In the Lu-FPR group, α v β3 integrin blockade reduced cellular uptake by approximately 1.3 times, FAP blockade reduced uptake by approximately 6.3 times, and the dual blockade group reduced uptake by approximately 14.8 times.

[0063] Similar results have also been seen in 177 Lu-FR and 177 Lu-FPNR group.

[0064] The above results indicate that all three dual-targeting probes can simultaneously target FAP and α. v β3 integrin undergoes specific binding.

[0065] (3) FAP combined with competition experiment like Figure 2 As shown in Figure C, a competitive binding assay was used to evaluate the binding affinity of the probe to FAP. U87-MG-FAP cells were seeded in 24-well plates at a seeding density of 1 × 10⁻⁶ cells / well. 5 Cells / wells were cultured overnight at 37 °C and 5% CO2. FAPI-2286 was used as a competing ligand, and the cells were compared with those cultured under different concentrations. 177 Cells were co-incubated with Lu-labeled probes (0, 0.01, 0.1, 1, 10, 100, 1000, and 10000 nM) in culture medium. The inhibition rate was calculated by measuring cell binding to radioactivity, and the IC50 was obtained by fitting the results. 50 value.

[0066] The results show: 177 Lu-FR: 30.97 ± 7.90 nM 177 Lu-FPR: 11.50 ± 1.44 nM 177 Lu-FPNR: 16.50 ± 2.62 nM in177 Lu-FPR exhibited the lowest IC 50 The value indicates that it has the strongest FAP binding capability.

[0067] (4) Cytotoxicity evaluation (non-radioactive precursor) like Figure 2 As shown in Figure D, the cytotoxicity of non-radioactive precursors (FR, FPR, and FPNR) was evaluated using the CCK-8 assay. U87-MG-FAP cells were seeded in 96-well plates at a seeding density of 8 × 10⁶ cells / well. 3 Cells / wells were prepared by adding different concentrations (up to 8 μM) of FR, FPR, and FPNR precursors and culturing at 37 °C for 48 h, with five replicates per group for the control. After incubation with CCK-8 reagent for 1 h, absorbance was measured at 450 nm, and cell viability was calculated. The results showed that the cell viability in each group was greater than 93%, indicating that the non-radioactive precursors have good biocompatibility.

[0068] (5) Evaluation of cellular radiotoxicity like Figure 2 As shown in Figure E, U87-MG-FAP cells were seeded in 96-well plates, and different doses of radioactive iodine (0, 0.185, 0.37, 0.74, or 1.48 MBq / well, 200 μL / well) were added. 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR. Cell viability was measured using the CCK-8 assay after 48 h of culture. The results showed that all probes exhibited dose-dependent cytotoxicity. At 0.74 MBq / well, the cell viability was as follows: 177 Lu-FR: Approximately 81% 177 Lu-FPR: Approximately 54% 177 Lu-FPNR: Approximately 72% The IC50 value was further calculated as follows: 177 Lu-FR: 1.47 MBq 177 Lu-FPR: 0.83 MBq 177 Lu-FPNR: 1.05 MBq in 177 Lu-FPR exhibited the strongest cell-killing effect.

[0069] (6) Renal clearance rate measurement like Figure 2As shown in F, the radiolabeled probe 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR was administered to healthy BALB / c mice via tail vein injection at a dose of 3.7 MBq / mouse (200 μL). Urine samples were collected from the animals after injection in separate metabolic cages.

[0070] Excreted urine was collected at 2 h, 4 h, 8 h, 24 h and 48 h after injection, and the volume was recorded. The radioactivity in the urine samples was determined using a gamma counter.

[0071] Renal clearance rate is calculated using the following formula: Renal clearance (%) = ne / ni × 100% in: ne represents the cumulative radioactivity in excreted urine; ni represents the total radioactivity injected.

[0072] The results show: All probes were primarily excreted via the kidneys, exhibiting clear renal clearance characteristics.

[0073] in: 177 Lu-FAPI-2286 exhibits a relatively fast clearance rate; 177 Lu-FR and 177 Lu-FPR exhibits similar renal clearance behavior; 177 The relatively slow clearance rate of Lu-FPNR suggests that its increased hydrophobicity may lead to delayed excretion in vivo.

[0074] (7) Determination of blood circulation half-life like Figure 2 As shown in G-2I, healthy BALB / c mice were used as experimental animals, and injections were administered via the tail vein. 177 Lu-FAPI-2286 177 Lu-FR, 177 Lu-FPR and 177 Lu-FPNR.

[0075] Peripheral blood samples were collected at predetermined time points following injection: 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, and 24 h. After weighing the blood samples at each time point, the radioactivity in the samples was determined using a gamma counter. The distribution of radioactivity in the blood was expressed as a percentage of the injected dose per gram of tissue (%ID / g). The blood concentration-time data were nonlinearly fitted, and a two-compartment pharmacokinetic model was used to analyze the in vivo behavior and calculate the plasma half-life.

[0076] The results show: 177 The plasma half-life of Lu-FAPI-2286 is 0.89 h; 177 The plasma half-life of Lu-FR is 1.29 h; 177 The plasma half-life of Lu-FPR is 1.41 h; 177 The plasma half-life of Lu-FPNR is 1.91 h.

[0077] The results indicate that, compared with single-target probes, the dual-target structure significantly prolongs blood circulation time, and the Linker structure has a regulatory effect on pharmacokinetic behavior.

[0078] Example 4: Micro-SPECT / CT Imaging and In Vivo Tumor Targeting Evaluation (1) Animal model establishment and administration method A subcutaneous U87-MG human glioblastoma model was established using BALB / c nude mice. To simulate the tumor microenvironment, U87-MG cells recruit and activate host fibroblasts in vivo, thereby forming a tumor microenvironment rich in cancer-associated fibroblasts (CAFs). All mice were injected via tail vein, with a single injection volume of 150 μL.

[0079] (2) Micro-SPECT / CT imaging When the tumor volume reaches 100–150 mm³, the radioactive probes are administered via tail vein injection: 177 Lu-FAPI-2286, 177 Lu-FR, 177 Lu-FPR, 177 Lu-FPNR, 7.4 MBq / animal, was subjected to micro-SPECT / CT imaging at 1 h, 2 h, 4 h, 24 h and 48 h respectively.

[0080] The results are shown in Figures 3A and 3B. All probes showed significant radioactive enrichment signals at the tumor site after 1 hour.177 Lu-FPR exhibited the highest tumor uptake level, at 1 h (9.18 ± 0.74 %ID / g), which was approximately [value missing]. 177 1.2 times that of Lu-FAPI-2286 177 1.7 times that of Lu-FR and 177 The 1.6-fold increase in Lu-FPNR indicates that its early tumor targeting efficiency is significantly superior to other control probes. As the time interval increased to 24 hours, the radioactive signal of all probes at the tumor site decreased to varying degrees, but their decay rates differed significantly. 177 Lu-FPR maintained a high tumor uptake level (3.76 ± 0.23 %ID / g), significantly higher than... 177 Lu-FAPI-2286 177 Lu-FR and 177 Lu-FPNR showed superior tumor retention ability. After 48 hours of imaging, all mice were dissected, and the radioactivity of tumors and vital organs was measured. Figure 3 As shown in Figure C, in the tissue activity results... 177 The uptake of Lu-FPR in tumor tissue remained at 1.90 ± 0.27 %ID / g, which was still significantly higher than the other three probes, suggesting that it has a more durable tumor enrichment effect and better target retention ability in vivo.

[0081] (3) Tumor / normal tissue ratio analysis Quantitative analysis was performed on the radioactivity distribution of each tissue at 48 h, and the ratios of tumor / heart (T / H), tumor / liver (T / L), tumor / kidney (T / K), tumor / muscle (T / M), and tumor / bone (T / B) were calculated.

[0082] The results show: 177 Lu-FPR demonstrated the best tumor-background contrast across all analytical metrics for the three dual-target probes, indicating its superior in vivo imaging clarity and target selectivity.

[0083] although 177 Lu-FAPI-2286 showed a high ratio in some tissues due to its faster clearance, but its tumor uptake and retention capacity was significantly lower than that of dual-targeting probes.

[0084] Example 5: Competitive Blocking Experiment (Verification of Dual-Target Specificity) To verify 177 Lu-FPR exhibits dual-target specificity, with the following blocking agents administered 30 minutes prior to probe injection: FAPI-2286 (FAP blocker, 125 μg / animal) c(RGDfK)(α v β3 integrin blocker 100 μg / animal) FAPI-2286 + c(RGDfK) (FAP blocker 125μg + α) v β3 integrin blocker 100μg / animal) combined blockade The blocking agents were all administered via tail vein injection, followed by injection. 177 Lu-FPR (7.4 MBq / animal) was administered, and imaging and quantitative analysis were performed 1 h later. Results showed that tumor uptake in the non-blocked group was 9.18 ± 0.74 %ID / g. After single-target blockade: In the FAP blockade group, uptake decreased to 0.39-fold; α v β3 blockade group: uptake decreased to 0.52-fold; dual-target combined blockade group further decreased to 0.25-fold (p<0.001). Among them, the FAP blockade group showed stronger inhibitory effects than the α group. v The β3 blockade group suggests that the FAP-related tumor microenvironment makes a more significant contribution to probe enrichment in this model.

[0085] Example 6: Radionuclide Therapy and Safety Evaluation Based on the aforementioned in vivo imaging and tumor uptake results, this embodiment selects the drug with the best targeting performance. 177 Lu-FPR was used as a candidate therapeutic probe, and physiological saline and 177 Lu-FAPI-2286 was used as a control group to systematically evaluate the therapeutic efficacy and in vivo safety of radionuclide therapy. U87-MG tumor-bearing mice in each group were administered the drug via a single tail vein injection at a dose of 14.8 MBq / mouse (150 μL). Tumor growth and the general condition of the animals were monitored dynamically over a long period after administration. Throughout the treatment observation period, no significant behavioral abnormalities or significant weight fluctuations were observed in any group of mice, indicating that the radioactive dose used was well tolerated under experimental conditions.

[0086] Tumor volume growth curve results ( Figure 5 (B, 5C) As shown, there was no significant difference in tumor volume among the groups before treatment, indicating good baseline consistency. Over time, the tumor in the saline group continued to grow rapidly, reaching 1454.29 ± 221.54 mm on day 11. 3 Tumor growth was significantly inhibited in both treatment groups, among which... 177 The Lu-FAPI-2286 group showed a tumor inhibition rate of approximately 55%, while 177 The Lu-FPR group showed a more significant tumor suppression effect, with an inhibition rate of approximately 75%, which was significantly better than the control treatment group (P<0.01), suggesting that it has stronger in vivo antitumor activity. Figure 5D). The tumor mass measurement results were consistent with the trend of volume change, further validating the differential efficacy of the two radiotherapy strategies. 177 The tumor weight was significantly reduced in the Lu-FPR group, reflecting its stronger destructive effect on tumor tissue. Figure 5 E).

[0087] Further pathological analysis of the tumor tissue showed that ( Figure 5 (F) Compared to the saline group, both treatment groups showed varying degrees of tumor tissue necrosis, among which... 177 The Lu-FPR group showed more extensive necrotic areas, accompanied by a significant decrease in cell density. Immunohistochemical staining further revealed a significant reduction in FAP-positive signal after treatment compared to the control group. 177 The Lu-FPR group showed the most significant decrease in FAP expression, a result consistent with its stronger tumor suppression effect, suggesting that it may further enhance the therapeutic effect by targeting tumor microenvironment-associated fibroblasts.

[0088] To systematically evaluate the safety of the treatment, peripheral blood analysis, serum biochemical tests, and histological assessment of major organs were performed on animals in each group. Results showed that peripheral blood cell counts (including white blood cells, red blood cells, hemoglobin, and platelets) remained within the normal range in all groups. Figure 6 No significant bone marrow suppression was observed. Serum biochemical analysis showed no significant abnormalities in liver function indicators such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST), or in kidney function indicators such as creatinine (CREA) and urea (UREA), with no statistically significant differences compared to the control group. Figure 7 ).

[0089] In addition, H&E staining of major organs such as the heart, liver, spleen, lungs, and kidneys showed that ( Figure 8 No significant tissue damage, inflammatory cell infiltration, or necrotic pathological changes were observed in any of the treatment groups, suggesting that the treatment did not cause significant damage to major organs. The combined hematological, biochemical, and histopathological results indicate that, under the dosage conditions used in this study, 177 Lu-FPR exhibits excellent anti-tumor effects without causing significant systemic toxicity, demonstrating good in vivo safety and therapeutic potential.

[0090] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0091] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A series of dual-targeting radiopharmaceutical precursors, characterized in that, The dual-targeting radiopharmaceutical precursors are FR, FPR, and FPNR. Each precursor includes an FAPI-2286 derivative, a cyclic RGDfK peptide, DOTA, and corresponding linker arms. FR is a direct linker, while FPR uses a PEG4 linker arm. The FAPI-2286 derivative is linked to the cyclic RGDfK peptide via the PEG4 linker arm, and the DOTA is linked to a dual-targeting intermediate formed by the FAPI-2286 derivative and the cyclic RGDfK peptide.

2. The dual-targeting radiopharmaceutical precursor according to claim 1, characterized in that, The FAPI-2286 derivative is a maleimide-modified FAPI-2286 derivative, and the cyclic RGDfK peptide is a thiol-modified cyclic RGDfK peptide. The modified linker group is linked to the lysine side chain of the cyclic RGDfK peptide, and a thiosuccinimide linker structure is formed between the FAPI-2286 derivative and the cyclic RGDfK peptide.

3. The dual-targeting radiopharmaceutical precursor according to claim 1, characterized in that, The PEG4 linker includes four ethylene glycol repeating units.

4. A dual-targeting radiopharmaceutical, characterized in that, The dual-targeting radiopharmaceutical includes the dual-targeting radiopharmaceutical precursor as described in any one of claims 1 to 3 and a radionuclide chelated with the DOTA.

5. The dual-targeting radiopharmaceutical according to claim 4, characterized in that, The radioactive nuclide is 177 Lu.

6. A method for preparing a dual-targeted radiopharmaceutical precursor according to any one of claims 1 to 3, characterized in that, Includes the following steps: Synthesize maleimide-modified FAPI-2286 derivatives; Synthesize cyclic RGDfK derivatives containing PEG4 linker arms and modified with thiol groups; The maleimide-modified FAPI-2286 derivative was subjected to a thiol-maleimide coupling reaction with the cyclic RGDfK derivative containing a PEG4 linker and modified with thiol to obtain a dual-targeting intermediate. The dual-targeting intermediate is reacted with NHS-DOTA to obtain the dual-targeting radiopharmaceutical precursor.

7. The preparation method according to claim 6, characterized in that, The cyclic RGDfK derivative containing a PEG4 linker and modified with thiol groups was prepared by Fmoc solid-phase peptide synthesis and obtained by Alloc deprotection, DPPA-mediated coupling, acid cleavage deprotection, and preparative high-performance liquid chromatography purification.

8. The preparation method according to claim 6, characterized in that, The maleimide-modified FAPI-2286 derivative was prepared by the following steps: FAPI-2286-related intermediates were prepared by 2-chlorotriphenylmethyl chloride resin and Fmoc solid-phase peptide synthesis method, and after acid cleavage and deprotection, they were reacted with 1,3,5-tris(bromomethyl)benzene, and then reacted with 3-(maleimide)propionic acid-N-hydroxysuccinimide ester.

9. A kind 177 A method for preparing Lu-labeled dual-targeting radiopharmaceuticals, characterized in that, Includes the following steps: Dissolve the dual-targeting radiopharmaceutical precursor according to any one of claims 1 to 3 in dimethyl sulfoxide to obtain a precursor stock solution; Will 177 LuCl3 was added to sodium acetate buffer, and the precursor stock solution was also added. The resulting reaction mixture was reacted at 90°C for 30 minutes to obtain... 177 Lu-labeled dual-targeting radiopharmaceutical; The sodium acetate buffer solution has a pH of 4.

5.

10. A kind 177 Lu-FPR in the preparation of FAP and α v Its application in diagnostic reagents, efficacy assessment reagents, or radionuclide therapy drugs for β3 integrin-related solid tumors is characterized by, The 177 Lu-FPR is a precursor composed of FAPI-2286 derivative, PEG4 linker, cyclic RGDfK peptide, and DOTA. 177 Complexes formed by Lu.

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