Radionuclide-labeled RANKL-targeted small-molecule inhibitor, precursor compound thereof, preparation method and application of radionuclide-labeled RANKL-targeted small-molecule inhibitor

Abstract

The invention discloses a radionuclide-labeled small-molecule inhibitor targeting RANKL, a precursor compound thereof, a preparation method and application, and belongs to the technical field of nuclear medicine. The invention discloses a precursor compound with a structure as shown in a formula I or pharmaceutically acceptable salt thereof, and a radionuclide-labeled small-molecule inhibitor targeting RANKL with a structure as shown in a formula II or pharmaceutically acceptable salt thereof. The invention also discloses a preparation method of the compounds as shown in the formula I and the formula II, and application of the compounds in preparation of tumor imaging agents. Or / and application in preparation of drugs for treating tumors. According to the invention, a chelating agent DOTA is creatively combined with a small molecule compound to synthesize a novel compound as shown in a formula I; according to the present invention, the nuclide-labeled compound represented by the formula I can effectively provide the RANKL / RANK signal pathway targeting potential, and can be used for imaging and treatment of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine technology, specifically relating to a radionuclide-labeled small molecule inhibitor targeting RANKL, its precursor compound, preparation method, and application. Background Technology

[0002] Bone metastases are commonly found in patients with malignant solid tumors such as lung cancer, prostate cancer, breast cancer, and kidney cancer. Once bone metastases occur, they often lead to skeletal-related events (SREs), significantly impacting patients' quality of life, predicting poor prognosis, and potentially increasing the risk of death. Local manifestations of SREs include bone pain, pathological fractures, vertebral compression fractures, and dysfunction of the bladder, rectum, and reproductive system; systemic changes may manifest as hypercalcemia and renal failure. Early diagnosis and timely treatment of solid bone metastases significantly improve patient prognosis and quality of life. Currently, treatment for bone metastases is primarily comprehensive. In addition to traditional radiotherapy and chemotherapy, targeted bone therapy has become one of the main management methods, mainly including the use of bisphosphonates and RANKL inhibitors.

[0003] Under physiological conditions, bone remodeling depends on the sustained biological activity of osteoblasts and osteoclasts, maintaining a dynamic balance through bone formation and resorption. When tumors metastasize to bone, metastatic cancer cells first activate the differentiation and maturation of osteoclasts, followed by osteoclast-mediated bone resorption, resulting in tumor-induced bone destruction and promoting its implantation. Receptor activator of nuclear factor κB (RANK) belongs to the tumor necrosis factor (TNF) receptor family and is widely expressed on the surface of various cells, including osteoclast precursors, mature osteoclasts, dendritic cells, breast epithelial cells, and breast and prostate cancer cells. RANKL is a related ligand of RANK, mainly secreted by osteoblasts and their precursor cells, T cells, B cells, and megakaryocytes. The binding of RANK to RANKL plays a crucial role in the survival, differentiation, and activation of osteoclasts. Therefore, targeting RANKL is a highly promising option in therapeutic strategies for bone metastases. Denosumab, as the first humanized monoclonal antibody targeting RANKL, can effectively relieve bone pain and reduce bone destruction. However, its application also has serious adverse reactions such as withdrawal symptoms and osteonecrosis of the mandible, which are difficult to manage. It is worth noting that RANKL is not only present in osteoclasts but also expressed in various tumor cells. Increasing research shows that the RANKL-RANK signaling pathway is not only involved in the process of tumor bone metastasis but also plays an important role in the development and progression of various malignant solid tumors such as breast cancer, lung cancer, prostate cancer, and melanoma. This pathway can inhibit tumor occurrence and metastasis by binding to RANK receptors on tumor cells, exhibiting a direct anti-tumor effect. Therefore, inhibiting RANKL can not only reduce bone destruction and decrease bone tumor burden but also possess certain anti-tumor potential. This has led to increasing attention being paid to the role of RANKL in the treatment of primary tumors.

[0004] With the continuous development of nuclear medicine, radionuclide therapy has provided cancer patients with new options in addition to traditional treatments, among which precise radionuclide-targeted therapy for tumors has become a research hotspot. Gallium-68 / lutetium-177 ( 68 Ga / 177 The integrated diagnosis and treatment approach (LU) has demonstrated good results in clinical applications. Existing studies have adopted... 177Lu-DOTA-ibandronic acid treatment for bone metastases from solid tumors achieved significant pain relief at relatively low doses with no obvious side effects observed. However, since the mechanism of action of bisphosphonates mainly relies on their targeting to bone, radionuclide-labeled bisphosphonates do not have significant anti-tumor effects on the primary tumor. RANKL plays an important role in the development and progression of various solid tumors and their bone metastases, and is highly expressed in both primary tumors and bone metastases. Therefore, targeting RANKL holds promise for simultaneously guiding radionuclides to both the primary tumor and bone metastases, thereby achieving dual therapeutic effects on both.

[0005] Currently, there are no reports on the use of radionuclide labeling or small molecule inhibitors targeting RANKL for the integrated diagnosis and treatment of malignant tumors and their bone metastases. Therefore, developing molecular probes with high specificity for the diagnosis and treatment of the RANKL / RANK signaling pathway is of great significance. Clinically, there is an urgent need to develop new diagnostic and therapeutic methods to diagnose primary solid tumors and bone metastases at an early stage, thereby improving patients' quality of life. Summary of the Invention One of the objectives of this invention is to provide a diagnostic and therapeutic probe targeting small molecule inhibitors of the RANKL / RANK signaling pathway, the structure of which is shown in Formula II. This probe can not only visualize primary solid tumors and bone metastases, but also treat the corresponding lesions and relieve bone pain.

[0006] The second objective of this invention is to provide a precursor compound for the diagnostic and therapeutic probe of the RANKL / RANK signaling pathway small molecule inhibitor, the structure of which is shown in Formula I.

[0007] A third objective of this invention is to provide a method for preparing the compound of formula I.

[0008] The fourth objective of this invention is to provide a method for preparing compound II.

[0009] The fifth objective of this invention is to provide applications of compounds of formula I and formula II.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention discloses a radionuclide-labeled precursor compound of a small molecule inhibitor targeting RANKL, or a pharmaceutically acceptable salt thereof, with the structure shown in Formula I.

[0011] Formula I X is a halogen; preferably, X is F or Cl.

[0012] The second aspect of this invention discloses a radionuclide-labeled small molecule inhibitor targeting RANKL, or a pharmaceutically acceptable salt thereof, the structure of which is shown in Formula II.

[0013] Formula II Where A is a nuclide, preferably 68 Ga、 64 Cu、 89 Zr、 90 Y、 111 In、 177 Lu、 225 Ac.

[0014] The third aspect of this invention discloses a method for preparing the precursor compound shown in Formula I, the synthetic route of which is as follows: , Wherein X is a halogen; preferably, X is F or Cl.

[0015] In some embodiments of the present invention, the method for preparing the precursor compound shown in I includes the following steps: S1. Compound 1 undergoes hydrogenation under catalytic conditions to produce compound 2; preferably, the catalyst is Pd(OH)2 or Co / Al2O3; S2. Compound 2 reacts with compound 3 to generate compound 4; preferably, an acid-binding agent is added to the reaction system, more preferably, the acid-binding agent is K2CO3 or Na2CO3; S3. Compound 4 and compound 5 undergo a sulfonation reaction to generate the anticomposite 6; preferably, an acid-binding agent is added to the reaction system, more preferably, the acid-binding agent is K2CO3 or Na2CO3; S4. Compound 6 undergoes deprotection to form compound 7; S5. Compound 7 reacts with Compound 8 to produce Compound I.

[0016] In some embodiments of the present invention, the reaction temperature in step S1 is 20~25℃.

[0017] In some embodiments of the present invention, in step S2, the molar ratio of compound 2 to compound 3 is 1:0.8~1.2; preferably 1:0.9; the molar ratio of compound 2 to acid-binding agent is 1:2~4; preferably 1:3; The temperature for step S2 is 80~90℃, preferably 85℃; The preferred reaction solvent for step S2 is N,N-dimethylformamide.

[0018] In some embodiments of the present invention, in step S3, the molar ratio of compound 4 to compound 5 is 1:0.8~1.2; preferably 1:0.9; the molar ratio of compound 4 to the acid-binding agent is 1:1~2; preferably 1:1.5; The preferred reaction solvent for step S3 is N,N-dimethylformamide, and the reaction is carried out under reflux.

[0019] In some embodiments of the present invention, in step S4, compound 6 is deprotected under acidic conditions at 20-25°C; preferably, the molar ratio of compound 6 to HCl is 1:10~15; more preferably, it is 1:12.

[0020] In some embodiments of the present invention, in step S5, compound 7 and compound 8 undergo a coupling reaction in the presence of DIEA (N,N-diisopropylethylamine); preferably, the molar ratio of compound 7 to compound 8 is 1:1.5~2.5; more preferably, it is 1:2; preferably, the molar ratio of compound 7 to N,N-diisopropylethylamine is 1:3~5; more preferably, it is 1:4. Preferably, the reaction temperature in step S5 is 20-25°C; the reaction solvent is N,N-diisopropylethylamine.

[0021] The fourth aspect of this invention discloses a method for preparing a small molecule inhibitor targeting RANKL labeled with a radionuclide as shown in Formula II, wherein a compound of Formula I is reacted with a radionuclide salt solution to obtain the radiolabeled substance shown in Formula II.

[0022] The fifth aspect of the present invention discloses the use of the precursor compound of Formula I or a salt thereof in the preparation of tumor imaging agents; and / or in the preparation of medicaments for treating tumors. The sixth aspect of this invention discloses the use of a radionuclide-labeled small molecule inhibitor targeting RANKL, as shown in Formula II, or a salt thereof, in the preparation of a tumor imaging agent; or / and in the preparation of a medicament for treating tumors.

[0023] The tumors include primary solid tumors and bone metastases, metastatic bone tumors, and giant cell tumors of bone.

[0024] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively combines the chelating agent DOTA with a small molecule compound to synthesize a novel compound represented by Formula I.

[0025] In a first aspect, the compound represented by Formula I of the present invention, when used as a precursor, has the advantages of low dosage (microgram level) and low toxicity when labeled with radionuclides.

[0026] Secondly, this invention provides various radionuclide-labeled compounds of formula I (such as... 68Ga、 64 Cu、 89 Zr、 111 In、 90 Y、 177 Lu、 225 Ac). These labeling methods are simple to operate, have moderate reaction time, and high labeling yield. The radiolabeled compound of formula I has dual functions: (1) Imaging function: it can be used for imaging diagnosis of primary solid tumors and bone metastases. (2) Therapeutic function: it can treat the above-mentioned lesions and effectively relieve bone pain.

[0027] Therefore, the labeled Formula I compounds can more effectively exert their potential to target the RANKL / RANK signaling pathway for the imaging and treatment of related diseases, including metastatic bone tumors, giant cell tumors of bone, and solid tumors related to the RANKL / RANK signaling pathway (such as breast cancer and melanoma). Attached Figure Description

[0028] Figure 1 This is the mass spectrum of compound DOTA-TRX01; Figure 2 The HPLC chromatogram of compound DOTA-TRX01 is shown below. Figure 3 The mass spectrum of compound DOTA-TRX02; Figure 4 The HPLC chromatogram of compound DOTA-TRX02 is shown below. Figure 5 To determine the markers using HPLC 68 Radiochemical purity results of Ga-DOTA-TRX01; Figure 6 To determine the markers using HPLC 68 Radiochemical purity results of Ga-DOTA-TRX02; Figure 7 for 68 Micro PET / CT images of Ga-DOTA-TRX01 in normal mice, where A is the image at 30 min, B is the image at 1 h, C is the image at 2 h, and D is the image at 4 h. Figure 8 for 68 Micro PET / CT images of Ga-DOTA-TRX01 in 4T1 bone metastasis tumor-bearing mice, where A is the 30-minute image, B is the 1-hour image, C is the 2-hour image, and D is the 4-hour image. Figure 9 for 68Micro PET / CT images of Ga-DOTA-TRX02 in normal mice, where A is the image at 30 min, B is the image at 1 h, C is the image at 2 h, and D is the image at 4 h. Figure 10 Inject normal mice 68 Dose distribution map of organs of interest at different time points after Ga-DOTA-TRX01; Figure 11 1 mCi was injected into the tail vein of normal mice. 68 Pathological images of organs and tissues after 28 days of Ga-DOTA-TRX01, with each small image corresponding to an organ: A: Heart, B: Liver, C: Spleen, D: Lung, E: Kidney, F: Stomach, G: Small Intestine, H: Muscle, I: Bone Marrow, J: Brain. Figure 12 for 68 The results of radiochemical purity of Ga-DOTA-TRX01 at various time points in PBS, FBS and NS. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0030] In this embodiment of the invention, FA condition refers to the condition containing 0.1v% formic acid.

[0031] Example 1 This embodiment discloses the preparation of the compound of formula I of the present invention, wherein X is F, and the compound obtained in this embodiment is named DOTA-TRX01; its synthetic route is as follows:

[0032] The specific preparation steps are as follows: S1: In a dry hydrogenation flask, dry Pd(OH)₂ (200 mg) was added under an Ar atmosphere, followed by the addition of MeOH (10.0 mL) to completely wet the Pd(OH)₂. Then, a MeOH solution (10.0 mL) of compound 1-1 (2.00 g, 14.5 mmol, 1.00 eq.) was slowly added under an Ar atmosphere. The resulting mixture was degassed and purged three times with H₂, and then stirred for 2 hr under an H₂ atmosphere (50 psi) at 20–25 °C. TLC (DCM: MeOH = 10:1, Rf = 0.2) showed that compound 1-1 was almost completely consumed and a major new spot was detected. The reaction mixture was carefully filtered under reduced pressure and an N₂ atmosphere. Purification by column chromatography (SiO₂, DCM: MeOH = 10:1) gave compound 2-1 as a white solid.

[0033] S2: Compound 2-1 (500 mg, 3.54 mmol, 1.00 eq.), compound 3 (706 mg, 3.19 mmol, 0.90 eq.), and K2CO3 (1.47 g, 10.5 mmol, 3.00 eq.) were placed in DMF (5.0 mL) and stirred at 85 °C for 12 h. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by pre-HPLC (0.1% TFA conditions) to give compound 4-1 as a red solid.

[0034] S3: A mixture of compound 4-1 (300 mg, 1.07 mmol, 1.00 eq.), compound 5 (432 mg, 965 μmol, 0.90 eq.), and K2CO3 (222 mg, 1.61 mmol, 1.50 eq.) in DMF (3.50 mL) was refluxed for 12 hours at 60 °C. The residue was purified by preparative-HPLC (TFA conditions) to give compound 6-1 as a red oil.

[0035] S4: A solution of compound 6-1 (50.0 mg, 83.1 μmol, 1.00 eq.) in HCl / dioxane (2.00 M, 500 μL, 12.0 eq.) was stirred for 1 hr at 20–25 °C. The reaction mixture was concentrated under reduced pressure to give compound 7-1 as a pale yellow oil.

[0036] S5: Add DIEA (20.6 mg, 159 μmol, 27.7 μL, 4.00 eq.) and compound 8 (39.9 mg, 79.7 μmol, 2.00 eq.) to a solution of compound 7-1 (20.0 mg, 39.8 μmol, 1.00 eq.) in DMF (0.20 mL). Stir at 20–25 °C for 2 hours. Concentrate the reaction mixture under reduced pressure to obtain a residue. LCMS and HPLC confirmed that DOTA-TRX01 is a red solid; its mass spectrum is attached. Figure 1 As shown, the HPLC chromatogram is attached. Figure 2 As shown.

[0037] Example 2 This embodiment discloses the preparation of the compound of formula I of the present invention, wherein X is Cl, and the compound obtained in this embodiment is named DOTA-TRX02; its synthetic route is as follows:

[0038] The specific preparation steps are as follows: S1: Co / Al₂O₃ (0.18 g, 8.47 mmol, 14% purity) was added to a solution of compounds 1-2 (1.30 g, 8.47 mmol, 1.00 eq.) in NH₃ / MeOH (78.0 mL). The mixture was stirred for 2 hr at 80 °C under H₂ (0.50 MPa). TLC (dichloromethane:methanol = 5:1) showed that compounds 1-2 (Rf = 0.80) were completely consumed, and a new spot with high polarity was detected (Rf = 0.20). The reaction mixture was filtered and NH₃ / MeOH was removed under vacuum. The residue was purified by column chromatography (SiO₂, dichloromethane:methanol = 1 / 0 ~ 0 / 1, dichloromethane:methanol = 5:1, Rf = 0.20). Compound 2-2 (0.91 g, 5.81 mmol, 68.5% yield) is a white solid, as confirmed by ¹H NMR.

[0039] S2: K₂CO₃ (1.05 g, 7.61 mmol, 3.00 eq.) and compound 3 (506 mg, 2.28 mmol, 0.90 eq.) were added to a DMF (4.00 mL) solution of compound 2-2 (0.40 g, 2.54 mmol, 1.00 eq.). The mixture was stirred at 60 °C for 12 hr. LCMS showed that compound 3 was completely consumed and the desired mass was detected (MS cal: 342.09, MS observed: [M+H]⁺ = 342.9) (Rt = 0.96 min). The reaction mixture was centrifuged, filtered, and the filtrate was purified. The residue was purified by preparative high-performance liquid chromatography (FA conditions). Compound 4-2 (0.11 g, 301 μmol, 11.8% yield, 93.9% purity) was identified by LCMS (Rt = 0.98 min), ¹H NMR, and Special ¹H NMR.

[0040] S3: Add K2CO3 (62.4 mg, 452 μmol, 1.50 eq.) and compound 5 (121 mg, 271 μmol, 0.90 eq.) to a DMF (1.10 mL) solution of compound 4-2 (0.11 g, 301 μmol, 1.00 eq.). Stir the mixture at 60 °C for 12 hr. LCMS showed that compound 4-2 was completely consumed and the desired mass was detected (MS cal: 617.26, MS observed: [M+H]+ = 618.7) (Rt = 1.25 min). Decenter the reaction mixture and filter and purify the filtrate. Purify the residue by preparative high performance liquid chromatography (FA conditions). Compound 6-2 (40.0 mg, 64.7 μmol, 21.4% yield, 100% purity) was identified by LCMS (Rt = 1.27 min), ¹H NMR, and Special ¹H NMR.

[0041] S4: A solution of compound 6-2 (40.0 mg, 64.7 μmol, 1.00 eq.) in HCl / dioxane (2.00 M, 0.40 mL, 12.3 eq.) was prepared. The mixture was stirred at 20 °C for 2 hr. LCMS showed that compound 6-2 was completely consumed and the desired mass was detected (MS cal: 517.21, MS observed: [M+H]+ = 518.4) (Rt = 0.84 min). The reaction mixture was concentrated under reduced pressure to remove HCl / dioxane. Compound 7-2 (30.0 mg, crude) was obtained as a red solid and used in the next step without further purification.

[0042] S5: DIEA (74.8 mg, 579 μmol, 100 μL, 4.00 eq.) and compound 8 (58.0 mg, 115 μmol, 2.00 eq.) were added to a DMF (0.80 mL) solution of compound 7-2 (30.0 mg, 57.9 μmol, 1.00 eq.). The mixture was stirred at 25 °C for 2 hr. LCMS showed that compound 7-2 was completely consumed and the desired mass was detected (MS cal: 903.39, MS observed: [M / 2+H]+ = 452.9) (Rt = 0.91 min). Purification was performed directly without further inspection. The residue was purified by preparative high-performance liquid chromatography (FA conditions). The white solid DOTA-TRX02 (5.50 mg, 6.08 μmol, yield 10.5%, purity 100%) was obtained by LCMS (Rt = 0.92 min) and HPLC (Rt = 1.34 min); its mass spectrum is attached. Figure 3 As shown, its chromatogram is attached. Figure 4 As shown.

[0043] Example 3 This embodiment discloses 68 Ga-marked DOTA-TRX01 ( 68 The preparation method of Ga-DOTA-TRX01 is as follows: The precursor compound DOTA-TRX01 was added to a sodium acetate solution with a concentration of 0.25 mol / L to prepare a DOTA-TRX01 solution with a concentration of 1 mg / mL.

[0044] Take freshly rinsed 68 Mix the GaCl3 eluent (5 mCi / mL) with an equal volume of 0.25 mol / L sodium acetate solution, measure the pH value, and adjust the pH of the solution to 7.0-7.4 using PBS (pH 7.4).

[0045] 4 mL of pH-adjusted solution was used. 68 Add 25 μL of DOTA-TRX01 solution to GaCl3 solution, mix well, incubate at 95℃ for 15 minutes, and then purify the reaction solution by extraction with a C18 column (Sep-Pak). 18 Before C-column separation and purification 18 The C-column was first activated sequentially with 5 ml of 75% ethanol and 5 ml of sterile saline. Then, 0.5 mL of 50% ethanol was used as the eluent, and the final product was diluted with sterile saline. Subsequently... 68Ga-DOTA-TRX01 uses ultraviolet and radioactive high-performance liquid chromatography (HPLC) for quality control of radioactive synthesis. Results are as follows: Figure 5 As shown. 68 HPLC quality control of Ga-DOTA-TRX01 showed that the prepared... 68 The radiochemical purity of Ga-DOTA-TRX01 is 97.36%.

[0046] Example 4 This embodiment discloses 68 Ga-marked DOTA-TRX02 ( 68 The preparation method of Ga-DOTA-TRX02 is as follows: The precursor compound DOTA-TRX02 was added to a sodium acetate solution with a concentration of 0.25 mol / L to prepare a DOTA-TRX02 solution with a concentration of 1 mg / mL.

[0047] Take freshly rinsed 68 The GaCl3 eluent (10 mCi / mL) was mixed thoroughly with an equal volume of 0.25 mol / L sodium acetate solution. The pH value was measured and the solution pH was adjusted to 7.0-7.4 using PBS (pH 7.4).

[0048] 4 mL of pH-adjusted solution was used. 68 Add 25 μL of DOTA-TRX02 solution to GaCl3 solution, mix well, incubate at 95℃ for 15 minutes, and then purify the reaction solution by extraction with a C18 column (Sep-Pak). 18 Before C-column separation and purification 18 The C-column was first activated sequentially with 5 ml of 75% ethanol and 5 ml of sterile saline. Then, 0.5 mL of 50% ethanol was used as the eluent, and the final product was diluted with sterile saline. Subsequently... 68 Ga-DOTA-TRX02 underwent quality control using ultraviolet and radioactive high-performance liquid chromatography (HPLC). Results are as follows: Figure 6 As shown. 68 HPLC quality control of Ga-DOTA-TRX02 showed that the prepared... 68 The radiochemical purity of Ga-DOTA-TRX02 is 98.09%.

[0049] Example 5 This embodiment discloses 177 Lu marked DOTA-TRX01 ( 177 The preparation method of Lu-DOTA-TRX01 is as follows: The precursor compound DOTA-TRX01 was added to a sodium acetate solution with a concentration of 0.25 mol / L to prepare a DOTA-TRX01 solution with a concentration of 1 mg / mL.

[0050] Take freshly rinsed 177 The LuCl3 eluent (20 mCi / mL) was mixed thoroughly with an equal volume of 0.25 mol / L sodium acetate solution. The pH value was measured and the solution pH was adjusted to 7.0-7.4 using PBS (pH 7.4).

[0051] The pH value was adjusted. 177 LuCl3 solution was added to DOTA-TRX01 solution, mixed well, and incubated at 95°C for 15 minutes. The reaction solution was then purified by extraction using a C18 column (Sep-Pak). 18 Before C-column separation and purification 18 The C-column was first activated sequentially with 5 ml of 75% ethanol and 5 ml of sterile saline. Then, 0.5 mL of 50% ethanol was used as the eluent, and the final product was diluted with sterile saline. Subsequently... 177 Lu-DOTA-TRX01 was prepared using ultraviolet and radioactive high-performance liquid chromatography (HPLC) for quality control of radioactive synthesis. The product obtained in this example... 177 The radiochemical purity of Lu-DOTA-TRX01 is 95.07%.

[0052] Experimental Example 1 This experimental example discloses the radionuclide-labeled DOTA-TRX of the present invention. 68 Imaging studies of Ga-DOTA-TRX01 in normal and tumor-bearing mice. This experimental case... 68 Ga-DOTA-TRX01 was prepared according to the method in Example 3.

[0053] 1. In vivo imaging study in normal mice Kunming mice were anesthetized and injected with 0.1 mCi via the tail vein. 68 Ga-DOTA-TRX01 was used for whole-body imaging using a miniature PET / CT scanner at 30 min, 1 h, 2 h, and 4 h post-injection. PET imaging parameters were as follows: time window 1.1 ns, energy range 350-650 keV, matrix 140×140, acquisition time 15 minutes. CT scanning used a tube voltage of 80 kV, a tube current of 0.5 mA, and a scan time of 2 minutes.

[0054] The results are attached. Figure 7 As shown. Marker 68Micro PET / CT images of Ga-DOTA-TRX01 in normal mice showed rapid clearance of the marker in normal tissues. Specifically, the SUVmax in the liver was 2.16 at 30 minutes, 2.02 at 1 hour, 1.68 at 2 hours, and 1.53 at 4 hours; a significant amount of radioactivity also accumulated in the bladder. These results indicate... 68 Ga-DOTA-TRX01 is excreted through both the hepatobiliary and urinary systems, with the urinary system being the primary route of excretion.

[0055] 2. In vivo imaging study in tumor-bearing mice A 4T1 bone transfer nude mouse model was established by injecting 0.1 mCi via the tail vein under anesthesia. 68 Ga-DOTA-TRX01 was used for whole-body imaging using a miniature PET / CT scanner at 30 min, 1 h, 2 h, and 4 h post-injection. PET imaging parameters were as follows: time window 1.1 ns, energy range 350-650 keV, matrix 140×140, acquisition time 15 minutes. CT scans used a tube voltage of 80 kV, a tube current of 0.5 mA, and a scan time of 2 minutes. For PET image quantization, regions of interest (ROIs) for tumors and organs were manually delineated on attenuation-corrected images.

[0056] The results are attached. Figure 8 As shown, in the 4T1 bone metastasis nude mouse model, radioactive uptake in the affected right lower limb was significantly higher than that in the healthy left lower limb at various time points. However, at 30 minutes, the uptake range of the tumor site was not yet clear; by 1 hour, the radioactive uptake of the tumor had significantly increased, and the imaging was more distinct.

[0057] Experimental Example 2 This experimental example discloses the radionuclide-labeled DOTA-TRX of the present invention. 68 Imaging study of Ga-DOTA-TRX02 in normal mice. Example 1 of the experimental procedure. This experimental example... 68 Ga-DOTA-TRX02 was prepared according to the method in Example 4. The results are attached. Figure 9 As shown. The results indicate... 68 Ga-DOTA-TRX02 is cleared relatively quickly from normal tissues and is also excreted through both the hepatobiliary and urinary systems, with the urinary system being the primary excretion pathway.

[0058] Experimental Example 3 This experimental example discloses the radionuclide-labeled DOTA-TRX01 ( 68 This experiment investigated the biodistribution of Ga-DOTA-TRX01 in normal mice. 68Ga-DOTA-TRX01 was prepared according to the method in Example 3.

[0059] Use physiological saline to label 68 After diluting Ga-DOTA-TRX01 and disinfecting the mouse tail vein skin with alcohol, 150 μL of the solution was administered using a 1 ml insulin needle. 68 Ga-DOTA-TRX01 (0.1 mCi) was injected into mice via the tail vein. Mice were sacrificed under anesthesia at 30 min, 1 h, 2 h, and 4 h. Heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, brain, salivary glands, and blood were collected. After weighing, the radioactivity was measured using a gamma radioimmunoassay counter. The biodistribution results (%ID / g) are expressed as a percentage of the injected dose per gram, and the results were analyzed using mean ± standard deviation. The results are attached. Figure 10 As shown.

[0060] Depend on Figure 10 It can be seen that, 68 Ga-DOTA-TRX01 exhibits rapid blood clearance, with a blood retention rate of only 0.63 ± 0.83 %ID / g after 4 hours. The liver... 68 Ga-DOTA-TRX01 exhibits high absorption, peaking at 30 minutes (4.22±0.76% ID / g), and gradually decreasing over time. Initially, uptake in the blood pool, heart, and lungs is slightly high, gradually decreasing over time, becoming almost undetectable at 4 hours. Mild uptake occurs in the thyroid gland, muscle, and bones, gradually decreasing over time. Due to… 68 Ga-DOTA-TRX01 is primarily excreted through the urinary tract, and its absorption by the kidneys is also very high.

[0061] Test Example 4 This experimental example discloses the radionuclide-labeled DOTA-TRX01 ( 68 Toxicity study of Ga-DOTA-TRX01 in normal mice.

[0062] Normal mice were randomly divided into 5 groups of 5 mice each: high-dose group, medium-dose group, low-dose group, blank control group, and precursor compound control group.

[0063] Mice in the high-dose, medium-dose, and low-dose groups were injected internally. 68 Ga-DOTA-TRX01 solution. The injection doses for each group were as follows: high-dose group: 1 mCi; medium-dose group: 0.5 mCi; low-dose group: 0.1 mCi. The blank control group was injected with an equal volume of physiological saline, and the precursor compound control group was injected with an equal volume of the precursor compound DOTA-TRX01.

[0064] This experimental example 68The Ga-DOTA-TRX01 solution was prepared according to the method in Example 3, namely: 68 After reacting GaCl3 solution and the precursor compound DOTA-TRX01 in sodium acetate solution, the mixture was purified by 18C column elution. To obtain the product, the column was eluted with 50% ethanol and then diluted with physiological saline.

[0065] Experimental results show that the marker 68 No mice died within 28 days of being injected with Ga-DOTA-TRX01. Throughout the observation period, the feed consumption and activity levels of the mice in each group remained stable without significant reduction, and no abnormalities such as vomiting or diarrhea occurred.

[0066] Furthermore, the body weight of all five groups of mice showed an increasing trend over time, and there was no significant difference in body weight change among the experimental groups (P>0.05). Routine blood tests revealed no obvious abnormalities.

[0067] After the observation period, the mice were dissected. The results showed no abnormalities in the size, color, morphology, or texture of the major tissues and organs in each group. Further paraffin embedding and sectioning of the tissues and organs, followed by HE staining for pathological observation, revealed no significant differences in cell size, morphology, and proportion in the tissue sections of the high-dose, medium-dose, and low-dose groups compared to the control group. No obvious pathological abnormalities such as degeneration or necrosis were found. Specific results are as follows: Figure 11 As shown.

[0068] Experimental Example 5 This experimental example discloses the marker of the present invention. 68 In vitro stability study of Ga-DOTA-TRX01. This test example... 68 Ga-DOTA-TRX01 was prepared according to the method in Example 3.

[0069] Take twelve 2mL EP tubes and add 250μL of fetal bovine serum (FBS, n=4), 250μL of PBS (n=4), and 250μL of normal saline (NS, n=4) to each tube, respectively. Add 250μL of a radiochemically pure solution with a specific activity of 1.6μCi / μL to each EP tube. 68 Ga-DOTA-TRX01 solution. Twelve EP tubes were incubated at 37°C for 30, 60, 120, and 240 minutes, respectively. After incubation, 500 μL of acetonitrile was added to each EP tube in the FBS group, and the mixture was shaken to precipitate the protein, followed by centrifugation. 15 μL of the supernatant was collected after centrifugation, and 15 μL was collected directly from the PBS and NS groups. The radiochemical purity of the samples at each time point was determined using high-performance liquid chromatography (HPLC). The results are as follows: Figure 12 As shown.

[0070] Depend on Figure 12 It can be seen that the labeling rate decreased slightly after 4 hours of incubation in physiological saline, fetal bovine serum, and PBS, reaching 96.96%, 97.12%, and 95.33% respectively, all greater than 95%. This indicates that... 68 Ga-DOTA-TRX01 exhibits good in vitro stability.

[0071] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A prodrug compound of a radionuclide-labeled small molecule inhibitor targeting RANKL, as shown in Formula I, or a pharmaceutically acceptable salt thereof. Formula I, Wherein X is a halogen; preferably, X is F or Cl.

2. A radionuclide-labeled small molecule inhibitor targeting RANKL or a pharmaceutically acceptable salt thereof, characterized in that, The structure is shown in Equation II. Formula II, Where A is a nuclide, preferably... 68 Ga、 64 Cu、 89 Zr、 90 Y、 111 In、 177 Lu、 225 Ac.

3. The method for preparing the precursor compound represented by Formula I according to claim 1, characterized in that, Its synthetic route is as follows: ， Wherein X is a halogen; preferably, X is F or Cl.

4. The method for preparing the precursor compound represented by Formula I according to claim 3, characterized in that, Includes the following steps: S1. Compound 1 undergoes hydrogenation under catalytic conditions to produce compound 2; preferably, the catalyst is Pd(OH)2 or Co / Al2O3; S2. Compound 2 reacts with compound 3 to generate compound 4; preferably, an acid-binding agent is added to the reaction system, more preferably, the acid-binding agent is K2CO3 or Na2CO3; S3. Compound 4 and compound 5 undergo a sulfonation reaction to generate the anticomposite 6; preferably, an acid-binding agent is added to the reaction system, more preferably, the acid-binding agent is K2CO3 or Na2CO3; S4. Compound 6 undergoes deprotection to form compound 7; S5. Compound 7 reacts with Compound 8 to produce Compound I.

5. The method for preparing the precursor compound represented by Formula I according to claim 4, characterized in that, In step S1, the reaction temperature is 20~25℃; In step S2, the molar ratio of compound 2 to compound 3 is 1:0.8~1.2; preferably 1:0.9; the molar ratio of compound 2 to the acid-binding agent is 1:2~4; preferably 1:3; The reaction temperature in step S2 is 80~90℃, preferably 85℃; The preferred reaction solvent for step S2 is N,N-dimethylformamide.

6. The method for preparing the precursor compound represented by Formula I according to claim 4, characterized in that, In step S3, the molar ratio of compound 4 to compound 5 is 1:0.8~1.2; preferably 1:0.9; the molar ratio of compound 4 to the acid-binding agent is 1:1~2; preferably 1:1.5; The preferred reaction solvent for step S3 is N,N-dimethylformamide, and the reaction is carried out under reflux. In step S4, compound 6 is deprotected at 20-25°C under acidic conditions; preferably, the molar ratio of compound 6 to HCl is 1:10~15; more preferably, it is 1:

12.

7. The method for preparing the precursor compound represented by Formula I according to claim 4, characterized in that, In step S5, in the presence of N,N-diisopropylethylamine, compound 7 and compound 8 undergo a coupling reaction; preferably, the molar ratio of compound 7 to compound 8 is 1:1.5~2.5; more preferably, it is 1:2; preferably, the molar ratio of compound 7 to N,N-diisopropylethylamine is 1:3~5; more preferably, it is 1:

4. Preferably, the reaction temperature in step S5 is 20-25°C; the reaction solvent is N,N-dimethylformamide.

8. The method for preparing a small molecule inhibitor targeting RANKL labeled with radionuclide as shown in Formula II according to claim 2, characterized in that, The compound of Formula I according to claim 1 is reacted with a radioactive nuclide salt solution to obtain the radioactive labeling substance shown in Formula II.

9. The use of the precursor compound of Formula I according to claim 1 or its salt in the preparation of tumor imaging agents; or / and in the preparation of medicaments for treating tumors.

10. The use of a small molecule inhibitor of RANKL labeled with radionuclide as shown in Formula II according to claim 2, or a salt thereof, in the preparation of a tumor imaging agent; or / and in the preparation of a medicament for treating tumors.

Images