A coupling compound, its preparation method and application

Abstract

This invention relates to a conjugate, its preparation method, and its applications. The conjugate has the structure of a compound as shown in Formula I or a pharmaceutically acceptable salt thereof. The conjugate can achieve conjugation with a target molecule while maintaining its original metal coordination ability, thereby constructing a radioactive probe for nuclear medicine diagnosis or treatment. Furthermore, the conjugate achieves clear imaging in PSMA-positive tumors and exhibits superior tumor-specific imaging contrast within 12 hours. Formula I.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry, specifically the field of nuclear medicine radiopharmaceutical chemistry, and relates to a conjugate, its preparation method, and its application. Background Technology

[0002] Nuclear medicine molecular imaging and radionuclide therapy have significant applications in the precision diagnosis and treatment of diseases such as tumors. Among these, those based on metallic radionuclides (such as…) 68 Ga、 90 Y and 177 Radiopharmaceuticals (such as Lu et al.) have been widely used in clinical practice and research due to their unique advantages. In metal radiopharmaceuticals, chelating agents are used to connect metal ions to target molecules, and their coordination ability and complexation stability directly affect the labeling efficiency and in vivo stability of the radiopharmaceutical. Currently, commonly used chelating agents in clinical practice are mainly cyclic polydentate ligands, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its derivatives. However, these cyclic chelating agents usually require high temperatures (e.g., 80-95℃) and long reaction times to achieve the desired effect on certain metal ions (e.g., 177Lu). 3+ Highly efficient labeling of molecule-targeting molecules. This demanding labeling condition is a significant limitation for heat-sensitive biological targets such as proteins or peptides, which may lead to structural denaturation or loss of activity.

[0003] Acyclic chelators typically exhibit faster complexation kinetics, achieving efficient metal complexation even under mild conditions, and have therefore attracted increasing attention in recent years. However, how to achieve rapid labeling while maintaining good coordination stability remains a crucial research challenge in this field. Furthermore, regardless of whether cyclic or acyclic chelators are used, constructing targeted radioactive probes requires further functionalization into bifunctional chelators (BFCs). An ideal BFC must possess both a coordinating group at one end capable of forming a stable complex with a specific metal ion, and a reactive group at the other end capable of efficiently and specifically coupling with functional groups (such as primary amine groups) on the biological target molecule.

[0004] Therefore, developing conjugates with both excellent coordination properties and efficient coupling ability for promising novel acyclic chelating agent skeletons is key to promoting their practical application in the field of radiopharmaceuticals. Summary of the Invention

[0005] The purpose of this invention is to provide a conjugate that can couple with a target molecule while maintaining its original metal coordination ability, thereby constructing a radioactive probe for nuclear medicine diagnosis or treatment. Furthermore, the conjugate achieves clear imaging in PSMA-positive tumors and exhibits superior tumor-specific imaging contrast within 12 hours.

[0006] This invention provides a conjugate selected from compounds of formula I or pharmaceutically acceptable salts thereof. Formula I Wherein, P is selected from residues of a targeting ligand that can specifically bind to any of the following targets: prostate-specific membrane antigen (PSMA), fibroblast activator protein (FAP), gastrin-releasing peptide receptor (GRPR), integrin αvβ3 / αvβ5, carbonic anhydrase IX (CAIX), folate receptor (FR), or somatostatin receptor (SSTR). n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0007] In some embodiments, P is selected from PSMA-targeting ligand residues; preferably, P is selected from... ; The n is selected from an integer from 5 to 10, preferably 6, 7, 8, or 9.

[0008] In some embodiments, the compound represented by Formula I is selected from the following compounds: .

[0009] The present invention also provides a method for preparing the coupling compound as described above, comprising: S2. React the compound of formula I-2 with a fluoride ion source to obtain the compound shown in formula I; Formula I-2 Formula I in, PG1 and PG2 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl, benzyl, p-methoxybenzyl, allyl, 2-(trimethylsilyl)ethyl or trichloroethyl; preferably selected from tert-butyl, benzyl or 2-(trimethylsilyl)ethyl; The fluoride ion source is selected from tetrabutylammonium fluoride, triethylamine trifluoride or cesium fluoride, preferably tetrabutylammonium fluoride; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; Preferably, the reaction is carried out in the presence of an organic solvent; more preferably, the organic solvent is a polar aprotic solvent, and even more preferably N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, or tetrahydrofuran.

[0010] In some embodiments, the method for preparing compound I-2 is as follows: S1. React compound I-1 with PSMA-targeting peptide to obtain compound I-2; Formula I-1 Formula I-2; Preferably, step S1 is carried out in the presence of a polar aprotic solvent selected from N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO), preferably N,N-dimethylacetamide; and / or PSMA-targeting peptides .

[0011] In some embodiments, the preparation method of the compound of formula I-1 is as follows: PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0012] The present invention also provides a compound, said compound being a compound of formula I-1, a compound of formula I-2, a compound of T1, a compound of T2, a compound of T3, and their pharmaceutically acceptable salts. PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; Preferably, the compound is a compound with the following structure and its pharmaceutically acceptable salts. ; ; ; ; ; .

[0013] The present invention also provides a radionuclide-labeled conjugate, wherein the radionuclide-labeled conjugate is a complex obtained by labeling a radionuclide with a compound having the structure shown in Formula I as a ligand. The radionuclides mentioned are selected from 177 Lu、 43 Sc、 44 Sc、 47 Sc、 55 Co、 57 Co、 68 Ga、 67 Ga、 62 Cu、 64 Cu、 67 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 89 Sr、 152 Gd, 153 Gd, 153 Sm、 149 Tb, 151 Tb, 161 Tb,166 Ho、 166 Dy、 186 Re、 188 Re、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 225 Ac、 226 Th or 227 Any one of Th; Preferably, the radionuclide-labeled conjugate is selected from compounds of formula II or their pharmaceutically acceptable salts. Formula II in, M is selected from 68 Ga、 64 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 177 Lu、 161 Tb, 166 Ho、 186 Re、 188 Re、 225 Ac、 213 Bi、 212 Pb, 223 Ra、 227Th or 153 Sm; preferably, M is selected from... 68 Ga、 177 Lu、 99m Tc, 111 In、 90 Y、 161 Tb, 225 Ac、 212 Pb; and preferably, M is selected from Pb; 177 Lu; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, preferably an integer from 5 to 10, and more preferably 6, 7, 8, 9; Preferably, the compound of formula II is selected from the compound of formula II-1. Formula II-1 More preferably, the compound of formula II is selected from the following compounds: .

[0014] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned conjugate or the aforementioned radiolabeled conjugate, and a pharmaceutically acceptable carrier.

[0015] The present invention also provides the use of the aforementioned radionuclide-labeled conjugate or the aforementioned pharmaceutical composition in radionuclide therapy and / or companion diagnostics of PSMA-positive tumors; Preferably, the aforementioned radionuclide-labeled conjugate or the aforementioned pharmaceutical composition is used as a diagnostic tracer or therapeutic agent; And / or, the aforementioned radionuclide-labeled conjugates or the aforementioned pharmaceutical compositions are used for positron emission tomography, computed tomography, positron emission tomography, single-photon emission tomography, or single-photon emission tomography. And / or, the aforementioned radionuclide-labeled conjugates or the aforementioned pharmaceutical compositions are used for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, and post-metastatic prostate cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The conjugate provided by this invention, while maintaining the original metal coordination ability, achieves conjugation with the target molecule and... 177 Lu enables efficient labeling, thereby enabling the construction of radioactive probes for nuclear medicine diagnosis or treatment.

[0017] 2. Animal experiments have shown that the conjugate of this patent has significant tumor targeting ability, achieves clear imaging in PSMA-positive tumors, and has better tumor-specific imaging contrast within 12 hours.

[0018] 3. The chelating agent of the present invention achieves coupling with the target molecule without affecting the coordination core structure, and has good in vitro stability. Attached Figure Description

[0019] Figure 1 ESI-HRMS spectrum of H4pyox-NHS; Figure 2 H4pyox-NHS 1 H NMR spectrum; Figure 3 ESI-HRMS spectrum of pyox-Nap-PSMA-TMSE; Figure 4 ESI-HRMS spectrum of pyox-Nap-PSMA; Figure 5 [ nat ESI-HRMS spectrum of Lu-pyox-Nap-PSMA; Figure 6 ESI-HRMS spectrum of pyox-PSMA-TMSE; Figure 7 ESI-HRMS spectrum of pyox-PSMA; Figure 8 Comparison of HPLC spectra of compound 1 and pyox-Nap-PSMA in stability experiments; Figure 9 [ 177 Lu]Lu-pyox-Nap-PSMA and [ 177 SPECT images of Lu-PSMA617 in 22RV1 tumor-bearing mice; Figure 10 [ 177 Lu]Lu-pyox-Nap-PSMA and [ 177 A comparison of the target / non-target ratio (T / M Ratio) of Lu-PSMA617 in 22RV1 tumor-bearing mice; Figure 11 [ 177 Lu]Lu-pyox-Nap-PSMA and [ 177 A comparison of tumor uptake (%ID / g) of Lu-PSMA617 in 22RV1 tumor-bearing mice; Figure 12 [ 177 Lu]Lu-pyox-Nap-PSMA and [ 177 Biodistribution of Lu]Lu-PSMA617 in 22RV1 tumor-bearing mice 100 h after administration. Detailed Implementation

[0020] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the operations involved in the embodiments are conventional techniques in the art.

[0021] This invention provides a conjugate selected from compounds of Formula I or pharmaceutically acceptable salts thereof. Formula I Wherein, P is a residue of the targeting ligand, which can specifically bind to any of the following targets: prostate-specific membrane antigen (PSMA), fibroblast activating protein (FAP), gastrin-releasing peptide receptor (GRPR), integrin αvβ3 / αvβ5, carbonic anhydrase IX (CAIX), folic acid receptor (FR), or somatostatin receptor (SSTR). n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0022] In some embodiments, P is selected from PSMA-targeting ligand residues; preferably, P is selected from... ; The n is selected from an integer from 5 to 10, more preferably 6, 7, 8, or 9.

[0023] In some embodiments, the compound represented by Formula I is selected from the following compounds: .

[0024] The present invention also provides a method for preparing the coupling compound as described above, comprising: S2. React the compound of formula I-2 with a fluoride ion source to obtain the compound shown in formula I; Formula I-2 Formula I in, PG1 and PG2 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl, benzyl, p-methoxybenzyl, allyl, 2-(trimethylsilyl)ethyl or trichloroethyl; preferably selected from tert-butyl, benzyl or 2-(trimethylsilyl)ethyl; The fluoride ion source is selected from tetrabutylammonium fluoride, triethylamine trifluoride or cesium fluoride, preferably tetrabutylammonium fluoride; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; In some embodiments, the reaction is carried out in the presence of an organic solvent, preferably a polar aprotic solvent, and even more preferably N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran, and even more preferably N,N-dimethylacetamide.

[0025] In some embodiments, the reaction is carried out at 0–40°C, preferably at room temperature. In some embodiments, the reaction time is 1–24 h, preferably 5–12 h, and more preferably 6–9 h.

[0026] In some embodiments, the method for preparing compound I-2 is as follows: S1. React compound I-1 with PSMA-targeting peptide to obtain compound I-2; Formula I-1 Formula I-2.

[0027] In some embodiments, step S1 is carried out in the presence of a polar aprotic solvent selected from N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO), preferably N,N-dimethylacetamide.

[0028] In some implementations, the PSMA-targeting peptide is .

[0029] In some embodiments, the reaction is carried out at 0–40°C, preferably at room temperature. In some embodiments, the reaction time is 1–24 h, preferably 8–16 h, more preferably 10–14 h. In some embodiments, the reaction is purified by semi-preparative high-performance liquid chromatography, the target fraction is collected, and then freeze-dried to obtain compound I-2.

[0030] In some embodiments, the preparation method of the compound of formula I-1 is as follows: PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0031] The present invention also provides a compound, said compound being a compound of formula I-1, a compound of formula I-2, a compound of T1, a compound of T2, a compound of T3, and their pharmaceutically acceptable salts. PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; In some embodiments, the compound is a compound with the following structure and its pharmaceutically acceptable salts. ; ; ; .

[0032] The present invention also provides a radionuclide-labeled conjugate, wherein the radionuclide-labeled conjugate is a complex obtained by labeling a radionuclide with a compound of Formula I as described above as a ligand; The radionuclides mentioned are selected from 177 Lu、 43 Sc、 44 Sc、 47 Sc、 55 Co、 57 Co、 68 Ga、 67 Ga、 62 Cu、 64 Cu、 67 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 89 Sr、 152 Gd, 153 Gd, 153 Sm、 149 Tb, 151 Tb, 161 Tb, 166Ho、 166 Dy、 186 Re、 188 Re、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 225 Ac、 226 Th or 227 Any one of Th.

[0033] In some embodiments, the radionuclide-labeled conjugate is selected from compounds of formula II or pharmaceutically acceptable salts thereof. Formula II in, M is selected from diagnostic or therapeutic radionuclides; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

[0034] In some embodiments, diagnostic radionuclides include 99m Tc, 111 In、 68 Ga、 64 Cu、 89 Zr、 86 Y; therapeutic radionuclides include 177 Lu、 90 Y、 161 Tb, 166 Ho、 186 Re、 188 Re、 153 Sm、 225 Ac、 213 Bi、 212 Pb, 227 Th.

[0035] In some implementations, M is selected from 68 Ga、 64 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 177 Lu、 161 Tb, 166 Ho、 186 Re、 188 Re、 225 Ac、 213 Bi、 212 Pb, 223 Ra、227Th or 153 Sm; preferably selected from 68 Ga、 177 Lu、 99m Tc, 111 In、 90 Y、 161 Tb, 225 Ac、 212 Pb.

[0036] In some implementations, n is selected from an integer from 5 to 10, more preferably 6, 7, 8, or 9.

[0037] In some embodiments, the compound of formula II is selected from compounds with the structure shown in formula II-1. Formula II-1, The definition of M is as described above.

[0038] In some embodiments, the compound of formula II is selected from the following compounds: .

[0039] The present invention also provides a method for preparing the radionuclide-labeled conjugate as described above, comprising the following steps: S3. React the compound shown in Formula I with M ions to obtain the compound shown in Formula II as described above; Formula I Formula II Where n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; The M ion is selected from the ions of diagnostic or therapeutic radionuclides; preferably, the M ion is... 177 Lu 3 + .

[0040] In some embodiments, the M ion exists in the form of a chloride salt; preferably, the M ion is used in the form of a chloride salt dissolved in water or a buffer solution; more preferably, the M ion is... 177 It is added in the form of LuCl3 solution.

[0041] In some implementations, n is selected from an integer from 5 to 10, more preferably 6, 7, 8, or 9.

[0042] In some embodiments, the reaction is a complexation reaction. In some embodiments, the compound shown in Formula I is reacted with... 177LuCl3 reacts in a buffer solution with a pH of 3-6 to give compound II. In some embodiments, the buffer solution is sodium acetate buffer. In some embodiments, the pH is 3-6, for example 3.5, 4, 4.5, 5, 5.5, preferably 4.5-5.5. In some embodiments, the reaction temperature is room temperature. In some embodiments, the reaction time is 2-30 min, for example 3 min, 5 min, 7 min, 9 min, 10 min, 12 min, 15 min, 16 min, 18 min, 20 min, 25 min, preferably 5-15 min.

[0043] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the conjugate as described above or the radiolabeled conjugate as described above, and a pharmaceutically acceptable carrier.

[0044] The present invention also provides the use of the radionuclide-labeled conjugate or the pharmaceutical composition as described above in the radionuclide therapy and / or companion diagnostics of PSMA-positive tumors; In some embodiments, the radionuclide-labeled conjugates or pharmaceutical compositions as described above are used as diagnostic tracers or therapeutic agents.

[0045] In some embodiments, the radionuclide-labeled conjugates or pharmaceutical compositions described above are used for positron emission tomography (PET), computed tomography (CT), single-photon emission tomography (SPECT), or single-photon emission tomography (SPECT).

[0046] In some embodiments, the radionuclide-labeled conjugates or pharmaceutical compositions as described above are used for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, and post-metastatic prostate cancer.

[0047] Example 1: Synthesis of compound H4pyox-NHS Step 1: Synthesis of Compound 2 Triisopropylchlorosilane (TIPSCl, 500 mg, 2.6 mmol, 1.5 equivalence) was added to a solution of compound 1 (300 mg, 1.7 mmol, 1.0 equivalence) and imidazole (236 mg, 3.5 mmol, 2.0 equivalence) in anhydrous dichloromethane (5 mL). The reaction was carried out at 45 °C for 5 h. TLC showed that compound 1 reacted completely and produced a new spot. Dichloromethane (10 mL) and water (10 mL) were added, and the mixture was separated. The organic phase was washed three times with saturated sodium bicarbonate (10 mL) and saturated brine (10 mL), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether:ethyl acetate = 10:1) to give compound 2 (704 mg, 99.9% yield), a bright yellow oil.

[0048] Step 2: Synthesis of Compound 4 Under ice bath conditions, NaBH(OAc)3 (3.9 g, 18.2 mmol, 1.2 equivalents) was slowly added to a methanol (50 mL) solution of compound 2 (2.9 g, 16.7 mmol, 1.1 equivalents) and compound 3 (5.0 g, 15.2 mmol, 1.0 equivalents). After compound 2 was completely consumed, dilute hydrochloric acid (1 M) was added dropwise until no gas was produced. The reaction solution was extracted with ethyl acetate (20 mL) and water (10 mL), and the organic phase was washed three times with saturated sodium bicarbonate (15 mL) and saturated brine (15 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a yellow oily residue. The residue was purified by rapid column chromatography (petroleum ether:ethyl acetate = 4:1) to give a yellow oily compound 4 (4.96 g, yield 66.8%).

[0049] Step 3: Synthesis of Compound 6: Potassium carbonate (6.50 g, 47.4 mmol, 2.0 eq) was added to a DMF (60 mL) solution of compound 5 (5.00 g, 23.7 mmol, 1.0 eq) and benzyl bromide (4.90 g, 28.4 mmol, 1.2 equivalences), and the reaction was carried out at 55 °C for 12 h. TLC showed that compound 5 was completely consumed and a new spot was formed. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated to dryness under reduced pressure using a rotary evaporator to give a white solid residue. The residue was purified by rapid column chromatography (petroleum ether:ethyl acetate = 1:1) to give a white solid product (5.46 g, yield 86.5%).

[0050] Step 4: Synthesis of Compound 7 Compound 6 (5.46 g, 18.1 mmol, 1.0 equivalent) and anhydrous tetrahydrofuran (60 mL) were added to a 250 mL single-necked round-bottom flask. Under nitrogen protection and an ice bath, the reaction mixture was cooled to 0 °C, and diisobutylaluminum hydride (72 mL, 108.8 mmol, 6.0 equivalent) was added dropwise over 20 min. The reaction mixture was stirred at 0 °C for 1 h, and TLC showed complete consumption of compound 6. The reaction was quenched by slowly adding methanol under an ice bath until no gas was produced. The pH of the system was adjusted to 4 with 1 M hydrochloric acid. The mixture was transferred to a separatory funnel, and extracted with ethyl acetate (200 mL) and water (100 mL). The aqueous phase was then extracted three more times with ethyl acetate (100 mL each time). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give an off-white solid. The crude product was purified by slurrying with a mixture of ethyl acetate / methanol (10:1) to give a white solid compound 7 (1.51 g, yield 33.8%).

[0051] Step 5: Synthesis of Compound 8: Phosphorus tribromide (PBr3, 2.91 g, 10.8 mmol, 2.0 equivalent) was added to a 1,4-dioxane (15 mL) solution of compound 7 (1.32 g, 5.4 mmol, 1.0 equivalent). The reaction mixture was stirred at 50 °C for 1 h, and TLC showed complete consumption of the starting material and formation of a new spot. After cooling the reaction mixture to room temperature, saturated sodium bicarbonate aqueous solution (5 mL) and ethyl acetate (10 mL) were slowly added dropwise, followed by extraction of the aqueous phase three times with ethyl acetate (5 mL each time). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether:ethyl acetate = 1:1) to give a white solid product (1.01 g, yield 50.1%).

[0052] Step 6: Synthesis of Compound 9 DIPEA (209 mg, 1.6 mmol, 3.0 equivalent) was added to a solution of compound 4 (580 mg, 1.2 mmol, 2.2 equivalent) and compound 8 (200 mg, 0.5 mmol, 1.0 equivalent) in acetonitrile (3 mL), and the reaction was carried out at room temperature for 10 h. TLC showed that compound 9 was completely consumed. The reaction mixture was extracted with dichloromethane (10 mL) and water (10 mL). The organic phase was washed three times with saturated ammonium chloride (10 mL) and saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by FLASH (petroleum ether:ethyl acetate = 8:1) to give a yellow oily compound 9 (623 mg, yield 97.3%).

[0053] Step 7: Synthesis of Compound 10: Compound 9 (300 mg, 0.51 mmol), 10% Pd / C (30 mg), and methanol (10 mL) were added to a 250 mL two-necked round-bottom flask. The reaction mixture was subjected to vacuum / hydrogen purging three times, and then reacted at 50 °C for 12 h under a hydrogen atmosphere. TLC showed complete consumption of compound 9. The reaction solution was cooled to room temperature, filtered through diatomaceous earth, and the filtrate was concentrated under reduced pressure to give 139 mg of a white solid, with a yield of 50.1%.

[0054] Step 8: Synthesis of Compound 11 Potassium carbonate (94 mg, 0.7 mmol, 1.5 eq) was added to a DMF (5 mL) solution of compound 10 (500 mg, 0.5 mmol, 1.0 eq) and benzyl 8-bromooctanoate (212 mg, 0.7 mmol, 1.5 eq), and the reaction was carried out at 50 °C for 12 h. LC-MS monitoring showed that compound 10 was completely consumed and the target product was formed. The reaction solution was cooled to room temperature and filtered. Dichloromethane (15 mL) and water (10 mL) were added to the filtrate, and the mixture was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by rapid column chromatography (petroleum ether:ethyl acetate = 10:1) to give a yellow oily compound 11 (171 mg, yield 33.7%).

[0055] Step 9: Synthesis of compound H4pyox-NHS.

[0056] Compound 11 (100 mg, 0.1 mmol), 10% Pd / C (10 mg), and THF (5 mL) were added to a 25 mL two-necked round-bottom flask. The reaction mixture was evacuated / purged with hydrogen three times, and then reacted at room temperature for 12 h under a hydrogen atmosphere. LC-MS monitoring showed that compound 11 was completely consumed, forming the debenzylidene ester product. After cooling to room temperature, the reaction solution was filtered through a diatomaceous earth filter; the filtrate was used directly for the next reaction without further purification.

[0057] EDCI (23 mg, 0.1 mmol, 1.2 equivalents) and NHS (23 mg, 0.2 mmol, 2.0 equivalents) were added to the above filtrate, and the reaction was carried out at room temperature for 6 h. LC-MS monitoring showed that the debenzylidene intermediate was completely consumed, and the target product was generated. Dichloromethane (15 mL) and water (10 mL) were added to the reaction solution for extraction. The organic phase was washed three times with water (10 mL each time), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was purified by semi-preparative high-performance liquid chromatography to obtain a yellow solid H4pyox-NHS (34 mg, total yield of 33.8% in both steps). The ESI-HRMS spectrum of H4pyox-NHS is shown below. Figure 1 As shown; H4pyox-NHS 1HNMR spectrum as follows Figure 2 As shown.

[0058] 1 H NMR (400 MHz, CDCl3) δ 8.70 (s, 2H), 8.05 (s, 2H), 7.60 (dd, J = 22.0, 7.5Hz, 4H), 7.50 – 7.34 (m, 4H), 4.99 (s, 4H), 4.70 (s, 4H), 4.37 – 4.07 (m, 6H), 3.82 (s, 4H), 2.86 (s, 4H), 2.61 (t, J = 7.2 Hz, 2H), 1.82 – 1.68 (m, 4H), 1.52 – 1.36 (m, 6H), 0.93 (t, J = 8.6 Hz, 4H), -0.00 (s, 18H). ESI-HRMS (m / z): Calculated value [C 54 H 72 N5O 11 Si2] + 1023.4795; measured value 1023.4738.

[0059] Example 2: Synthesis of pyox-Nap-PSMA Step 1: Synthesis of pyox-Nap-PSMA-TMSE H4pyox-NHS (2 mg), PSMA-targeting peptide (2 mg), and DMA (1 mL) were added to a 1.5 mL sample vial. After complete dissolution, the mixture was allowed to stand at room temperature for 12 hours. HPLC analysis confirmed complete consumption of H4pyox-NHS. The reaction solution was purified by semi-preparative liquid chromatography (A: water containing 0.1% formic acid, B: acetonitrile containing 0.1% formic acid; isocratic elution with 35% A phase for 15 minutes at a flow rate of 2 mL / min). The eluent was collected and freeze-dried to obtain pyox-Nap-PSMA-TMSE as a yellow solid (2.9 mg).

[0060] The ESI-HRMS spectrum of pyox-Nap-PSMA-TMSE was analyzed as follows: Figure 3 As shown.

[0061] ESI-HRMS (m / z): Calculated value [C 82 H 112 N 10 O 17 Si2] 2+782.3867; measured value 782.3735.

[0062] Step 2: Synthesis of pyox-Nap-PSMA Add pyox-Nap-PSMA-TMSE (2.9 mg), DMA (1 mL), and TBAF (100 μL) to a 1.5 mL sample vial. After standing at room temperature for 8 hours, HPLC analysis was performed to detect complete consumption of pyox-Nap-PSMA-TMSE. The reaction solution was purified by semi-preparative liquid chromatography (A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid; from 70% A to 5% A over 20 minutes, flow rate 2 mL / min). The eluent was collected and freeze-dried to obtain a yellow solid (1.5 mg).

[0063] The ESI-HRMS spectrum of pyox-Nap-PSMA was analyzed as follows: Figure 4 As shown.

[0064] ESI-HRMS (m / z): Calculated value [C 72 H 87 N 10 O 17 ] + 1363.6245; Measured value 1363.6217.

[0065] Example 3 [ 177 Synthesis of Lu-pyox-Nap-PSMA [ 177 Lu]Lu-pyox-Nap-PSMA The synthesis steps are as follows: Using pyox-Nap-PSMA compound as a precursor, it was formulated into a 10 -4 Prepare an aqueous solution of the precursor of M for later use; take 10 μL of the above precursor solution and add it to 90 μL of acetate-sodium acetate buffer (pH 4.5-5), then add 40 MBq. 177 LuCl3 solution (3 μL), mixed well, reacted at room temperature for 10 minutes to obtain the labeled compound [ 177 Lu]Lu-pyox-Nap-PSMA, with a radiochemical purity greater than 99%.

[0066] Example 4 [ nat Synthesis of Lu-pyox-Nap-PSMA Take 10 μL of pyox-Nap-PSMA precursor solution (10 -4M), added to 90 μL of acetate-sodium acetate buffer (pH 4.5-5), then added nat LuCl3 solution (3 μL, 10) -3 M), after mixing, react at room temperature for 10 minutes to obtain [ nat Lu]Lu-pyox-Nap-PSMA.

[0067] After testing, [ nat The ESI-HRMS spectrum of Lu-pyox-Nap-PSMA is as follows: Figure 5 As shown.

[0068] ESI-HRMS: m / z calculated value: C 72 H 85 LuN 10 O 17 [M+2H] 2+ 768.2745, Measured value: 768.2634.

[0069] Preparation of Comparative Compound 1 (Comparative Example 1) Step 1: Synthesis of pyox-PSMA-TMSE: H4pyox-NHS (2 mg), Glu-urea-Lys (0.5 mg), and DMA (1 mL) were added to a 1.5 mL sample vial and dissolved completely. The mixture was then allowed to stand at room temperature for 12 hours. Complete consumption of H4pyox-NHS was detected by HPLC (A: water + 0.04% TFA, B: acetonitrile + 0.02% TFA; elution from 70% A to 5% A over 25 minutes, flow rate 1 mL / min). The reaction solution was purified by semi-preparative HPLC (A: water containing 0.1% formic acid, B: acetonitrile containing 0.1% formic acid; isocratic elution with 35% A phase for 15 minutes, flow rate 2 mL / min). The eluent was collected and freeze-dried to obtain pyox-PSMA-TMSE as a yellow solid (1.7 mg).

[0070] The ESI-HRMS spectrum of pyox-PSMA-TMSE was analyzed as follows: Figure 6 As shown.

[0071] ESI-HRMS (m / z): Calculated value [C 61 H 86 N8O 15 Si2] + 1227.5824; measured value 1227.5647.

[0072] Step 2: Compare the synthesis of compound 1 pyox-PSMA-TMSE (1.7 mg), DMA (1 mL), and TBAF (100 μL) were added to a 1.5 mL sample vial. After standing at room temperature for 8 hours, HPLC (method as above) was used to detect complete consumption of pyox-PSMA-TMSE. The reaction solution was purified by semi-preparative liquid chromatography (A: water + 0.1% formic acid, B: acetonitrile + 0.1% formic acid; from 70% A to 5% A within 20 minutes, flow rate 2 mL / min). The eluent was collected and freeze-dried to obtain a yellow solid (1.5 mg). LC-MS analysis showed that the product contained a small amount of TBAF residue. The yellow solid was dissolved in 2 mL of pure water and purified by CM column (Waters Sep-Pak Plus Light CM) to remove the residual TBAF, yielding the final product, comparative compound 1 (1.4 mg, yellow solid).

[0073] The ESI-HRMS spectrum of pyox-PSMA was analyzed as follows: Figure 7 As shown.

[0074] ESI-HRMS (m / z): Calculated value [C 51 H 62 N8O 15 Si2] + 1027.4407; Measured value 1027.4455.

[0075] Experiment Example 1: Stability Test After incubating comparative compound 1 and pyox-Nap-PSMA at room temperature for one week, samples were taken for HPLC analysis. Changes in the HPLC chromatograms of comparative compound 1 and pyox-Nap-PSMA before and after incubation were measured, such as... Figure 8 As shown.

[0076] The HPLC detection conditions are as follows: Comparative compound 1: Mobile phase A was water + 0.1% TFA, and mobile phase B was acetonitrile + 0.1% TFA; the flow rate was reduced from 95% A to 45% A within 15 minutes at a flow rate of 1 mL / min.

[0077] Pyox-Nap-PSMA: Mobile phase A is water + 0.1% TFA, and mobile phase B is acetonitrile + 0.1% TFA; the flow rate is reduced from 75% A to 45% A within 15 minutes at a rate of 1 mL / min.

[0078] Depend on Figure 8It can be seen that, compared with compound 1, a significant degradation peak appeared after being placed at room temperature for one week, indicating that it has poor stability at room temperature and is prone to decomposition; while the HPLC chromatogram of pyox-Nap-PSMA remained basically consistent before and after placement, without significant degradation peaks, showing excellent room temperature stability.

[0079] Experimental Example 2 [ 177 SPECT / CT Imaging Experiment with Lu-pyox-Nap-PSMA Experimental steps: (1) Establishment of 22RV1 tumor animal model: 22RV1 cells expressing PSMA were cultured in RPMI 1640 medium containing 10% FBS. The culture conditions were 37 ℃, 5% CO2, and saturated humidity. After the cells grew to 70-80%, they were digested with trypsin, centrifuged, and then inoculated.

[0080] (2) After anesthetizing BALB / c-nu nude mice, subcutaneous inoculation was performed under their armpits, with each BALB / c-nu nude mouse receiving 5 cells. 10 6 0.1 mL / cell. Wait until the tumor grows to 300-400 mm. 3 It can be used for imaging experiments.

[0081] (3) In this experimental example, [ 177 Lu-pyox-Nap-PSMA was prepared according to the method in Example 3. 21 MBq of [ 177 Lu]Lu-pyox-Nap-PSMA or [ 177 Lu-PSMA617 was injected via tail vein into 22RV1 tumor-bearing mice (n = 3) that highly expressed PSMA. SPECT / CT imaging was performed at 1 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h, and 96 h post-injection. The results are shown in the figure. Figure 9 .

[0082] (4) Biodistribution experiment: Four hours after the completion of the SPECT / CT imaging, mice were euthanized after deep anesthesia with isoflurane, and organs (including heart, lungs, liver, spleen, kidneys, intestines, muscles, blood, etc.) and tumor tissue were rapidly dissected and collected. After weighing the collected samples, the radioactivity count was determined using a gamma counter. The in vivo distribution results are expressed as the percentage of injected dose per gram of tissue (%ID / g). Each set of data is reported as mean ± standard error (mean ± SEM, n=3).

[0083] Experimental results: [ 177 Lu]Lu-pyox-Nap-PSMA and [177 SPECT / CT imaging results of Lu-PSMA617 are as follows: Figure 9 As shown; its target / non-target ratio (T / M ratio), tumor uptake rate (%ID / g), and biodistribution data are respectively shown in [reference needed]. Figure 10 , Figure 11 and Figure 12 .

[0084] Depend on Figure 9 It can be seen that, [ 177 Lu-pyox-Nap-PSMA can be specifically taken up at the 22RV1 tumor site, and its retention time at the tumor site is similar to that of the positive control drug. 177 Lu-PSMA617 is readily available and cleared via both hepatic and renal pathways. Figure 10 It can be seen that, [ 177 The target / non-target ratio of Lu-pyox-Nap-PSMA is higher than that of [ 177 The Lu-PSMA617, especially at the 12-hour time point, demonstrates superior tumor-specific imaging contrast. Figure 11 It can be seen that, [ 177 Tumor uptake of Lu-pyox-Nap-PSMA (%ID / g) and [ 177 The levels of Lu]Lu-PSMA617 are comparable, and remain high at the 96-hour time point, which is beneficial for therapeutic dose deposition.

[0085] Depend on Figure 12 It can be seen that, [ 177 The hepatic uptake value of Lu-pyox-Nap-PSMA was 0.33 ± 0.10 %ID / g, which was significantly higher than that of [ 177 The concentration of Lu-PSMA617 at 0.11 ± 0.01 %ID / g was consistent with the liver radioactivity observed in SPECT images, confirming its significant presence in the liver metabolic pathway. 177 The renal uptake value of Lu-pyox-Nap-PSMA was 0.13 ± 0.02 %ID / g, which is lower than [ 177 The uptake of Lu-PSMA617 (0.19 ± 0.03 %ID / g) is beneficial in reducing radiotoxicity. The uptake values ​​of the two probes in tumor tissue were 0.43 ± 0.12 %ID / g and 0.56 ± 0.11 %ID / g, respectively. Furthermore, the uptake of both probes in the heart, muscle, and blood was low (all <0.05 %ID / g), indicating low non-specific uptake, rapid blood clearance, and good biocompatibility.

[0086] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A coupling agent, characterized in that, The conjugate is selected from compounds of Formula I or pharmaceutically acceptable salts thereof. Formula I Wherein, P is selected from residues of a targeting ligand that can specifically bind to any of the following targets: prostate-specific membrane antigen (PSMA), fibroblast activator protein (FAP), gastrin-releasing peptide receptor (GRPR), integrin αvβ3 / αvβ5, carbonic anhydrase IX (CAIX), folate receptor (FR), or somatostatin receptor (SSTR). n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

2. The coupling according to claim 1, characterized in that, P is selected from PSMA-targeting ligand residues; preferably, P is selected from... ; The n is selected from an integer from 5 to 10, preferably 6, 7, 8, or 9.

3. The coupling according to claim 1, characterized in that, The compound represented by Formula I is selected from the following compounds. 。 4. A method for preparing the coupling compound according to any one of claims 1-3, characterized in that, include S2. React the compound of formula I-2 with a fluoride ion source to obtain the compound shown in formula I; Formula I-2 Formula I in, PG1 and PG2 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl, benzyl, p-methoxybenzyl, allyl, 2-(trimethylsilyl)ethyl or trichloroethyl; preferably selected from tert-butyl, benzyl or 2-(trimethylsilyl)ethyl; The fluoride ion source is selected from tetrabutylammonium fluoride, triethylamine trifluoride or cesium fluoride, preferably tetrabutylammonium fluoride; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; Preferably, the reaction is carried out in the presence of an organic solvent; more preferably, the organic solvent is a polar aprotic solvent, and even more preferably N,N-dimethylacetamide, N,N-dimethylformamide, dimethyl sulfoxide, or tetrahydrofuran.

5. The preparation method according to claim 4, characterized in that, The preparation method of compound I-2 is as follows: S1. React compound I-1 with PSMA-targeting peptide to obtain compound I-2; Formula I-1 Formula I-2; Preferably, step S1 is carried out in the presence of a polar aprotic solvent selected from N,N-dimethylacetamide (DMA), N,N-dimethylformamide (DMF), or dimethyl sulfoxide (DMSO), preferably N,N-dimethylacetamide; and / or PSMA-targeting peptides .

6. The preparation method according to claim 5, characterized in that, The preparation method of the compound of formula I-1 is as follows: PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

7. A compound, characterized in that, The compounds are compounds of formula I-1, I-2, T1, T2, T3, and their pharmaceutically acceptable salts. PG1, PG2, and PG3 may be the same or different, and are independently selected from methyl, ethyl, tert-butyl (t-Bu), benzyl (Bn), p-methoxybenzyl (PMB), allyl (Alloc), 2-(trimethylsilyl)ethyl (TMSE), or trichloroethyl; preferably tert-butyl, benzyl, or 2-(trimethylsilyl)ethyl; PG4 and PG5 may be the same or different, and are independently selected from trimethylsilyl (TMS), tert-butyldimethylsilyl (TBS), tert-butyldiphenylsilyl (TBDPS), triisopropylsilyl (TIPS), benzyl (Bn), p-methoxybenzyl (PMB), tetrahydropyranyl (THP), methoxymethyl (MOM), acetyl (Ac) or benzoyl (Bz), preferably TBS, TIPS, Bn or THP; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20; Preferably, the compound is a compound with the following structure and its pharmaceutically acceptable salts. ; ； ； ； ； 。 8. A radionuclide-labeled conjugate, characterized in that, The radionuclide-labeled conjugate is a complex obtained by labeling a radionuclide with a compound of the structure shown in Formula I as described in any one of claims 1-7. The radionuclides mentioned are selected from 177 Lu、 43 Sc、 44 Sc、 47 Sc、 55 Co、 57 Co、 68 Ga、 67 Ga、 62 Cu、 64 Cu、 67 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 89 Sr、 152 Gd, 153 Gd, 153 Sm、 149 Tb, 151 Tb, 161 Tb, 166 Ho、 166 Dy、 186 Re、 188 Re、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 225 Ac、 226 Th or 227 Any one of Th; Preferably, the radionuclide-labeled conjugate is selected from compounds of formula II or their pharmaceutically acceptable salts. Formula II in, M is selected from 68 Ga、 64 Cu、 89 Zr、 86 Y、 90 Y、 99m Tc, 111 In、 177 Lu、 161 Tb, 166 Ho、 186 Re、 188 Re、 225 Ac、 213 Bi、 212 Pb, 223 Ra、 227Th or 153 Sm; preferably, M is selected from... 68 Ga、 177 Lu、 99m Tc, 111 In、 90 Y、 161 Tb, 225 Ac、 212 Pb; and preferably, M is selected from Pb; 177 Lu; n is selected from 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, preferably an integer from 5 to 10, and more preferably 6, 7, 8, 9; Preferably, the compound of formula II is selected from the compound of formula II-1. Formula II-1 More preferably, the compound of formula II is selected from the following compounds: 。 9. A pharmaceutical composition comprising a therapeutically effective amount of the conjugate of any one of claims 1 to 7 or the radiolabeled conjugate of claim 8, and a pharmaceutically acceptable carrier.

10. The use of a radionuclide-labeled conjugate of claim 8 or a pharmaceutical composition of claim 9 in radionuclide therapy and / or companion diagnostics for PSMA-positive tumors; Preferably, the radionuclide-labeled conjugate of claim 8 or the pharmaceutical composition of claim 9 is used as a diagnostic tracer or therapeutic agent; And / or, the radionuclide-labeled conjugate of claim 8 or the pharmaceutical composition of claim 9 may be used for positron emission tomography, computed tomography, positron emission tomography, single-photon emission tomography or single-photon emission tomography. And / or, the radionuclide-labeled conjugate of claim 8 or the pharmaceutical composition of claim 9 may be used for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, and post-metastatic prostate cancer.

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