Multi-target molecular ligand as well as preparation method and application thereof

Abstract

The invention relates to a multi-target molecular ligand as well as a preparation method and application thereof. The multi-targeting molecular ligand disclosed by the invention aims at prostate cancer and bone metastasis thereof, and the ligand simultaneously contains a polypeptide chain Glu-urea-Lys (ACUPA) capable of being recognized by a prostate specific membrane antigen (PSMA), is used for targeting diphosphoric acid of hydroxyapatite nanocrystals in bones, and is also combined with an anisidine fragment which is specifically combined with a Sigma-1 receptor. The multi-target molecular ligand can efficiently mark radionuclide and has excellent radiation stability, cell and animal experiments prove that the multi-target molecular ligand can be efficiently taken by PSMA positive prostate cancer RM-1 cells, RM-1 subcutaneous tumor models and RM-1 tibial metastatic tumor models, and can be used for developing diagnosis and treatment of PSMA positive prostate cancer and bone metastasis thereof, and diagnosis and treatment integration is achieved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a multi-target molecular ligand, its preparation method, and its application. Background Technology

[0002] Prostate cancer is one of the most common cancers of the male genitourinary system. Currently, treatment methods for prostate cancer mainly include surgery, radiotherapy, endocrine therapy, and chemotherapy. Small molecule peptides have shown potential for precise targeting, multimodal integration, and high modifiability in the diagnosis and treatment of prostate cancer, particularly in PSMA-targeted imaging and therapy, overcoming drug resistance, and immune regulation. However, the specific uptake and stability of these drugs in vivo need improvement, and some may even cause toxic damage to non-tumor tissues. Specifically: First, low bioavailability: Peptides typically have a molecular weight of 1-5 kDa, smaller than proteins, but they may still be rapidly excreted by the kidneys through glomerular filtration or non-specifically taken up by the reticuloendothelial system in the liver, spleen, etc., resulting in short retention times and a narrow effective uptake window at the tumor site. Second, insufficient targeting and interference from non-specific uptake: Although small molecule peptides can improve selectivity for prostate cancer cells by designing targeting sequences (such as targeting PSMA), non-specific uptake problems still exist. Third, PSMA expression in prostate cancer is heterogeneous; PSMA expression is decreased in some primary tumors, especially metastatic tumors. This can also be the case with bone metastases, especially as the heterogeneity becomes more pronounced with disease progression. This means that PSMA expression in prostate cancer bone metastases may decrease as the disease progresses. Therefore, dual-targeting ligands that simultaneously target both PSMA and bone metastases may simplify the detection of prostate cancer bone metastases and more directly treat PSMA-positive primary lesions as well as metastatic diseases in the bone, achieving additional non-PSMA-dependent treatment for bone metastases. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a multi-targeting molecular ligand, its preparation method, and its application. This ligand can not only efficiently label radionuclides but also simultaneously and specifically target PSMA and bone metastases. It can be used for the imaging diagnosis and treatment of PSMA-positive prostate cancer bone metastases, achieving an integrated diagnostic and therapeutic effect.

[0004] This invention provides the following technical solutions:

[0005] The first aspect of this invention provides a multi-targeting molecular ligand, the structural formula of which is as follows:

[0006]

[0007] Where n is any integer from 1 to 10.

[0008] Furthermore, the multi-targeting molecular ligand has the following structure:

[0009]

[0010] Furthermore, the multi-targeting molecular ligand can be labeled with a radionuclide, preferably a radionuclide. 68 Ga、 89 Zr、 64 Cu、 86 Y、 99m Tc, 111 In、 90 Y、 67 Ga、 177 Lu、 161 Tb, 211 At、 153 Sm、 186 Re、 188 Re、 67 Cu、 203 Pb, 225 Ac、 213 Bi、 223 Ra、 212 Bi、 212 Pb and 227 One or more of Th.

[0011] A second aspect of the present invention provides a method for preparing the aforementioned multi-target molecular ligand, comprising the following steps:

[0012] (1) 3-mercaptopropionic acid was reacted with an amide condensing agent by stirring to obtain a mixture containing intermediates;

[0013] (2) Add an inorganic base to an aqueous solution of sodium alendronate to adjust the pH to 6.5-7.5, and then add it to the mixture containing the intermediate prepared in step (1) and stir to react. After the reaction is completed, remove the solvent by rotary evaporation, precipitate, and wash the precipitate with anhydrous acetonitrile to obtain the compound shown in formula (I).

[0014] (3) Dissolve the compound shown in formula (I) and the compound shown in formula (II) in water, add an alkaline reagent to adjust the pH of the solution to 6.5-7.5, and then stir the reaction under an inert atmosphere to obtain the multi-target molecular ligand;

[0015] The structures of equations (I) and (II) are shown below:

[0016]

[0017] Where n is any integer from 1 to 10.

[0018] Further, in step (1), the amide condensing agent is dicyclohexylcarbodiimide and N-hydroxysuccinimide. Preferably, the molar ratio of dicyclohexylcarbodiimide, N-hydroxysuccinimide and 3-mercaptopropionic acid is 1.2:1.2:1. The reaction temperature is 10-30℃, more preferably 18-26℃, and the reaction time is 2-12h.

[0019] More preferably, 3-mercaptopropionic acid is dissolved in a solvent, which includes but is not limited to acetonitrile, and then dicyclohexylcarbodiimide and N-hydroxysuccinimide are added. The mixture is stirred at 18-26°C for 2-12 hours. After the reaction is completed, the dicyclohexylurea precipitate is removed by filtration through a filter membrane. The filtered solution is then rotary evaporated to obtain a solid, which is then dissolved in dichloromethane and washed with water for extraction. The organic layer is collected. A drying agent, such as anhydrous sodium sulfate, is then added to the organic layer to remove trace amounts of water. The dichloromethane is then removed by rotary evaporation to obtain an intermediate.

[0020] Further, in step (2), the inorganic base is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, anhydrous sodium carbonate, sodium bicarbonate, potassium carbonate, and sodium bicarbonate. An inorganic base solution is added to the sodium alendronate aqueous solution to adjust the pH of the solution to 6.5-7.5. The reaction temperature is 10-30℃, more preferably 18-26℃, and the reaction time is 6-24h.

[0021] More preferably, the intermediate prepared in step (1) is dissolved in acetonitrile and slowly added dropwise to an aqueous solution of sodium alendronate with a pH of 6.5-7.5. The mixture is stirred at 18-26°C for 6-24 hours. After the reaction is complete, the solvent is removed by rotary evaporation. After a large amount of precipitate is precipitated, the precipitate and the remaining liquid are collected and freeze-dried. The precipitate is then washed with anhydrous acetonitrile to obtain a mixture containing alendronate and the compound shown in formula (I).

[0022] Further, in step (3), the alkaline reagent is an inorganic or organic base, such as potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, triethylamine, triethylenediamine, 1,8-diazabicycloundec-7-ene, 4-dimethylaminopyridine, etc.; the reaction temperature is 10-30℃, more preferably 18-26℃, and the reaction time is 1-8h.

[0023] Furthermore, the preparation method also includes a process of purifying the reaction product prepared in step (3). After the reaction is completed, the reaction solution is concentrated by rotary evaporation, filtered using a filter membrane, and then purified and separated by preparative high performance liquid chromatography to obtain the purified multi-target molecular ligand.

[0024] A third aspect of the present invention provides the use of the aforementioned multi-targeting molecular ligand in the preparation of medicaments for the diagnosis and / or treatment of cancer.

[0025] Furthermore, the multi-target molecular ligand drug is used for the diagnosis and / or treatment of PSMA-overexpressing tumors; the PSMA-overexpressing tumors include solid tumors such as prostate cancer, non-small cell lung cancer, esophageal cancer, colorectal cancer, and renal cell carcinoma, as well as their bone metastases.

[0026] The technical solution of the present invention has the following advantages compared with the prior art:

[0027] 1. This invention provides a multi-targeting molecular ligand, its preparation method, and its applications. The multi-targeting molecular ligand comprises a polypeptide chain Glu-urea-Lys that can be recognized by prostate-specific membrane antigens, a bisphosphonic acid that can target hydroxyapatite nanocrystals in bone, and an anisamide fragment that specifically binds to the Sigma-1 receptor, thereby achieving multiple targeting. Furthermore, the molecular ligand contains a twelve-membered tetrazolium heterocyclic macrocyclic ligand that can coordinate with radionuclides, thus allowing for efficient labeling by radioactive elements (labeling efficiency >95%) and excellent stability after labeling, making it suitable for radiodiagnosis and therapy. Additionally, by introducing maleimide acetic acid of different chain lengths into the molecular ligand, its pharmacokinetic characteristics can be effectively improved.

[0028] 2. Cell and animal experiments have demonstrated that the multi-target molecular ligands provided by this invention can be efficiently taken up by PSMA-positive prostate cancer RM-1 cells, RM-1 subcutaneous tumor models, and RM-1 tibial metastasis models, and can be used for the imaging diagnosis of PSMA-positive prostate cancer and its bone metastases.

[0029] 3. The multi-targeting molecular ligands prepared by this invention can be efficiently labeled with radionuclides and exhibit excellent stability after labeling. They can also specifically target prostate cancer cells and prostate cancer bone metastases, thus enabling their use in the diagnosis and treatment of prostate cancer and prostate cancer bone metastases, achieving integrated diagnosis and treatment. Attached Figure Description

[0030] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0031] Figure 1 The 1H NMR spectrum of the multi-targeting molecular ligand PSMA-ALN-4;

[0032] Figure 2 The phosphorus NMR spectrum of the multi-targeting molecular ligand PSMA-ALN-4;

[0033] Figure 3 Mass spectrometry results for the multi-targeting molecular ligand PSMA-ALN-4;

[0034] Figure 4 radioactive ligands177 Lu-PSMA-ALN-2 177 Radio-TLC spectrum of Lu-PSMA-ALN-4;

[0035] Figure 5 radioactive ligands 177 Lu-PSMA-ALN-2 177 Stability test results of Lu-PSMA-ALN-4 in PBS and 10% FBS;

[0036] Figure 6 radioactive ligands 177 Lu-PSMA-ALN-2 177 The binding rate of Lu-PSMA-ALN-4 to PSMA-positive / negative RM-1 cells in prostate cancer at different time points;

[0037] Figure 7 radioactive ligands 177 Lu-PSMA-ALN-2 177 Results of in vitro binding experiments of Lu-PSMA-ALN-4 with hydroxyapatite;

[0038] Figure 8 radioactive ligands 177 Lu-PSMA-ALN-2 177 SPECT / CT images of Lu-PSMA-ALN-4 administered via tail vein to PSMA-positive RM-1 subcutaneous tumor-bearing mice at different time points.

[0039] Figure 9 radioactive ligands 177 Lu-PSMA-ALN-2 177 SPECT / CT images of Lu-PSMA-ALN-4 administered via tail vein to mice with PSMA-negative RM-1 tibial metastases at different time points.

[0040] Figure 10 radioactive ligands 177 Lu-PSMA-ALN-2 177 SPECT / CT images of Lu-PSMA-ALN-4 administered via the tail vein to mice with PSMA-positive RM-1 tibial metastases at different time points. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0042] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0043] Example 1

[0044] This embodiment relates to the synthesis of the multi-targeting molecular ligand PSMA-ALN-4, and the specific preparation process is as follows:

[0045] 1. Synthesis of PSMA-4

[0046] The synthesis route for PSMA-4 is as follows:

[0047]

[0048] The specific steps for synthesizing PSMA-4 are as follows:

[0049] (1) Bonding resin

[0050] Resin preparation: Place 1 eq resin (0.36 mmol, 300 mg), 2 eq Fmoc-Glu(OtBu)-OH (0.72 mmol, 325.8 mg), and 2.4 eq DIPEA (0.86 mmol, 143 μL) in a solid-phase reaction tube, add 5 ml anhydrous DMF, shake for 70-90 rpm, and incubate for 8-10 h.

[0051] End sealing: Blow out the reaction solution and wash three times with DMF; dissolve 300 μL of methanol and 350 μL of DIPEA in 5 mL of DMF and add to the reaction tube, shake for 10 min; blow out the reaction solution and wash three times with DMF.

[0052] (2) Activation of amino groups: under N2 protection, at 0℃, 10 eq triphosgene (3.6 mmol, 1.065 g) was dissolved in 5 mL of anhydrous DCM and stirred for 10 min.

[0053] 2 eq H-Lys(Fmoc)-OtBu (0.72 mmol, 213 mg) and 18 eq DIPEA (6.5 mmol, 1.042 mL) were dissolved in 5 mL of anhydrous DCM and slowly injected into the solution using a syringe (the solution turned yellow). The mixture was stirred for 4–6 h. The solvent was removed by evaporation under reduced pressure, yielding a yellow precipitate, which was the activated product, to obtain substance 1.

[0054] (3) Connect H-Lys(Fmoc)-OtBu

[0055] Defomics removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin, detect ninhydrin, and the resin turns blue after heating to obtain substance 2.

[0056] H-Lys(Fmoc)-OtBu: Add 2.4 eq DIPEA (142 μL) to the solid-phase reaction tube in an ice bath and dissolve it in 3 mL of anhydrous DCM; dissolve the activated product 1 obtained in step (2) in 5 mL of anhydrous DCM (pretreated at 0 °C), slowly add it to the solid-phase reaction tube, and shake for 4-8 h.

[0057] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin turns blue after heating, the reaction has failed. Repeat this step. If the resin does not turn blue after heating, the reaction has succeeded and substance 3 is obtained, which can be used for the next reaction.

[0058] (4) Connect Fmoc-2-NaI-OH

[0059] Fomc removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin and test for ninhydrin; the resin turns blue after heating.

[0060] Add Fmoc-2-NaI-OH: Dissolve 1.5 eq Fmoc-2-NaI-OH (0.54 mmol, 236 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) in 5 mL DMF, add to a solid-phase reaction tube, and shake for 4-8 h.

[0061] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 4 is obtained, which can proceed to the next step.

[0062] (5) Inoculate with isoquinoline (substance X3)

[0063] Fomc removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin and test for ninhydrin; the resin turns blue after heating.

[0064] Isoquinoline inoculation: 1.5 eq isoquinoline (0.54 mmol, 216 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) were dissolved in 5 mL DMF, added to a solid-phase reaction tube, and shaken for 4-8 h.

[0065] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 5 is obtained, which can proceed to the next step.

[0066] The synthetic route for substance X3 is as follows:

[0067]

[0068] The specific steps for synthesizing X3 are as follows:

[0069] Amino compound (X1, 1 eq) and FMOC-ONSu (X2, 1.2 eq) were dissolved in 1,4-dioxane (8 mL) and water (4 mL), and sodium carbonate (1.85 eq) was added. The mixture was stirred overnight at 25 °C, and TLC was used to detect the complete reaction of the starting materials. The solvent was concentrated by rotary evaporation, and the pH was adjusted to 3-4 with 0.1 mol / L ammonium chloride solution. Extraction was performed with a large amount of ethyl acetate, and the reaction was repeated twice. The product was dried over anhydrous sodium sulfate and concentrated by rotary evaporation. The crude product was purified by preparative HPLC to obtain the corresponding Fmoc-protected compound X3.

[0070] (6) Connect to Dde-Lys(Fmoc)-OH

[0071] Fomc removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin and test for ninhydrin; the resin turns blue after heating.

[0072] For Dde-Lys(Fmoc)-OH: 1.5 eq Dde-Lys(Fmoc)-OH (0.54 mmol, 288 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) were dissolved in 5 mL DMF, added to a solid-phase reaction tube, and shaken for 4-8 h.

[0073] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 6 is obtained, which can proceed to the next step.

[0074] (7) Connecting material Y4

[0075] Fomc removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin and test for ninhydrin; the resin turns blue after heating.

[0076] Y4: 1.5 eq Y4 (0.54 mmol, 196 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) were dissolved in 5 mL DMF, added to a solid-phase reaction tube, and shaken for 4-8 h.

[0077] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 7 is obtained, which can proceed to the next step.

[0078] The synthetic route for substance Y4 is as follows:

[0079]

[0080] The specific steps for synthesizing Y4 are as follows:

[0081] p-Methoxybenzoic acid (1 eq, 1 mmol, 152 mg), HBTU (1.2 eq, 1.2 mmol, 456 mg), and DIPEA (1.2 eq, 1.2 mmol, 203 μL) were added to 30 mL of DCM. After reacting at room temperature for 0.5 h, 4-N-(2-aminoethyl)-1-N-BOC-piperidine (1 eq, 1 mmol, 229 mg) was added, and the mixture was stirred for 8 h. TLC analysis showed the reaction was complete, and Y1 was purified. Y1 was dissolved in 10 mL of DCM / TFA (1 / 1) solution, reacted at room temperature for 1 h, and the pH was adjusted to approximately 7 with sodium bicarbonate. The supernatant was concentrated by centrifugation and rotary evaporation to obtain crude Y2. HPLC analysis showed its purity was suitable for direct use in the next step. Monotert-butyl succinate (1 eq, 1 mmol, 174 mg), HBTU (1.2 eq, 1.2 mmol, 456 mg), and DIPEA (1.2 eq, 1.2 mmol, 203 μL) were added to 30 mL of DCM. After reacting at room temperature for 0.5 h, crude Y2 was added, and the mixture was stirred for 8 h. TLC analysis showed the reaction was complete, and Y3 was purified to obtain Y3. Y3 was dissolved in 10 mL of DCM / TFA (1 / 1) solution, reacted at room temperature for 2 h, concentrated by rotary evaporation, precipitated with diethyl ether, and centrifuged to obtain crude Y4. Pure Y4 was obtained after HPLC separation.

[0082] (8) Connect to Fmoc-Lys(Dde)-OH

[0083] De-Dde removal: Wash the resin with 10 mL of 2% hydrazine hydrate / DMF solution for 3 min, repeat three times, wash three times with DMF, take a small amount of resin, and test for ninhydrin. The resin turns blue after heating.

[0084] For Fmoc-Lys(Dde)-OH: 1.5 eq Fmoc-Lys(Dde)-OH (0.54 mmol, 288 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) were dissolved in 5 mL DMF, added to a solid-phase reaction tube, and shaken for 4-8 h.

[0085] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 8 is obtained, which can proceed to the next step.

[0086] (9) Connect to DOTA

[0087] Fomc removal: Add 10 mL of 20% piperidine / DMF solution to the solid-phase reaction tube and shake for 10 min; repeat 3 times; wash 3 times with DMF; take a small amount of resin and test for ninhydrin; the resin turns blue after heating.

[0088] For DOTA: Dissolve 1.5 eq DOTA (0.54 mmol, 309 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) in 5 mL DMF, add to a solid-phase reaction tube, and shake for 4-8 h.

[0089] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful and substance 9 is obtained, which can proceed to the next step.

[0090] (10) Attach 6-maleimide

[0091] De-Dde removal: Wash the resin with 10 mL of 2% hydrazine hydrate / DMF solution for 3 min, repeat three times, wash three times with DMF, take a small amount of resin, and test for ninhydrin. The resin turns blue after heating.

[0092] For 6-maleimide: 1.5 eq 6-maleimide (0.54 mmol, 114 mg), 1.8 eq HOBT (0.648 mmol, 87.5 mg), HBTU (0.648 mmol, 246 mg), and DIPEA (0.648 mmol, 110 μL) were dissolved in 5 mL DMF, added to a solid-phase reaction tube, and shaken for 4–8 h.

[0093] Amino detection: Blow out the reaction solution, wash three times with DMF, take a small amount of resin, and test with ninhydrin. If the resin does not turn blue, the reaction is successful, and substance 10 is obtained, which can proceed to the next step.

[0094] (11) Cut resin

[0095] The resin was washed three times in sequence with anhydrous methanol, petroleum ether, and DCM. 10 mL of 1% TFA / DCM solution was added to the solid-phase reaction tube, and the liquid was collected after shaking for 10 min until the resin turned blackish-purple. This process was repeated twice.

[0096] After the liquid is concentrated by rotary evaporation, the concentrated liquid is added to ice-cold ether for precipitation, centrifuged, and the precipitate is collected.

[0097] (12) Deprotection

[0098] Dissolve the precipitate from the previous step in 10 mL of TFA / DCM solution (1 / 1) and stir for 6 h. Add a large amount of DCM and concentrate by rotary evaporation, precipitate with diethyl ether, centrifuge, collect the precipitate, and dry under vacuum.

[0099] (13) Identification and purification

[0100] The target product was detected by mass spectrometry, purified by preparative HPLC, and lyophilized to obtain substance 11, PSMA-4.

[0101] 2. Connecting alendronate sodium (ALN):

[0102] The synthetic route for PSMA-ALN-4 is as follows:

[0103]

[0104] The specific steps for synthesizing PSMA-ALN-4 are as follows:

[0105] (1) Preparation of crude HS-ALN product

[0106] 3-Mercaptopropionic acid (2.0 mmol, 2.0 eq), dicyclohexylcarbodiimide (2.4 mmol, 2.4 eq), and N-hydroxysuccinimide (2.4 mmol, 2.4 eq) were dissolved in 5 mL of acetonitrile and stirred at room temperature for 4 h. After the reaction was complete, the mixture was filtered through a filter membrane to remove dicyclohexylurea precipitate. The solvent was removed by rotary evaporation, and then 15 mL of dichloromethane was added to dissolve the precipitate. The mixture was washed with ultrapure water, and the organic layer was collected. The dichloromethane was removed by rotary evaporation to obtain the crude intermediate product. Sodium alendronate trihydrate (1.0 mmol, 1.0 eq) was dissolved in 5 mL of ultrapure water and added to a reaction flask. The pH was adjusted to 7 with 1 M NaOH solution. The crude intermediate product was dissolved in 5 mL of acetonitrile, and the solution was slowly added dropwise to the reaction flask. The mixture was stirred at room temperature for 24 h. After the reaction was completed, the reaction solution was concentrated by rotary evaporation and added to methanol (35 mL) for precipitation. After precipitation, the solution was centrifuged to remove the supernatant. The precipitate was placed in a freeze dryer to obtain a white solid compound, namely the crude HS-ALN product.

[0107] (2) Connect HS-ALN

[0108] PSMA-4 (1.0 nmol, 1.0 eq) and the crude product of compound HS-ALN (10.0 nmol, 10.0 eq) were dissolved in 1 mL of ultrapure water, respectively. 1 M NaOH was added dropwise to the HS-ALN solution until the pH reached 7.4. The solutions were then injected into a nitrogen-filled reaction flask and stirred at room temperature for 6 h. After stirring, samples were taken for mass spectrometry analysis. Preparative HPLC purification and separation were performed, and the product was lyophilized to obtain a white solid product, yielding PSMA-ALN-4 (0.81 mg, yield 36.9%).

[0109] 3. Product identification:

[0110] The prepared product PSMA-ALN-4 was analyzed by NMR, and the 1H NMR spectrum is shown below. Figure 1 As shown, in the 1H NMR spectrum, peaks 1-7 and 20-26 are characteristic peaks of the naphthalene ring, isoquinoline ring, and benzene ring in substance Y4; peaks 8-11 are characteristic peaks of the carboxyl branch of the chelating agent DOTA; peaks 12-16 are characteristic peaks of hydrogen on the core carbon atom in lysine and glutamic acid; peaks 17 and 18 are characteristic peaks of HS-ALN; and peak 30 is a multiplet characteristic peak of hydrogen on the carbon atom connected to the sulfur atom. The 1H NMR spectrum is as follows: 1H NMR (400MHz, Deuterium Oxide) δ 8.44-8.21 (m, 6H), 8.01 (s, 5H), 7.76 (s, 1H), 7.54 (d, J = 8.6Hz, 2H), 5.24 (d, J = 17.7Hz, 2H), 4.59 (s, 1H), 4.46 (s, 1H), 4.36 (s, 3H), 4.31 (d, J = 5.3Hz, 2H), 4.28-4.13 (m, 6H), 3.82 (p, J = 1.6Hz, 8H), 3.17 (s, 2H), 3.07 (t, J = 6.1Hz, 2H);

[0111] Phosphorus spectrum Figure 2 As shown, A is the phosphorus spectrum of alendronic acid and B is the phosphorus spectrum of PSMA-ALN-4, proving that the synthesized product contains alendronic acid.

[0112] The prepared product PSMA-ALN-4 was analyzed by mass spectrometry, and the mass spectrometry results are as follows: Figure 3 As shown, M = 2194.31, m / z: 1097.9 = (M + 2H) 2+ / 2, proving that the ligand was successfully synthesized.

[0113] Example 2

[0114] This embodiment involves radionuclide-labeled multi-target molecular ligands. 177The preparation and stability study of Lu-PSMA-ALN-4 were conducted, with a comparative study of PSMA-ALN-2 (see patent CN118221764A for this compound); details are as follows:

[0115] 177 Lu-PSMA-ALN-2 177 Preparation of Lu-PSMA-ALN-4: The ligand was labeled using a high-temperature labeling method to... 177 Labeling was performed using LuCl3:DOTA at a ratio of 1:100. 1 mg of PSMA-ALN-2 and PSMA-ALN-4 were weighed and dissolved in 1 mL of ultrapure water, resulting in a precursor concentration of 1 mg / mL. 1 mL of... 177 Add LuCl3 to the bottom of a 1.5 mL centrifuge tube, then add 8.73 μL of LPSMA-ALN-2 and 11.3 μL of PSMA-ALN-4 solution to the bottom of the centrifuge tube. Next, add 0.4 M sodium acetate standard solution with pH = 4.5 to bring the total volume to 50-100 μL. Place the centrifuge tube in a constant temperature stirrer and set the temperature to 95℃, 800 rpm, and time to 30 min.

[0116] The radiolabeling efficiency and radiochemical purity of the compounds were determined using a radio-TLC scanner. A silica gel TLC plate was used as the stationary phase, and 1% EDTA solution was used as the developing solvent to determine the radiolabeled and free radioactive compounds. 177 The specific shift (R) of Lu, for radiolabeled substances Rf = 0.1-0.2, 177 The Rf of LuCl3 is 0.9–1.0. The labeling results are as follows: Figure 4 As shown, the radiolabeling efficiency of the ligand is >98%.

[0117] Marked 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 was divided into two groups, with three samples in each group. 0.5 mL of phosphate-buffered saline (PBS) and 10% fetal bovine serum (FBS) were added to one group, respectively. The peak area of ​​the radiolabeled substances was detected using a Radio-TLC scanner on days 1, 2, 3, 4, 5, 6, and 7. 177 The peak area of ​​LuCl3 was used to study its labeling stability. The detection results are as follows: Figure 5 As shown, the ligands all exhibited very good radiolabeling stability, and no radionuclide shedding occurred.

[0118] Example 3

[0119] This embodiment investigated the binding ability of radionuclide-labeled multi-targeting molecular ligands to PSMA-positive prostate cancer RM-1 cells, and used PSMA-negative prostate cancer RM-1 cells as a control for specificity studies. The specific procedures are as follows:

[0120] PSMA-positive / negative prostate cancer RM-1 cells were cultured normally in DMEM medium containing 10% FBS and 1% penicillin / streptomycin under a constant temperature incubator at 37℃ and 5% CO2. When the cells reached the logarithmic cell cycle, they were digested with trypsin and then seeded into 24-well plates at approximately 1 × 10⁶ cells per well. 5 Cells were cultured in a constant temperature incubator for 12 hours to allow them to adhere. After cell adhesion, the culture medium was replaced, and the same radioactive count (10 μCi) was added to each well. 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 (n=3). Cells were incubated for 1 h, 4 h, 8 h, and 24 h. At each time point, the culture medium was aspirated, and the cells were washed three times with PBS, with the PBS collected. The culture medium and PBS were mixed to obtain a radioactive supernatant, and 1 mL of 2N NaOH solution was added to lyse the cells. After 10 min, the lysate was collected, and the wells were washed three times with PBS, with the PBS collected. The lysate was mixed with PBS to obtain cell lysate, and the radioactivity counts of each supernatant and lysate were measured using a gamma counter. The cell binding rate was calculated as: [radioactivity count in lysate / (radioactivity count in lysate + radioactivity count in supernatant)] × 100%.

[0121] Depend on Figure 6 It can be seen that the specific uptake of radionuclide-labeled multi-target molecular ligands by PSMA-positive RM-1 cells gradually increases over time. Furthermore, the binding rate with PSMA-negative RM-1 cells remains essentially unchanged over time, with a maximum binding rate of <5%, significantly lower than that with PSMA-positive cells, indicating that... 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 has PSMA targeting specificity.

[0122] Example 4

[0123] This embodiment investigated the targeting ability of radionuclide-labeled multi-targeting molecular ligands to bone. Hydroxyapatite (HAP) was selected for in vitro experiments, and the specific procedures are as follows:

[0124] Add 1.5 mL of physiological saline to a centrifuge tube containing 0.5 mg hydroxyapatite, then add 50 μL (20 μCi, n = 3) of [unclear - possibly a specific ingredient or solution]. 177 Lu-PSMA-ALN-2 177For Lu-PSMA-ALN-4, centrifuge tubes were placed in a thermostatic mixer, set to 37°C and 1000 rpm, and shaken for 1, 5, 30 min, 1, 4, 8, and 24 h. After centrifugation, the supernatant was collected and washed twice with physiological saline. The supernatant was then collected, and the radioactivity count of the precipitate was measured using a gamma radioimmunoassay counter. The supernatant was collected and the radioactivity count was measured. The binding rate with HAP was calculated as: [precipitate radioactivity count / (precipitate radioactivity count + supernatant radioactivity count)] × 100%.

[0125] Depend on Figure 7 It can be known that radioactive ligands 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 rapidly bound to HAP after co-incubation for 1 min, with binding rates of all ligands exceeding 90%, and no significant increase in binding rate over time. The ligands modified with ALN... 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 has an extremely strong ability to bind hydroxyapatite.

[0126] Example 5

[0127] This embodiment investigates radionuclide-labeled multi-target molecular ligands. 177 Lu-PSMA-ALN-2 177 The tumor uptake of Lu-PSMA-ALN-4 in PSMA-positive RM-1 cell subcutaneous tumor mice and tibial metastatic tumor mice was investigated as follows:

[0128] First, a mouse subcutaneous PSMA-positive RM-1 cell tumor model was established. A 1:1 mixture of matrix gel and PSMA-positive RM-1 cells was used to form a cell suspension, which was then subcutaneously injected into the right rib region of nude mice (male, 5 weeks old, 18-20g). Tumor size and mouse weight were recorded. Tumors were allowed to develop until they reached a volume of 100-200 mm². 3 The experiment was conducted on the left and right sides.

[0129] Then, a mouse model of PSMA-positive / negative RM-1 cell tibial metastases was established. A cell suspension was prepared by mixing matrix gel with PSMA-positive / negative RM-1 cells at a 1:1 ratio. Using a 1mL syringe needle, the needle was inserted into the knee joint space of the right leg of a nude mouse, entering through the tibial head fossa and slowly drilling along the long axis of the tibial shaft through the cancellous bone to reach the medullary cavity, creating a wound. The cell suspension (10-15μL) was then injected using a microsyringe. Tumor growth and the mouse's condition were monitored. Experiments were conducted 7-10 days after tumor implantation, when a significant protrusion appeared on the tumor-bearing leg.

[0130] To monitor tumor uptake of radioligands in various tumor model mice, SPECT imaging was performed. Each group of mice (n=3) was injected intravenously with 200 μCi (200 μL) of labeled ligand. Whole-body scans of the mice were performed using microSPECT / CT at 1, 4, 8, 24, 48, 72, and 96 h post-injection. The scan results were quantitatively analyzed using PMOD software.

[0131] 177 Lu-PSMA-ALN-2 177 SPECT / CT imaging results of subcutaneously tumor-bearing mice after tail vein administration of Lu-PSMA-ALN-4 are as follows: Figure 8 As shown, 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 exhibits high uptake in tumors, and the remaining radioligands are rapidly metabolized by the kidneys, while also being specifically uptaken in active bone tissue.

[0132] 177 Lu-PSMA-ALN-2 177 SPECT / CT imaging results of mice with PSMA-negative and positive tibial metastases after tail vein administration of Lu-PSMA-ALN-4 are as follows: Figure 9 , Figure 10 As shown, 177 Lu-PSMA-ALN-2 177 Lu-PSMA-ALN-4 exhibits high uptake at metastatic sites. Ligand uptake in PSMA-negative bone metastases is significantly higher than that in contralateral healthy bone of mice, verifying the specific targeting and binding ability of the ligand to bone metastases. Ligand uptake in PSMA-positive bone metastases is significantly higher than that in contralateral healthy bone of mice and PSMA-negative bone metastases, verifying the specific targeting and binding ability of the ligand to PSMA-positive bone metastases. Furthermore, the remaining radioligand is rapidly metabolized by the kidneys.

[0133] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A multi-targeting molecular ligand, characterized in that, The structure of the multi-target molecular ligand is as follows: wherein n is any integer from 1 to 10.

2. The multi-targeted molecular ligand of claim 1, wherein, The multi-target molecular ligand is as follows:

3. The multi-targeting molecular ligand according to claim 1 or 2, characterized in that, The multi-target molecular ligand can be labeled with a radionuclide.

4. The multi-targeted molecular ligand of claim 3, wherein, The radionuclide comprises 68 Ga, 89 Zr, 64 Cu, 86 Y, 99m Tc, 111 In, 90 Y, 67 Ga, 177 Lu, 161 Tb, 211 At, 153 Sm, 186 Re, 188 Re, 67 Cu, 212 Pb, 225 Ac, 213 Bi, 223 Ra, 212 Bi, 203 Pb, and 227 Th.

5. A method of preparing a multi-targeting molecular ligand according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: (1) stirring and reacting 3-mercaptopropionic acid with an amide condensing agent to obtain a mixture containing an intermediate; (2) adding an inorganic base to an aqueous sodium alendronate solution to adjust the pH to 6.5-7.5, then adding the mixture containing the intermediate prepared in step (1) to stir and react, after the reaction is completed, removing the solvent by rotary evaporation, precipitating, and washing to obtain the compound shown in formula (I); (3) dissolving the compound shown in formula (I) and the compound shown in formula (II) in water, adding a base reagent to adjust the pH of the solution to 6.5-7.5, then stirring and reacting under an inert atmosphere to obtain the multi-target molecular ligand; The structures of formula (I) and formula (II) are as follows: wherein n is any integer from 1 to 10.

6. The production method according to claim 5, wherein In step (1), the amide condensing agent is dicyclohexyl carbodiimide and N-hydroxysuccinimide; The temperature of the reaction is 10-40℃, and the reaction time is 4-8h.

7. The preparation method according to claim 5, characterized in that, In step (2), the inorganic base is selected from one or more of sodium hydroxide, potassium hydroxide, calcium hydroxide, anhydrous sodium carbonate, sodium bicarbonate, potassium carbonate, and sodium bicarbonate; The temperature of the reaction is 10-40℃, and the reaction time is 8-24h; The anti-solvent is selected from one or more of water, methanol, acetonitrile, and acetone.

8. The preparation method according to claim 5, characterized in that, In step (3), the base reagent is an inorganic base or an organic base; The inorganic base is selected from one or more of potassium hydroxide, calcium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, and potassium bicarbonate; The organic base is selected from one or more of triethylamine, triethylenediamine, 1,8-diazabicycloundec-7-ene, and 4-dimethylaminopyridine; The temperature of the reaction is 10-40℃, and the reaction time is 4-12h.

9. Use of the multi-target molecular ligand of any one of claims 1-4 in the preparation of a medicament for the diagnosis and / or treatment of cancer.

10. Use according to claim 9, characterized in that, The cancer is a tumor overexpressing PSMA.

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