Psma-targeting small molecules, nuclide-labeled probes, pharmaceutical compositions, and lyophilized kits and uses thereof

Abstract

The present application relates to the technical field of biological medicine, and more particularly to a PSMA-targeting small molecule, a nuclide-labeled probe, a pharmaceutical composition, a freeze-dried kit and applications thereof. The PSMA-targeting small molecule provided by the present application has the advantages of high target lesion uptake, long retention time, rapid clearance of non-target organs, low background, high target / non-target ratio, and is suitable for nuclide imaging and treatment of PSMA high-expression tumors, and can construct a diagnosis and treatment integrated system, and has good clinical popularization value and safety advantages.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to PSMA-targeting small molecules, radionuclide-labeled probes, pharmaceutical compositions and lyophilized kits and their applications. Background Technology

[0002] Prostate-specific membrane antigen (PSMA) is highly overexpressed on the surface of over 90% of prostate cancer cells (100-1000 times higher than in normal tissues), and its expression level is closely related to tumor invasiveness, metastasis, and castration resistance, making it one of the most valuable biomarkers in cancer diagnosis and treatment. In normal tissues, PSMA is expressed in small amounts only in salivary glands, lacrimal glands, and proximal renal tubules, making it an ideal diagnostic and therapeutic target with high sensitivity and specificity. In recent years, PSMA-targeting radionuclide-labeled probes, especially drugs represented by small molecule inhibitors (such as the classic glutamate-urea-lysine series), combined with positron emission tomography (PET) or single-photon emission computed tomography (SPECT) technologies, have shown revolutionary potential in the early and accurate diagnosis, staging, efficacy evaluation, and targeted radioligand therapy of cancer. Small molecule radioactive probes based on PSMA (such as Ga-PSMA-11, Lu-PSMA-617, etc.) have shown great potential in radionuclide imaging and therapy. However, existing probes still have the following technical bottlenecks: (1) High retention in non-target organs: High non-specific uptake and slow clearance in tissues such as kidneys, salivary glands, and liver, resulting in high imaging background noise and low detection rate of small lesions; (2) Insufficient tumor uptake and retention: The imaging of lesions with low PSMA expression is unclear, and the cumulative dose of therapeutic radionuclides at the tumor site is insufficient, affecting the efficacy; (3) Lack of optimization of the linker arm design: Existing small molecule PSMA inhibitors mostly adopt the classic modular design of "pharmacophore (glutamate-urea-lysine) + linker arm + chelating agent", but the physicochemical properties of the linker arm (such as hydrophilicity-hydrophobicity balance, charge distribution, and metabolizability) and the systematic structure-activity relationship of functionalized fragment modification are not yet fully studied, which makes it difficult to achieve optimal regulation of the biodistribution of the probe in vivo, and it is difficult to achieve a balance between the retention time of non-target tissues and the retention time of target lesions. In summary, existing PSMA-based small molecule radioactive probes are difficult to precisely regulate biological properties (including target lesion uptake, retention time, non-target organ clearance, and high target / non-target ratio). Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide PSMA-targeting small molecules, radionuclide-labeled probes, pharmaceutical compositions, and lyophilized kits, as well as their applications. The PSMA-targeting small molecules simultaneously possess advantages such as high uptake at target lesions, long retention time, rapid clearance from non-target organs, low background, and a high target / non-target ratio. They are suitable for radionuclide imaging and treatment of PSMA-overexpressing tumors and can be used to construct an integrated diagnostic and therapeutic system, demonstrating good clinical application value and safety advantages.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a PSMA-targeting small molecule having the structure shown in Formula I: Formula I; In Equation I, A is , , , or ; The and X1, X2, and X3 are all -CH=, or one of X1, X2, and X3 is independently -N=, and the remaining two are -CH=; The and The Y in the equation can be -O-, -NH-, or -N(CH3)-. The In this context, m is an integer from 1 to 5; The n is an integer from 1 to 5; The Z in , , , , , , , or ; In Z, x, y, e, j, f, v, and k are independent integers from 0 to 5, and G is independent -H or -CH3; In Equation I, L may or may not exist. When L exists, L is: , , , , , , , , , , , or ; In L, t, q, and p are independent integers from 1 to 3, and M is -CH= or -N=; In Formula I, T is the group to be labeled, and the structural formula of T is: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0005] This invention also provides a method for preparing the PSMA-targeting small molecule described in the above technical solution, comprising the following steps: The active substance T and a compound having the structure shown in Formula 1 were subjected to a substitution reaction to obtain the PSMA-targeting small molecule. The T-active substance contains an activating group; Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule.

[0006] Preferably, the activating group is an N-hydroxysuccinimide activating group, an acid anhydride, a pentafluorophenyl ester group, or an isothiocyanate group.

[0007] The present invention also provides a radionuclide-labeled probe having the structure shown in Formula II or Formula III: Formula II; Formula III; The types of A and L in Formulas II and III are the same as those in Formula I; In Formula II, R is a nuclide labeling group, which is a complex obtained by coordinating T with a nuclide in Formula I. In equation (III), Q is... ; The In this context, a is an integer from 0 to 5; Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

[0008] Preferably, the nuclide includes 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb、 128 Ba、 139 La、 140 La、 142 Pr, 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd,161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 One or more of Ac.

[0009] This invention also provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: The PSMA-targeting small molecule described in the above technical solution is coordinated with a nuclide to obtain a nuclide-labeled probe with the structure shown in Formula II. Alternatively, an amidation reaction can be carried out between a compound having the structure shown in Formula 1 and an active compound of Q to obtain a compound having the structure shown in Formula III. Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule; The active compound of Q is a compound having the structure shown in Formula 2 or Formula 3: Equation 2, Formula 3; In equations 2 and 3, 'a' is an independent integer from 0 to 5, and Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

[0010] The present invention also provides pharmaceutically acceptable forms of the PSMA-targeting small molecules or the radionuclide-labeled probes described in the above technical solutions, wherein the pharmaceutically acceptable forms include one or more of salts, stereoisomers, racemates, hydrates and solvates.

[0011] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; The active component includes one or more of the PSMA-targeting small molecule described in the above technical solution, the radionuclide-labeled probe described in the above technical solution, and pharmaceutically acceptable forms of the PSMA-targeting small molecule or the radionuclide-labeled probe described in the above technical solution.

[0012] The present invention also provides a lyophilized kit comprising a precursor and pharmaceutically acceptable excipients; The precursor includes one or more of the PSMA-targeting small molecules described in the above technical solutions or pharmaceutically acceptable forms of the PSMA-targeting small molecules described in the above technical solutions.

[0013] The present invention also provides the use of the PSMA-targeting small molecule described in the above-mentioned technical solutions, the radionuclide-labeled probe described in the above-mentioned technical solutions, the pharmaceutically acceptable form of the PSMA-targeting small molecule or the radionuclide-labeled probe described in the above-mentioned technical solutions, the pharmaceutical composition described in the above-mentioned technical solutions, or the lyophilized kit described in the above-mentioned technical solutions in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for PSMA protein-mediated diseases.

[0014] This invention provides a PSMA-targeting small molecule with the structure shown in Formula I. The invention introduces a novel, self-optimized linker L between the targeting group and the coordination structure, which can adjust the distance and molecular structure between the targeting group and the coordination structure, reducing the influence of the coordination structure on the compound's activity. Rather than a simple replacement of traditional linker arms, this linker is systematically optimized based on physicochemical parameters such as hydrophilicity-hydrophobicity balance, charge distribution, and in vivo metabolizability, enabling efficient bridging of glutamic acid-urea-lysine PSMA-targeting pharmacophores with radionuclide chelators and various functional groups. By precisely controlling the linker chain length, rigid-flexible configuration, lipid-water partitioning characteristics, and charge arrangement, precise regulation of the in vivo biological behavior of the targeting probe can be achieved. Simultaneously, this structure, by linking with different types of radionuclide chelating groups, alters the physicochemical and pharmacokinetic properties of the radionuclide-labeled probe, accelerating the clearance rate of the PSMA-targeting small molecule compound or radionuclide-labeled probe in non-target tissues, increasing the uptake of the target molecule and / or probe in PSMA-overexpressing lesions, and increasing the target / non-target ratio.

[0015] Compared with existing technologies, the PSMA-targeting small molecule described in this invention has several outstanding technical advantages: 1) Strong tumor-targeting uptake ability and long-term retention in lesions can effectively improve molecular binding affinity and cell internalization efficiency, meet the imaging needs of tumor lesions with low expression levels, and at the same time facilitate the full accumulation of therapeutic radionuclides at the lesion site. 2) The metabolic clearance rate of non-target tissues is fast, which can significantly reduce the non-specific radioactive accumulation in normal tissues such as kidneys and salivary glands, reduce radiation toxicity side effects, and greatly improve imaging clarity; 3) It can obtain an excellent target / non-target tissue radioactivity ratio, achieving the ideal in vivo distribution characteristics of high tumor enrichment and low background interference, and its comprehensive performance far exceeds that of existing similar probes. 4) It has a wide range of clinical applications and can be adapted to various conditions. 177 Lu、 225 Ac and other therapeutic radionuclide markers can be used for precise imaging diagnosis of PSMA-positive tumors, and can also be used to build an integrated diagnostic and therapeutic application platform. They are highly practical in clinical use and have high safety in use.

[0016] The present invention also provides a method for preparing the PSMA-targeted small molecule described in the above technical solution. The preparation method is simple (simple synthesis and operation), low in production cost, and suitable for industrial production.

[0017] The present invention also provides a radionuclide-labeled probe, which has the characteristics of convenient labeling, strong labeling ability, short labeling time, high labeling yield and good stability, which is conducive to the application and clinical promotion of radionuclide-labeled probes. Attached Figure Description

[0018] Figure 1 HPLC chromatograms of compounds DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1, and Nota-YD2; Figure 2 Here are the HPLC chromatograms of compounds DEPF and NEPF; Figure 3 Here are the HPLC chromatograms of compounds PB1 and PB2; Figure 4 for[ 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2 and [ 68 Radio-HPLC chart of Ga]Ga-DOTA-YD3; Figure 5 for[ 68 Radio-HPLC chromatogram of Ga]Ga-DEPF; Figure 6 for[ 68 Ga]Ga-PB1 and [ 68 Radio-HPLC chromatogram of Ga]Ga-PB2; Figure 7 for[ 18 F]AlF-NOTA-YD1 and [ 18 Radio-HPLC chromatogram of F]AlF-NOTA-YD2; Figure 8 for[ 18 Radio-HPLC chromatogram of F]AlF-NEPF; Figure 9 for[ 177 Radio-HPLC chromatogram of Lu-Lu-DEPF; Figure 10 for 131 Radio-HPLC chromatogram of IPB-IEPF; Figure 11 Based on the PSMA crystal structure (PDB ID: 5O5T), the binding conformations of DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1 and Nota-YD2 with PSMA were determined and compared with PSMA-617. Figure 12 The docking scores and key hydrogen bonds of DOTA-YD1, DOTA-YD2, DOTA-YD3, NOTA-YD1, and NOTA-YD2 with PSMA were compared with those of PSMA-617. Figure 13 for[ 177 Lu]Lu-DEPF (left) and 131 Cell uptake and inhibition results of IPB-IEPF (right) in PSMA-positive PC3-PIP cells at different incubation times; Figure 14 for[ 18 [F]Stability of AlF-NEPF in mouse urine and blood (radio-HPLC analysis chromatogram); Figure 15 for[ 18 Biodistribution of F]AlF-NEPF in tumor-bearing mice at different time points; Figure 16 for[ 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2 and [ 68 PET imaging results of Ga]Ga-DOTA-YD3 at different time points in tumor-bearing mice; Figure 17 for[ 68 Ga]Ga-PB1 and [ 68 PET imaging of Ga-PB2 in tumor-bearing mice; Figure 18 for[ 68 PET images of Ga]Ga-DEPF at different time points in tumor-bearing mice expressing PSMA-positive (PC3-PIP) and PSMA-negative (PC3); Figure 19 for[ 18 F]AlF-NOTA-YD1 and [18 PET imaging results of F]AlF-NOTA-YD2 at different time points in tumor-bearing mice; Figure 20 for[ 18 F]AlF-NEPF PET imaging results at different time points in normal mice; Figure 21 for[ 18 PET images of F]AlF-NEPF at different time points in PSMA-positive (PC3-PIP) and PSMA-negative (PC3) tumor-bearing mice; Figure 22 for[ 18 F]AlF-NEPF PET imaging at different time points in PSMA-positive hepatocellular carcinoma-bearing mice; Figure 23 for[ 18 PET images of F]AlF-NOTA-YD2 at different time points in prostate cancer patients (with systemic bone metastases), and compared with [ 18 Comparison of PET images of F]PSMA-1007 2 hours after injection; Figure 24 for[ 18 PET images of F]AlF-NOTA-YD2 in prostate cancer patients, and compared with [ 18 Compare the PET images of F]PSMA-1007; Figure 25 For PET imaging, [ 18 F]AlF-NOTA-YD2 and [ 18 Comparison of uptake (SUV) of PSMA-1007 in human tissues and organs; Figure 26 for[ 18 F]AlF-NEPF PET imaging in prostate cancer patients (with systemic bone metastases), and compared with [ 18 Compare the PET images of F]PSMA-1007; Figure 27 for[ 18 F]AlF-NEPF PET imaging in a liver cancer patient. Detailed Implementation

[0019] This invention provides a PSMA-targeting small molecule having the structure shown in Formula I: Formula I; In Equation I, A is , , , or ; The and X1, X2, and X3 are all -CH=, or one of X1, X2, and X3 is independently -N=, and the remaining two are -CH=; The and The Y in the equation can be -O-, -NH-, or -N(CH3)-. The In this context, m is an integer from 1 to 5; The n is an integer from 1 to 5; The Z in , , , , , , , or ; In Z, x, y, e, j, f, v, and k are independent integers from 0 to 5, and G is independent -H or -CH3; In Equation I, L may or may not exist. When L exists, L is: , , , , , , , , , , , or ; In L, t, q, and p are independent integers from 1 to 3, and M is -CH= or -N=; In Formula I, T is the group to be labeled, and the structural formula of T is: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0020] In this invention, the -CO- end (carbonyl end) of A is connected to the -NH- of the lysine residue in Formula I to form an amide bond; when L is absent, the -NH- end of A is directly connected to T; when L is present, the -NH- end of A is connected to the -CO- of L, and the -NH- end of L is then connected to T.

[0021] In this invention, the PSMA-targeting small molecule preferably has the structure shown in Formula I-1 to Formula I-5: Formula I-1; Formula I-2; Formula I-3; Formula I-4; Formula I-5.

[0022] In this invention, the preferred types and designations of the Z, L, and T components of the PSMA-targeting small molecule having the structure shown in Formula I-1 are as shown in Table 1: Table 1. Types and names of Z, L, and T of PSMA-targeting small molecules with the structure shown in Formula I-1.

[0023] In this invention, the preferred types and names of the L and T of the PSMA-targeting small molecules having the structures shown in Formulas I-2 to I-3 are as shown in Table 2: Table 2. Types and names of L and T values ​​for PSMA-targeting small molecules with structures shown in Formulas I-2 to I-3.

[0024] In this invention, the preferred types and names of the L and T of the PSMA-targeting small molecules having the structures shown in Formulas I-4 to I-5 are as shown in Table 3: Table 3. Types and names of L and T values ​​for PSMA-targeting small molecules with structures shown in Formulas I-4 to I-5.

[0025] This invention also provides a method for preparing the PSMA-targeting small molecule described in the above technical solution, comprising the following steps: The active substance T and a compound having the structure shown in Formula 1 were subjected to a substitution reaction to obtain the PSMA-targeting small molecule. The T-active substance contains an activating group; Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule.

[0026] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0027] In this invention, the molar ratio of the compound having the structure shown in Formula 1 and the active substance T is preferably 1:(1~10), more preferably 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0028] In this invention, the T active substance has an activating group, which is preferably an N-hydroxysuccinimide (NHS) activating group, an acid anhydride, a pentafluorophenyl ester group, or an isothiocyanate group.

[0029] In this invention, the T-active substance is preferably: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

[0030] In this invention, the substitution reaction is preferably carried out in a dispersion medium, which preferably includes one or more of an organic solvent, water, and PBS. The organic solvent preferably includes one or more of N-methylpyrrolidone (NMP), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). The pH of the PBS is preferably 6-12, more preferably 6, 7, 8, 9, 10, 11, or 12. This invention does not impose any special limitations on the amount of dispersion medium used; any amount well-known to those skilled in the art that ensures the smooth progress of the substitution reaction can be used.

[0031] In this invention, the substitution reaction is preferably carried out in the presence of an alkaline reagent, which preferably includes an organic base, and the organic base preferably includes triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA). When the organic base is TEA and DIPEA, this invention does not impose any special limitation on the ratio of TEA and DIPEA, and they can be mixed in any ratio.

[0032] In this invention, the molar ratio of the compound having the structure shown in Formula 1 and the basic reagent is preferably 1:(0.1~1000), more preferably 1:0.1, 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:50, 1:100, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900 or 1:1000.

[0033] In this invention, the temperature of the substitution reaction is preferably 15~60℃, more preferably 15℃, 20℃, 30℃, 40℃, 50℃ or 60℃; the time of the substitution reaction is preferably 0.5~48h, more preferably 0.5h, 1h, 2h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h or 48h.

[0034] After the substitution reaction is completed, the present invention preferably further includes: subjecting the reaction solution obtained from the substitution reaction to high-performance liquid chromatography (HPLC) purification and freeze-drying sequentially. The present invention does not have specific limitations on the temperature and time of the freeze-drying; freeze-drying to constant weight (i.e., lyophilization) is sufficient. The present invention does not have specific limitations on the HPLC purification conditions; any HPLC purification conditions that achieve the required compound purity are acceptable.

[0035] The present invention also provides a radionuclide-labeled probe having the structure shown in Formula II or Formula III: Formula II; Formula III; The types of A and L in Formulas II and III are the same as those in Formula I; In Formula II, R is a nuclide labeling group, which is a complex obtained by coordinating T with a nuclide in Formula I. In equation (III), Q is... ; The In this context, a is an integer from 0 to 5; Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

[0036] In this invention, the value of 'a' is preferably 0, 1, 2, 3, 4 or 5.

[0037] In this invention, the nuclide preferably includes 18 F,43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb, 128 Ba、 139 La、 140 La、 142 Pr、 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 One or more of Ac.

[0038] In this invention, the compound having the structure shown in Formula III is preferably having any one of the structures shown in Formulas III-1 to III-5: Formula III-1; Formula III-2; Formula III-3; Formula III-4; Formula III-5.

[0039] In this invention, the types of Z, L, and Q in the radionuclide-labeled probe with the structure shown in Formula III-1, and the corresponding names and numbers of the radionuclide-labeled probes, are preferably as shown in Table 4: Table 4. Types of Z, L, and Q in the nuclide-labeled probes with the structure shown in Formula III-1, and the corresponding names and numbers of the nuclide-labeled probes.

[0040] In this invention, the types of L and Q in the nuclide-labeled probes having the structures shown in Formulas III-2 to III-3, and the corresponding names and numbers of the nuclide-labeled probes, are preferably as shown in Table 5: Table 5. Types of L and Q in nuclide-labeled probes with structures shown in Formulas III-2 to III-3, and the corresponding names and numbers of the nuclide-labeled probes.

[0041] In this invention, the types of X3, Y, and Q in the radionuclide-labeled probes having the structures shown in Formulas III-4 to III-5, and the corresponding names and numbers of the radionuclide-labeled probes, are preferably as shown in Table 6: Table 6 lists the types of X3, Y, and Q in the nuclide-labeled probes with the structures shown in Formulas III-4 to III-5, along with their corresponding names and numbers.

[0042] This invention also provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: The PSMA-targeting small molecule described in the above technical solution is coordinated with a nuclide to obtain a nuclide-labeled probe with the structure shown in Formula II. Alternatively, an amidation reaction can be carried out between a compound having the structure shown in Formula 1 and an active compound of Q to obtain a compound having the structure shown in Formula III. Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule; The active compound of Q is a compound having the structure shown in Formula 2 or Formula 3: Equation 2, Formula 3; In equations 2 and 3, 'a' is an independent integer from 0 to 5, and Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

[0043] In this invention, unless otherwise specified, all raw materials used in the preparation are commercially available products well known to those skilled in the art.

[0044] The preparation method of the present invention includes coordinating the PSMA-targeting small molecule described in the above technical solution with a nuclide to obtain a nuclide-labeled probe having the structure shown in Formula II.

[0045] In this invention, the coordination reaction process is preferably carried out by mixing the solution of the PSMA-targeted small molecule with the solution of the radionuclide, performing the coordination reaction, and then diluting to obtain a reaction solution containing the radionuclide-labeled probe (wet labeling method).

[0046] The present invention does not have any particular limitation on the solvent in the solution of the PSMA-targeting small molecule, and any solvent well known to those skilled in the art (e.g., buffer solution, water and one or more organic solvents) may be used; the buffer solution preferably includes PBS, acetic acid-acetate solution or aluminum chloride-acetate solution; the organic solvent preferably includes one or more of acetonitrile, ethanol, DMSO and DMF.

[0047] In this invention, when the nuclide is 18 When F, the solvent for the solution of the nuclide preferably includes an acetic acid-acetate solution or an aluminum chloride-acetate solution; when the nuclide is 68 Ga、 177 Lu or 64 When using metallic nuclides such as Cu, the preferred solvent for the solution of the nuclide includes an acetate-acetate buffer solution. In this invention, the concentration of the PSMA-targeting small molecule solution is preferably 0.001~1000 mg / mL, more preferably 0.01~1 mg / mL.

[0048] In this invention, the ratio of the mass of the PSMA-targeting small molecule in the solution to the radioactivity of the radionuclide in the solution is preferably 20-400 µg: 1 kBq-1000 GBq, more preferably 20-400 µg: 0.037-74000 MBq, and even more preferably 20-200 µg: 0.037-7400 MBq. This invention does not have a particular limitation on the solution of the radionuclide; any radionuclide solution well known to those skilled in the art can be used, specifically as follows: 68 GaCl3 hydrochloric acid solution, 18 F - Solution 177 LuCl3 solution or Na 99m TcO4 eluent, the 68 The GaCl3 hydrochloric acid solution is preferably obtained by rinsing from a germanium-gallium generator, wherein the Na 99m The TcO4 eluent is preferably obtained by rinsing from a molybdenum technetium generator.

[0049] In this invention, the temperature of the coordination reaction is preferably 25-100°C, more preferably 80-100°C, and the time of the coordination reaction is preferably 10-60 min, more preferably 20-30 min. When the temperature of the coordination reaction is higher than room temperature, the invention preferably further includes cooling the obtained coordination reaction system to room temperature after the coordination reaction. This can be done by dilution according to actual needs, sterile membrane filtration, to obtain a radionuclide-labeled probe solution. This invention does not have any special limitations on the cooling process; any cooling method well known to those skilled in the art can be used, such as natural cooling.

[0050] In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the concentration of the radionuclide-labeled probe solution is preferably 0.037~3700 MBq / mL.

[0051] In this invention, when the nuclide is 18 In case F, the coordination reaction is preferably carried out under mixed media of co-coordinated ions, buffer solution and organic solvent; the co-coordinated ions are preferably metal ions, more preferably aluminum ions; the buffer solution is preferably an aluminum chloride-acetate solution; and the organic solvent is preferably acetonitrile.

[0052] Alternatively: In this invention, the coordination reaction process is preferably as follows: The solution containing PSMA-targeting small molecules was freeze-dried and then sealed to obtain a freeze-dried drug box. Solvent is added to the lyophilized kit to dissolve the sample, and then a radionuclide solution is added to perform a coordination reaction and then diluted to obtain a radionuclide-labeled probe solution (lyophilized labeling method).

[0053] This invention involves freeze-drying a solution containing PSMA-targeting small molecules and then sealing it to obtain a freeze-dried drug box.

[0054] In this invention, the freeze-drying preferably includes dispensing the solution containing PSMA-targeting small molecules into a freeze-drying container and then freeze-drying it; this invention does not have any special limitations on the freeze-drying conditions, and freeze-drying conditions well known to those skilled in the art can be used.

[0055] The present invention does not impose any special limitations on the sealing process; any process known to those skilled in the art can be used.

[0056] After obtaining the lyophilized kit, the present invention adds a solvent to the lyophilized kit for dissolution, then adds a radionuclide solution for coordination reaction and then dilutes it to obtain a radionuclide-labeled probe solution.

[0057] In this invention, the solvent is not particularly limited and any solvent well known to those skilled in the art (e.g., one or more of buffer solutions, water, and organic solvents) may be used; the buffer solution preferably includes PBS or acetate solution.

[0058] In this invention, there is no special limitation on the ratio of the freeze-dried medicine box to the solvent, as long as it is sufficient to meet the requirements of dissolution and coordination reaction of the compound in the medicine box.

[0059] In this invention, the concentration of the radionuclide solution is not particularly limited, as long as it is sufficient to allow the radionuclide labeling reaction to occur.

[0060] In this invention, the ratio of the mass of the PSMA-targeting small molecule in the lyophilized kit to the radioactivity of the radionuclide in the solution is preferably 20~400µg:1kBq~1000GBq, more preferably 20~400µg:0.037~74000MBq, and even more preferably 20~200µg:0.037~7400MBq.

[0061] In this invention, the temperature of the coordination reaction is preferably 25-100°C, more preferably 80-100°C, and the time of the coordination reaction is preferably 10-60 min, more preferably 20-30 min. When the temperature of the coordination reaction is higher than room temperature, the invention preferably further includes cooling the obtained coordination reaction system to room temperature after the coordination reaction. This can be done by dilution according to actual needs, sterile membrane filtration, to obtain a radionuclide-labeled probe solution. This invention does not have any special limitations on the cooling process; any cooling method well known to those skilled in the art can be used, such as natural cooling.

[0062] In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the concentration of the radionuclide-labeled probe solution is preferably 0.037~3700 MBq / mL.

[0063] In this invention, when the radiochemical purity of the radionuclide-labeled probe solution prepared by the wet labeling method or the lyophilization method is less than 95%, it is preferable to further purify the radionuclide-labeled probe solution. The purification preferably includes column separation purification, and the chromatographic column used for column separation purification preferably includes a Sep-Pak C18 separation column. The Sep-Pak C18 separation column is preferably activated and eluted sequentially with anhydrous ethanol and water before use. In this invention, the eluent used for column separation purification is preferably water and anhydrous ethanol sequentially. The eluent of anhydrous ethanol is collected and the solvent is removed, and then diluted to obtain a high-purity radionuclide-labeled probe solution. In this invention, the dilution is preferably performed using physiological saline or water for injection. In this invention, the radioactivity concentration of the high-purity radionuclide-labeled probe solution is preferably 0.037~3700 MBq / mL.

[0064] In this invention, the method for preparing the radionuclide-labeled probe includes the following steps: The compound having the structure shown in Formula 1 is subjected to an amidation reaction with the active compound Q to obtain the compound having the structure shown in Formula III.

[0065] In this invention, the molar ratio of the compound having the structure shown in Formula 1 to the active compound of Q is preferably 1:(0.0000001~10), more preferably 1:0.0000001, 1:0.000001, 1:0.00001, 1:0.0001, 1:0.001, 1:0.1, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0066] In this invention, the amidation reaction is preferably carried out in a dispersion medium, which is preferably one or more of an organic solvent, water, and PBS; the organic solvent preferably includes one or more of N-methylpyrrolidone (NMP), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). This invention does not impose any particular limitations on the amount of organic solvent used, the temperature of the amidation reaction, or the time, as long as the amidation reaction proceeds smoothly.

[0067] After the amidation reaction is completed, the present invention preferably includes post-treatment, which preferably includes purification by preparative or semi-preparative HPLC.

[0068] The preparation method provided by this invention has the advantages of simple and readily available labeling, good stability of the obtained radionuclide-labeled probe, and high tumor uptake, making it suitable for industrial production and clinical application.

[0069] The present invention also provides pharmaceutically acceptable forms of the PSMA-targeting small molecules or the radionuclide-labeled probes described in the above technical solutions, wherein the pharmaceutically acceptable forms include one or more of salts, stereoisomers, racemates, hydrates and solvates.

[0070] In this invention, the salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. This invention does not specifically limit the tautomer, racemate, hydrate, or solvate; any tautomer, racemate, hydrate, or solvate well known to those skilled in the art can be used.

[0071] The present invention does not impose any particular limitation on the preparation method of the tautomer, racemate, hydrate, solvate or salt, and any preparation method of tautomer, racemate, hydrate, solvate or salt known to those skilled in the art can be used.

[0072] The present invention also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; The active component includes one or more of the PSMA-targeting small molecule described in the above technical solution, the radionuclide-labeled probe described in the above technical solution, and pharmaceutically acceptable forms of the PSMA-targeting small molecule or the radionuclide-labeled probe described in the above technical solution.

[0073] In this invention, there are no special limitations on the mass ratio of the active ingredient and the pharmaceutically acceptable excipients; any ratio known to those skilled in the art can be used.

[0074] In this invention, the pharmaceutically acceptable excipients preferably include at least one of the following: excipients, antioxidants, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. This invention does not have any particular limitation on the types of the above-mentioned pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0075] In this invention, when the active component includes a radionuclide-labeled probe and / or a pharmaceutically acceptable form of the radionuclide-labeled probe, the dosage form of the pharmaceutical composition is preferably an injection, preferably administered by intravenous injection; when the active component does not contain a radionuclide-labeled probe and / or a pharmaceutically acceptable form of the radionuclide-labeled probe, this invention does not have any particular limitation on the dosage form and administration method of the pharmaceutical composition, and dosage forms and administration methods well known to those skilled in the art can be used.

[0076] The present invention also provides a lyophilized kit comprising a precursor and pharmaceutically acceptable excipients; The precursor includes one or more of the PSMA-targeting small molecules described in the above technical solutions or pharmaceutically acceptable forms of the PSMA-targeting small molecules described in the above technical solutions.

[0077] In this invention, there are no special limitations on the mass ratio of the precursor and the pharmaceutically acceptable excipient; any ratio known to those skilled in the art can be used.

[0078] In this invention, the pharmaceutically acceptable excipients preferably include at least one of the following: excipients, antioxidants, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, glidants, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. This invention does not have any particular limitation on the types of the above-mentioned pharmaceutically acceptable excipients, and any pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0079] The present invention also provides the use of the PSMA-targeting small molecule described in the above-mentioned technical solutions, the radionuclide-labeled probe described in the above-mentioned technical solutions, the pharmaceutically acceptable form of the PSMA-targeting small molecule or the radionuclide-labeled probe described in the above-mentioned technical solutions, the pharmaceutical composition described in the above-mentioned technical solutions, or the lyophilized kit described in the above-mentioned technical solutions in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for PSMA protein-mediated diseases.

[0080] In this invention, the PSMA protein-mediated diseases preferably include tumors and / or their metastases; the tumors preferably include one or more of the following: prostate cancer, breast cancer, ovarian cancer, liver cancer, lung cancer, colorectal cancer, bone sarcoma, connective tissue sarcoma, renal cell carcinoma, gastric cancer, pancreatic cancer, nasopharyngeal carcinoma, head and neck cancer, neuroendocrine tumors, skin melanoma, and bone metastases, more preferably prostate cancer. In this invention, the diagnostic methods of the diagnostic reagents and diagnostic drugs preferably include one or more of single-photon emission computed tomography (SPECT) and positron emission tomography (PET); the treatment methods of the therapeutic reagents and therapeutic drugs preferably include radionuclide targeted therapy.

[0081] The PSMA-targeting small molecule, the PSMA-targeting small molecule and its pharmaceutically acceptable form, and the radionuclide-labeled probe and its pharmaceutically acceptable form provided by this invention all have excellent in vivo biological properties. They exhibit excellent uptake and long retention time in lesions with high PSMA protein expression, while being rapidly cleared and exhibiting low signal background in non-target organs such as normal tissues. Therefore, they have a high target / non-target ratio and are particularly suitable for use in radionuclide therapy and imaging of tumors. They can improve the efficacy of PSMA-targeted radionuclide diagnosis and treatment and have the potential for widespread clinical application.

[0082] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0083] Examples 1-2 Synthesis of DOTA-YD1 (Example 1) and DOTA-YD2 (Example 2): The synthesis process is as follows: Reactant 1 (5.45 µmol), DOTA-NHS (5.47 mg, 10.9 µmol), and N,N-diisopropylethylamine (DIPEA, 3.52 mg, 27.25 µmol) were weighed and dissolved in dimethyl sulfoxide (DMSO, 1 mL). The reaction mixture was shaken and reacted at room temperature for 12 hours. After the reaction was complete, the mixture was separated by semi-preparative high-performance liquid chromatography (HPLC) and then lyophilized to obtain DOTA-YD1 or DOTA-YD2 lyophilized powder. The mobile phase was 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile solution (B), with a gradient elution program of 5% B increasing to 95% B over 20 minutes at a flow rate of 3 mL / min. Purity was determined using analytical HPLC with 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile solution (B) as the mobile phase. The gradient elution program was as follows: 5% B was increased to 95% B over 20 minutes at a flow rate of 1 mL / min.

[0084] DOTA-YD1: ESI MS [M+H] + for C 45 H 62 N 10 O 15 , calcd 983.44, found 983.70. DOTA-YD2: ESI MS [M+H]+ for C 48 H66 N 10 O 15 , calcd 1023.48, found 1023.73. Examples 3-4 Synthesis of NOTA-YD1 (Example 3) and NOTA-YD2 (Example 4): Prepared according to the same method as DOTA-YD1 or DOTA-YD2, replacing DOTA-NHS with NOTA-NHS to obtain NOTA-YD1 or NOTA-YD2. The compounds were then sampled and identified by HPLC and mass spectrometry.

[0085] NOTA-YD1: ESI MS [M+H] + for C 41 H 55 N9O 13 , calcd 882.95, found 882.64. NOTA-YD2: ESI MS [M+H] + for C 44 H 59 N9O 13 , calcd 922.43, found 922.67. Example 5 DOTA-YD3's crafting recipe: DOTA-YD3 was prepared by replacing reactant 2 with reactant 1, following the same preparation method as DOTA-YD1 or DOTA-YD2. The compound was then sampled and identified by HPLC and mass spectrometry.

[0086] DOTA-YD3: ESI MS [M+H] + for C 53 H 73 N 11 O 16 , calcd 1120.53, found 1120.80. Figure 1 The HPLC chromatograms of compounds DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1, and Nota-YD2 are shown below. Figure 1 It can be seen that the purity of the compound is greater than 95%.

[0087] Example 6 DEPF synthesis: DEPF was prepared according to the preparation methods of DOTA-YD1 or DOTA-YD2, by replacing reactant 1 with reactant 5. The compound was then sampled and identified by HPLC and mass spectrometry.

[0088] DEPF: ESI MS [M+K] + for C 46 H 69 KN 11 O 15 , calcd 1054.20, found 1054.41. Example 7 Synthesis of NEPF: NEPF was prepared by replacing DOTA-NHS with Nota-NHS according to the DEPF preparation method. The compound was then identified by HPLC and mass spectrometry analysis.

[0089] NEPF: ESI MS [M+H] + for C 42 H 62 N 10 O 13 , calcd 915.02, found 915.27. Figure 2 Here are the HPLC chromatograms of compounds DEPF and NEPF, from... Figure 2 It can be seen that the purity of the compound is greater than 95%.

[0090] Example 8 Synthesis of PB1: Prepared according to the preparation methods of DOTA-YD1 or DOTA-YD2, reactant 1 was replaced with reactant 4 to obtain PB1. The compound was sampled and identified by HPLC and mass spectrometry.

[0091] PB1: ESI MS [M+H] + for C 46 H 69 N9O 16 , calcd 1004.11, found 1004.8. Example 9 Synthesis of PB2: Prepared according to the preparation methods of DOTA-YD1 or DOTA-YD2, reactant 1 was replaced with reactant 3 to obtain PB2. The compound was sampled and identified by HPLC and mass spectrometry.

[0092] PB2: ESI MS [M+H] + for C 47 H 71 N9O 16 , calcd 1018.13, found 1018.9. Figure 3 Here are the HPLC chromatograms of compounds PB1 and PB2, from... Figure 3 It can be seen that the purity of the compound is greater than 95%.

[0093] Example 10 68 Ga-labeled nuclide-labeled probes[ 68 Synthesis of Ga]Ga-DOTA-YD1: The labeling method is as follows: 50 μg of compound DOTA-YD1 prepared in Example 1 was dissolved in 1 mL of 0.25 M acetate-sodium acetate buffer solution, and then added to approximately 740 MBq of 4 mL eluent from the germanium-gallium generator. 68 The GaCl3 hydrochloric acid solution was placed in a vial and shaken at 95°C for 15 minutes. After cooling to room temperature, it was diluted with physiological saline or water for injection and filtered through a sterile filter membrane to obtain the radionuclide. 68 Ga-marked [ 68 Ga]Ga-DOTA-YD1 solution.

[0094] The radionuclide-labeled probes were identified and purified by radio-HPLC using a reversed-phase C18 column (4.6 × 250 mm). Mobile phase A: water + 0.1 v / v% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1 v / v% TFA. Gradient elution program: 0–20 min, with the volume fraction of mobile phase B increasing from 5% to 95%; mobile phase flow rate: 1 mL / min; radioactivity was detected by a radiodetector.

[0095] Examples 11-15 According to the probe [ 68 The Ga-DOTA-YD1 was prepared by replacing the PSMA-targeted small molecule compound DOTA-YD1 with DOTA-YD2 (Example 11), DOTA-YD3 (Example 12), DEPF (Example 13), PB1 (Example 14), and PB2 (Example 15), respectively, to obtain nuclide-labeled probes. 68 Ga]Ga-DOTA-YD2、[68 Ga]Ga-DOTA-YD3、[ 68 Ga]Ga-DEPF, [ 68 Ga]Ga-PB1, [ 68 Ga]Ga-PB2.

[0096] The radio-HPLC results of the radionuclide-labeled probes prepared in Examples 10-15 are shown below. Figures 4-6 ,Depend on Figures 4-6 It can be seen that the synthesized by this invention 68 The radiochemical purity of Ga-labeled probes is greater than 95%.

[0097] Example 16 18 F-labeled nuclide-labeled probes [ 18 Synthesis of F]AlF-NOTA-YD1 80 μg of the precursor compound NOA-YD1 dissolved in 20 μL of dimethyl sulfoxide was mixed with 100 μL of 0.1 M acetate-sodium acetate buffer solution (pH=4), 200 μL of acetonitrile, and 3 μL of 2 mM AlCl3 solution (prepared from 0.1 M acetate-sodium acetate buffer solution at pH=4), and then 1.11 MBq of radionuclide obtained from accelerator production was added. 18 F - The solution was reacted with shaking at 100℃ for 20 min. A Sep-Pak C18 separation column was activated and rinsed successively with 10 mL of anhydrous ethanol and 10 mL of water. The labeled solution was diluted with 8 mL of pure water and then loaded onto the separation column. The C18 column was rinsed with 8 mL of pure water to remove unreacted reagents. 18 F - Ions. The radionuclide is prepared by rinsing with 1.5 mL of anhydrous ethanol solution, removing the organic solvent by nitrogen blowing, diluting with physiological saline or water for injection, and filtering through a sterile filter membrane. 18 F-labeled complexes [ 18 F]AlF-NOTA-YD1 solution.

[0098] Examples 17-18 Referring to Example 16, the difference is that: Nota-YD1 is replaced with Nota-YD2 and NEPF, respectively, to obtain probes [ 18 F]AlF-NOTA-YD2 and [ 18 F]AlF-NEPF.

[0099] Samples of each probe prepared in Examples 16-18 were analyzed and identified by HPLC. The HPLC results are shown in the figure. Figures 7-8 ,Depend on Figures 7-8 It can be seen that the present invention prepares 18The radiochemical purity of the F-labeled nuclide-labeled probes was greater than 95%.

[0100] Example 19 177 Lu-labeled nuclide-labeled probes[ 177 Synthesis of Lu-DEPF 370MBq 177 LuCl3 solution was added to a centrifuge tube containing 0.2 mL of 0.5 M, pH 4.0 acetate-sodium acetate solution (50 μg) of DEPF. The mixture was incubated at 95 °C for 25 min, then cooled to room temperature. The solution was diluted with physiological saline or water for injection and sterile filtered to obtain the radionuclide-labeled probe. 177 The Lu]Lu-DEPF solution was analyzed and identified by radio-HPLC according to the conditions of Example 18. Figure 9 for[ 177 The radio-HPLC chromatogram of Lu-DEPF was obtained from... Figure 9 It can be seen that the present invention prepares 177 Lu-marked [ 177 The radiochemical purity of Lu-Lu-DEPF is greater than 90%.

[0101] Example 20 131 I-labeled nuclide-labeled probe 131 Synthesis of I-IEPF: Dissolve 500 μg of reactant 5 in 200 μL of anhydrous DMSO. 131 After mixing IPBA-NHS with reactant solution 5, 5–10 μL of N,N-diisopropylethylamine was added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the product was purified by Radio-HPLC, and its radiochemical purity was further analyzed.

[0102] The radionuclide-labeled probes were identified and purified by radio-HPLC. A reversed-phase C18 column (4.6 × 250 mm) was used. Mobile phase A: water + 0.1 v / v% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1 v / % TFA; gradient elution program: 0–20 min, increasing the volume fraction of mobile phase B from 5% to 95%; 20–22 min, maintaining the volume fraction of mobile phase B at 95%; 22–25 min, decreasing the volume fraction of mobile phase B from 95% to 5%; mobile phase flow rate: 1 mL / min. The radioactive signal was detected by a radiodetector. Figure 5 probe 131 HPLC chromatogram of IPB-IEPF Figure 10 for131 The radio-HPLC chromatogram of IPB-IEPF, by Figure 10 It can be known that radioactivity 131 The radiochemical purity of IPB-IEPF is greater than 90%.

[0103] Test Example 1 Molecular docking predicts ligand-protein binding: Molecular docking was performed using AutoDock 4.2.6, with PSMA crystal structure (PDB: 5O5T) as the acceptor. The binding conformations of DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1, and Nota-YD2 with PSMA were determined and compared with PSMA-617. Water molecules and co-crystallized ligands were removed in PyMOL (v3.1.0), and polar hydrogen atoms were added for protein pretreatment. The ligands were generated, protonated, and energy minimized to obtain multiple low-energy conformations for docking. The structures of the resulting complexes were visualized and analyzed in PyMOL. Figure 11 Based on the PSMA crystal structure (PDB ID: 5O5T), the binding conformations of DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1, and Nota-YD2 with PSMA were determined and compared with PSMA-617. Figure 11 As can be seen from the molecular docking of PSMA (PDB: 5O5T), these ligands occupy binding pockets that are highly consistent with PSMA-617, and exhibit conserved interactions with key residues such as Tyr700, Arg536 and Lys514. Figure 12 The docking scores and key hydrogen bond bindings of DOTA-YD1, DOTA-YD2, DOTA-YD3, Nota-YD1, and Nota-YD2 with PSMA were analyzed and compared with PSMA-617. Figure 12 It is evident that the introduction of the acridine moiety enhances aromatic interactions within the binding pocket, thereby improving ligand-acceptor affinity. Predicted binding energy (kcal·mol⁻¹) -1 In the ranking, DOTA-YD3 (-10.6), NOTA-YD2 (-10.1), NOTA-YD1 (-9.8), and DOTA-YD1 (-9.8) are all better than PSMA-617 (-9.5).

[0104] Test Example 2 Lipid-water distribution coefficient (log P Measurement: 100 μL of the radionuclide-labeled probe solution diluted with PBS (pH=7.4) was added to a centrifuge tube containing a mixture of 0.9 mL PBS (pH=7.4) and 1 mL n-octanol. The mixture was vortexed for 3 min, then centrifuged at 10000 rpm for 3 min. 100 μL of liquid was taken from both the aqueous and n-octanol phases, and the radioactivity count was determined using a γ-counter. The experiment was repeated three times, and the average value was taken. P The calculation formula is shown in formula (1): P =I 有机相 / I 水相 Equation (1); Among them, I 有机相 I represents the radioactivity count measured in the organic phase. 水相 This represents the radioactivity count measured in the aqueous phase. 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2、[ 68 Ga]Ga-DOTA-YD3、[ 68 Ga]Ga-DEPF, [ 18 F]AlF-NOTA-YD1、[ 18 F]AlF-NOTA-YD2、[ 18 F]AlF-NEPF and [ 68 The results of the lipid-water distribution coefficient of the Ga]Ga-DEPF probe are shown in Table 7.

[0105] Table 7. Lipid-water distribution coefficient of radionuclide-labeled probes

[0106] As shown in Table 7, 68 Ga、 18 F-labeled probes are all hydrophilic and have good water solubility. This property helps to reduce the retention of probes in organs such as the liver, gallbladder, and intestines, and avoids non-specific uptake of radioactivity by normal tissues.

[0107] Test Example 3 Receptor affinity assay of the probe in PSMA-positive PC3-PIP cells: Affinity test method: Inoculate approximately 2 × 10⁶ cells per well in a 24-well plate 48 hours in advance. 5PC3-PIP cells were used. At the start of the experiment, the old culture medium was removed, and cells were washed in each well with 500 μL of cold PBS (pH 7.4, containing 0.2% BSA). In one 24-well plate, 100 μL of unlabeled PSMA-617 (10 μg / well, prepared in serum-free RPMI-1640 medium) was added to each well, and an equal volume of culture medium was added to another plate. 100 μL of radiolabeled radioligands at different concentrations (0.156–400 nM) were added to each plate, with each concentration replicated in 3 wells. After incubation at 37°C for 0.5 h, cells were washed twice with cold PBS (500 μL / well) containing 0.2% BSA. Cells were then lysed with 1 M NaOH (500 μL / well), and the radioactivity of the lysate was measured using a gamma counter.

[0108] [ 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2、[ 68 Ga]Ga-DOTA-YD3、[ 68 Ga]Ga-DEPF, [ 18 F]AlF-NOTA-YD1、[ 18 F]AlF-NOTA-YD2 and [ 18 K of F]AlF-NEPF probe d The values ​​are shown in Table 8: Table 8. K of the radionuclide-labeled probes d value

[0109] As can be seen from Table 8, the [ 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2、[ 68 Ga]Ga-DOTA-YD3、[ 68 Ga]Ga-DEPF, [ 18 F]AlF-NOTA-YD1、[ 18 F]AlF-NOTA-YD2 and [ 18 The F]AlF-NEPF probe exhibits good affinity for PSMA.

[0110] Test Example 4 Assay on probe uptake and inhibition in PSMA-positive PC3-PIP cells: PC3-PIP cells with high PSMA expression were seeded in 24-well plates containing culture medium and cultured for 48 hours. At the start of the uptake experiment, the original culture medium was aspirated, and the cells were washed with PBS. An equal volume of the target radionuclide-labeled probe, diluted with culture medium free of fetal bovine serum and antibiotics, was added to each well. The cells were incubated at 37°C for different time points. After incubation at each time point, the culture medium was aspirated, and 500 µL (1 M) of sodium hydroxide (NaOH) solution was added to each well to lyse the cells. After 5 min, the lysed cells were transferred to disposable centrifuge tubes, and the radioactivity count was measured. The cell uptake percentage was obtained by dividing the count by the total amount of radioactivity added. To investigate the PSMA targeting specificity of the radionuclide-labeled probe, an inhibition group was also set up, in which an appropriate amount of the inhibitor PSMA-617 was added to each well before adding the radionuclide-labeled probe.

[0111] Figure 13 for[ 177 Lu]Lu-DEPF and 131 Cell uptake and inhibition results of IPB-IEPF in PSMA-positive PC3-PIP cells at different incubation times. Figure 13 It can be seen that at each time point, the radionuclide-labeled probe showed significant uptake in PSMA-positive cells. The uptake of the probe in PSMA-positive cells was inhibited by PSMA-617, indicating that the […] provided by this invention… 177 Lu]Lu-DEPF and 131 The IPB-IEPF probe is specific for targeting the PSMA protein.

[0112] Test Example 5 Validation of the probe's metabolic stability in mice: Pick[ 18 F]AlF-NEPF injection solution was administered via tail vein injection into normal mice. After in vivo metabolic circulation, urine and blood samples were collected from mice at different time points and analyzed using radio-HPLC. The area of ​​the main radioactive peak was recorded, and the radiochemical purity was calculated. HPLC chromatograms of the stability of each probe in urine and blood at different time points are attached. Figure 14 As shown. By Figure 14 It can be seen that at each test time point, the probe [ 18 The radiochemical purity of F]AlF-NEPF in urine and blood at different time points was greater than 95%, indicating that it can remain stable during in vivo metabolism.

[0113] Test Example 6 Biodistribution experiment of radionuclide labeled probes: The radionuclide-labeled probe prepared by tail vein injection in tumor-bearing mice ([ 18[F]AlF-NEPF) injection solution. Mice were sacrificed at different time points after injection, and tumor and other organ tissue samples were obtained by dissection, weighed, and radioactivity counts were measured using a gamma counter. Results are expressed as percentage uptake dose per gram of tissue or organ (%ID / g).

[0114] Figure 15 For nuclide-labeled probes [ 18 Biodistribution results of F]AlF-NEPF in tumor-bearing mice. Figure 15 It is known that radionuclide-labeled probes exhibit high uptake at tumor sites and some retention in the kidneys. In normal tissues, radionuclide-labeled probes all exhibit low background signal characteristics, which is highly advantageous for PSMA-targeted PET imaging.

[0115] Test Example 7 PET imaging experiments with radionuclide-labeled probes: Preparation of Examples 68 Ga or 18 After diluting the F-labeled probe with physiological saline, 0.2 mL (approximately 7.4 MBq) of the radionuclide-labeled probe injection solution was injected via the tail vein of mice. MicroPET imaging was performed at different time points, and regions of interest (ROIs) were delineated on the images. The distribution values ​​of the radionuclide-labeled probes were then calculated.

[0116] Figures 16-18 The probes are respectively 68 Ga]Ga-DOTA-YD1、[ 68 Ga]Ga-DOTA-YD2、[ 68 Ga]Ga-DOTA-YD3、[ 68 Ga]Ga-PB1, [ 68 Ga]Ga-PB2 and [ 68 PET imaging of Ga-DEPF. Figures 16-18 It was found that the radionuclide-labeled probe exhibited high uptake at the tumor site. A slight radioactive signal was observed in the kidney at early time points, but this quickly diminished over time. The background values ​​of the radionuclide-labeled probe in other non-target organs were very low, and the absolute uptake and target / non-target ratio of the tumor significantly increased over time. In a PC3-bearing model with PSMA-negative expression, [ 68 The Ga]Ga-DEPF probe showed low uptake values ​​in tumors, demonstrating the probe's specificity for PSMA.

[0117] Figures 19-22 These are nuclide-labeled probes [ 18 F]AlF-NOTA-YD1、[ 18 F]AlF-NOTA-YD2 and [ 18PET imaging of PSMA-positive (PC3-PIP) tumor-bearing mice, normal mice, PSMA-negative (PC3) tumor-bearing mice, and hepatocellular carcinoma-bearing mice using [F]AlF-NEPF. Figures 19-22 It is evident that the radionuclide-labeled probe exhibits high uptake at the tumor site. The radioactive signal at the kidney location is rapidly cleared over time. The background values ​​of the radionuclide-labeled probe in other non-target organs are very low, and the absolute uptake by the tumor is well maintained over time, contributing to better imaging quality. In a PC3-bearing model with PSMA-negative expression, [ 18 The low uptake value of the F]AlF-NEPF probe in tumors demonstrates the probe's specificity for PSMA. In normal mice, […]. 18 Rapid clearance of the F]AlF-NEPF probe from the kidneys is crucial for ensuring imaging quality and the safety of radiopharmaceuticals. 18 The F]AlF-NEPF probe also showed significant tumor uptake in liver cancer-bearing mice, demonstrating that the probe's indications are not limited to prostate cancer, but can also play a diagnostic and therapeutic role in other PSMA-expressing lesions.

[0118] Test Example 8 Nucleotide-labeled probes[ 18 Human PET Imaging Experiment with F]AlF-NOTA-YD2 Figure 23 For nuclide-labeled probes [ 18 F]AlF-NOTA-YD2 and classic probes[ 18 F]Comparison of PET imaging of PSMA-1007 (self-made, procedure as in Example 10) in patients with bone metastases from prostate cancer. Figure 23 It can be seen that, compared to [ 18 F]PSMA-1007, [ 18 The F]AlF-NOTA-YD2 probe significantly improves PET imaging quality, absolute tumor uptake, and target / non-target ratio. This indicates that the probe of this invention performs well not only in the diagnosis and treatment of primary lesions but also in the treatment of metastatic lesions.

[0119] Figure 24 For nuclide-labeled probes [ 18 F]AlF-NOTA-YD2 and classic probes[ 18 Comparison of PET imaging of PSMA-1007 (self-made) in prostate cancer patients. Figure 24 It can be seen that, [ 18 F]AlF-NOTA-YD2 and [ 18 F]PSMA-1007 successfully identified both the primary tumor and lymph node metastases. It is noteworthy that, [ 18F]PSMA-1007 PET scan revealed suspected bone metastases in [ 18 F]AlF-NOTA-YD2 was not observed in imaging, and this was subsequently confirmed as a false positive result by multimodal imaging and postoperative PSA follow-up. These results indicate that, 18 F]AlF-NOTA-YD2 may improve diagnostic specificity by reducing nonspecific uptake and false positives, which has direct clinical significance for accurate staging and treatment decisions.

[0120] Figure 25 for[ 18 F]AlF-NOTA-YD2 and classic probes[ 18 F] Comparison of PSMA-1007 retention in normal human organs. Figure 25 It can be seen that both probes have good blood clearance rates and low blood pool background, which is beneficial for distinguishing lesions from the background. 18 The non-specific uptake of F]AlF-NOTA-YD2 in the salivary glands is significantly reduced, which can reduce the interference of glandular imaging on adjacent structures (such as head and neck lymph nodes) and also reduce the potential risk of long-term radiation damage to the salivary glands. 18 [F]AlF-NOTA-YD2 has lower uptake in the kidneys, which can reduce signal obstruction by the kidneys of lesions in the abdominal and pelvic regions (such as pelvic lymph nodes and bone metastases), thus improving image quality. 18 [F]AlF-NOTA-YD2 has lower uptake in the reticuloendothelial system, reducing background interference in the liver and spleen regions and making it more favorable for assessing peritoneal metastases. Furthermore, [ 18 F]AlF-NOTA-YD2 in multiple locations [ 18 F]PSMA-1007 reduces uptake in high-background organs, allowing for clearer visualization of micrometastases in the head, neck, and abdominal / pelvic regions, thus reducing false positives and false negatives. Reduced radiation dose to the salivary glands and kidneys decreases the potential side effects of long-term use, resulting in better patient tolerance.

[0121] Test Example 9 Nucleotide-labeled probes[ 18 Human PET imaging experiments with F]AlF-NEPF: Figure 26 For nuclide-labeled probes [ 18 F]AlF-NEPF and classic probes[ 18 Comparison of PET imaging of PSMA-1007 (self-made) in patients with bone metastases from prostate cancer. Figure 26 It can be seen that, compared to [ 18 F]PSMA-1007, [ 18 The F]AlF-NOTA-YD2 significantly improves PET imaging quality, absolute tumor uptake, and target / non-target ratio.

[0122] Figure 27 For nuclide-labeled probes [ 18 PET imaging of patients with in situ hepatocellular carcinoma using [F]AlF-NEPF. Figure 27 It can be seen that, [ 18 F]AlF-NEPF and were able to successfully identify the liver. It is worth noting that, [ 18 F]PSMA-1007 PET scan revealed suspected bone metastases in [ 18 F]AlF-NOTA-YD2 was not observed in imaging, and this was subsequently confirmed as a false positive result by multimodal imaging and postoperative PSA follow-up. These results indicate that, 18 F]AlF-NOTA-YD2 may improve diagnostic specificity by reducing nonspecific uptake and false positives, which has direct clinical significance for accurate staging and treatment decisions.

[0123] As can be seen, as a novel structure developed in-house, this patented probe retains the high specificity of PSMA targeting probes while solving the core problem of high background in traditional PSMA probes, and has the potential to become a better alternative.

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PSMA-targeting small molecule having the structure shown in Formula I: Formula I; In Equation I, A is , , , or ; The and X1, X2, and X3 are all -CH=, or one of X1, X2, and X3 is independently -N=, and the remaining two are -CH=; The and The Y in the equation can be -O-, -NH-, or -N(CH3)-. The In this context, m is an integer from 1 to 5; The n is an integer from 1 to 5; The Z in , , , , , , , or ; In Z, x, y, e, j, f, v, and k are independent integers from 0 to 5, and G is independent -H or -CH3; In Equation I, L may or may not exist. When L exists, L is: , , , , , , , , , , , or ; In L, t, q, and p are independent integers from 1 to 3, and M is -CH= or -N=; In Formula I, T is the group to be labeled, and the structural formula of T is: , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or .

2. The method for preparing the PSMA-targeted small molecule according to claim 1, characterized in that, Includes the following steps: The active substance T and a compound having the structure shown in Formula 1 were subjected to a substitution reaction to obtain the PSMA-targeting small molecule. The T-active substance contains an activating group; Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule.

3. The preparation method according to claim 2, characterized in that, The activating group is an N-hydroxysuccinimide activating group, an acid anhydride, a pentafluorophenyl ester group, or an isothiocyanate group.

4. A radionuclide-labeled probe, characterized in that, It has the structure shown in Formula II or Formula III: Formula II; Formula III; The types of A and L in Formulas II and III are the same as those in Formula I; In Formula II, R is a nuclide labeling group, which is a complex obtained by coordinating T with a nuclide in Formula I. In equation (III), Q is... ; The In this context, a is an integer from 0 to 5; Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

5. The radionuclide-labeled probe as described in claim 4, characterized in that, The nuclides include 18 F, 43 Sc、 44 Sc、 47 Sc、 51 Cr 55 Co、 57 Co、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 72 As、 72 Se、 89 Zr、 86 Y、 89 Sr、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 103 Pd, 109 Pd, 111 In、 119 Sb, 128 Ba、 139 La、 140 La、 142 Pr, 149 Pm, 149 Tb, 151 Tb, 151 Eu、 153 Eu、 169 Eu、 153 Sm、 152 Gd, 153 Gd, 157 Gd, 159 Gd, 161 Tb, 165 Dy、 166 Ho、 169 Er、 175 Yb、 177 Lu、 186 Re、 188 Re、 197 Hg, 198 Au、 201 Tl、 203 Pb, 211 At、 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 One or more of Ac.

6. The method for preparing the radionuclide-labeled probe according to claim 4 or 5, characterized in that, Includes the following steps: The PSMA-targeting small molecule of claim 1 is coordinated with a nuclide to obtain a nuclide-labeled probe with the structure shown in Formula II. Alternatively, an amidation reaction can be carried out between a compound having the structure shown in Formula 1 and an active compound of Q to obtain a compound having the structure shown in Formula III. Formula 1; In Formula 1, the types of A and L are the same as those of A and L in the PSMA-targeted small molecule; The active compound of Q is a compound having the structure shown in Formula 2 or Formula 3: Equation 2, Formula 3; In equations 2 and 3, 'a' is an independent integer from 0 to 5, and Q1 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At; Q2 is -CH= or -N=.

7. The pharmaceutically acceptable form of the PSMA-targeting small molecule of claim 1 or the radionuclide-labeled probe of claim 4 or 5, characterized in that, The pharmaceutically acceptable forms include one or more of the following: salt, stereoisomer, racemate, hydrate, and solvate.

8. A pharmaceutical composition, characterized in that, Including active ingredients and pharmaceutically acceptable excipients; The active component comprises one or more of the pharmaceutically acceptable forms of the PSMA-targeting small molecule of claim 1, the radionuclide-labeled probe of claim 4 or 5, and the PSMA-targeting small molecule or the radionuclide-labeled probe of claim 7.

9. A lyophilized reagent kit, characterized in that, Including precursors and pharmaceutically acceptable excipients; The precursor includes one or more of the pharmaceutically acceptable forms of the PSMA-targeting small molecule of claim 1 or the PSMA-targeting small molecule of claim 7.

10. The use of the PSMA-targeting small molecule of claim 1, the radionuclide-labeled probe of claim 4 or 5, the PSMA-targeting small molecule of claim 7 or the radionuclide-labeled probe in a pharmaceutically acceptable form, the pharmaceutical composition of claim 8, or the lyophilized kit of claim 9 in the preparation of therapeutic agents, therapeutic drugs, diagnostic agents, or diagnostic drugs for PSMA protein-mediated diseases.

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