PSMA-targeting small molecule compound, preparation method therefor, and use thereof, nuclide-labeled probe, preparation method therefor, and use thereof, pharmaceutical composition and use thereof, and kit and use thereof

Abstract

The present application relates to biomedicine and provides a PSMA-targeting small molecule compound, a preparation method therefor, and a use thereof, a nuclide-labeled probe, a preparation method therefor, and a use thereof, a pharmaceutical composition and a use thereof, and a kit and a use thereof. The PSMA-targeting small molecule compound and the nuclide-labeled probe thereof provided by the present application both have excellent in vivo biological performance, exhibiting excellent lesion uptake in lesions having high expression of the PSMA protein and a long retention time, and being rapidly cleared in non-targeted organs such as normal tissues and having a low signal background, thereby having the characteristic of a high target / non-target ratio. Therefore, the PSMA-targeting small molecule compound and the nuclide-labeled probe thereof are particularly suitable for nuclide therapy and imaging of tumors, can improve the effect of PSMA-targeting nuclide theranostics, and have potential for clinical promotion and application.

Description

A PSMA-targeting small molecule compound and its preparation method and application, a radionuclide-labeled probe and its preparation method and application, a pharmaceutical composition and its application, and a reagent kit and its application.

[0001] This application claims priority to Chinese Patent Application No. CN2024119092367, filed on December 24, 2024, entitled "A PSMA-targeting small molecule compound and its preparation method and application, a radionuclide-labeled probe and its preparation method and application, a pharmaceutical composition and its application, and a reagent kit and its application", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biomedical technology, specifically to a PSMA-targeting small molecule compound and its preparation method and application, a radionuclide-labeled probe and its preparation method and application, a pharmaceutically acceptable form of the PSMA-targeting small molecule compound and the radionuclide-labeled probe and its application, a pharmaceutical composition and its application, and a kit and its application. Background Technology

[0003] Prostate-specific membrane antigen (PSMA) expression is highly positively correlated with the progression of prostate cancer, and it is overexpressed on the surface of more than 90% of prostate cancer cells, with even higher expression levels in advanced and castration-resistant prostate cancer patients. In contrast, PSMA is expressed in small amounts in normal tissues such as salivary glands, lacrimal glands, and proximal renal tubules. Therefore, PSMA has become an ideal target for the diagnosis and treatment of many tumors, including prostate cancer, due to its high sensitivity and specificity. Positron emission tomography (PET) and single-photon emission computed tomography (SPECT) in the field of nuclear medicine molecular imaging offer high sensitivity, but both rely on radionuclide-labeled drugs. In recent years, radionuclide-labeled drugs targeting PSMA have received widespread attention and made promising progress, providing strong support for the diagnosis and treatment of prostate cancer.

[0004] 111ProstaScint, an In-labeled monoclonal antibody, was the first PSMA-targeting radiodiagnostic drug for prostate cancer approved by the FDA in the United States. However, its large molecular size negatively impacts cell membrane permeability and internalization rate. Small-molecule radiopharmaceuticals, on the other hand, offer better cell membrane permeability, faster pharmacokinetic rates, and are easier to synthesize, showing great potential in radionuclide-targeted therapy for prostate cancer. Clinical and preclinical studies have shown that existing PSMA-targeting small-molecule radiopharmaceuticals (e.g., 68 Ga-PSMA-617 177 Lu-PSMA-617 18 F-labeled PSMA-1007 exhibits some non-target organ retention, particularly in the kidneys, liver, and normal glands, which to some extent affects treatment outcomes, and its retention time at the lesion site is limited. Therefore, developing new drugs that can reduce the retention of radiopharmaceuticals in non-target tissues and increase their retention time at the lesion site is of great significance. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide PSMA-targeting small molecule compounds, their preparation methods and applications, radionuclide-labeled probes, their preparation methods and applications, pharmaceutical compositions and their applications, and kits and their applications. The PSMA-targeting small molecule compounds and their pharmaceutically acceptable forms, as well as the radionuclide-labeled probes and their pharmaceutically acceptable forms provided in this application, all possess PSMA-targeting properties, exhibiting high uptake in target organs, long retention time, low background in non-target organs, and a high target / non-target ratio.

[0006] To achieve the above-mentioned objectives, this application provides the following technical solution:

[0007] This application provides a PSMA-targeting small molecule compound having the structure shown in Formula I:

[0008] In Equation I, n is an integer from 0 to 5;

[0009] R1 includes any of the following structures:

[0010] R2 may or may not exist, and when R2 exists, it includes any of the following structures:

[0011] In R2, a, b, and t are independent integers from 0 to 5;

[0012] R3 is the group to be labeled, including any one of the following chelate structures:

[0013] This application also provides a method for preparing the PSMA-targeted small molecule compound described in the above technical solution, including method 1, method 2 or method 3;

[0014] Method 1 includes the following steps: reacting compound 1 with an R3 active compound bearing an activated ester group or an anhydride group via a substitution reaction to generate an amide bond, thereby obtaining the PSMA-targeting small molecule compound; the R3 active compound bearing an activated ester group or an anhydride group includes any one of the following structures:

[0015] Method 2 includes the following steps: reacting an R3 active compound with an isothiocyanate group to form a thiourea bond via a substitution reaction, thereby obtaining the PSMA-targeting small molecule compound; the R3 active compound with the isothiocyanate group includes any one of the following structures:

[0016] Method 3 includes the following steps: using an R3 chelating reagent with a carboxyl group to perform an amidation reaction with compound 1 under condensation reagent conditions to generate an amide bond, followed by removal of the protecting group to obtain the PSMA-targeting small molecule compound; the R3 chelating reagent with a carboxyl group includes any one of the following structures, wherein A is a carboxyl protecting group:

[0017] R1, R2, and n in compound 1 are the same as R1, R2, and n in formula I.

[0018] This application also provides a radionuclide-labeled probe having the structure shown in Formula II or Formula III:

[0019] Wherein, R1, R2, and n are defined in the same way as R1, R2, and n in Formula I; R4 is a radionuclide labeling group, obtained by coordinating the R3 labeling group in the PSMA-targeted small molecule compound described in the above technical solution with a radionuclide; R5 includes any one of the following structures:

[0020] Where y and z are independent integers from 0 to 5; X is carbon, hydrogen, or nitrogen; R6 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At.

[0021] Preferably, the nuclide includes 18 F, 43 Sc、 44 Sc、 47Sc、 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, 86 Y、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 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 At least one of Ac.

[0022] This application also provides a method for preparing the radionuclide-labeled probe described in the above technical solution.

[0023] The method for preparing the nuclide-labeled probe having the structure shown in Formula II includes the following steps: coordinating the PSMA-targeting small molecule compound described in the above technical solution with a nuclide to obtain the nuclide-labeled probe having the structure shown in Formula II;

[0024] The method for preparing the nuclide-labeled probe having the structure shown in Formula III includes the following steps: subjecting compound 1 to an amidation reaction with an active compound to obtain a nuclide-labeled probe having the structure shown in Formula III; the active compound includes compound 2 or compound 3;

[0025] This application also provides a pharmaceutically acceptable form of existence, including the pharmaceutically acceptable form of the PSMA-targeting small molecule compound described in the above technical solutions and / or the pharmaceutically acceptable form of the radionuclide-labeled probe described in the above technical solutions, wherein the pharmaceutically acceptable form of existence includes one or more of salts, stereoisomers, racemates, hydrates and solvates.

[0026] This application also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprising one or more of the PSMA-targeting small molecule compounds described in the above-described technical solutions, the radionuclide-labeled probes described in the above-described technical solutions, and pharmaceutically acceptable forms described in the above-described technical solutions.

[0027] This application also provides a lyophilized kit comprising a precursor compound and pharmaceutically acceptable excipients, wherein the precursor compound comprises one or more of the PSMA-targeting small molecule compounds described in the above-described technical solutions and pharmaceutically acceptable forms of the PSMA-targeting small molecule compounds described in the above-described technical solutions.

[0028] This application also provides the use of the PSMA-targeting small molecule compounds, the radionuclide-labeled probes, the pharmaceutically acceptable forms, the pharmaceutical compositions, or the lyophilized kits described above in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for PSMA protein-mediated diseases.

[0029] Preferably, the diseases mediated by the PSMA protein include tumors and / or their metastases;

[0030] The tumors 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.

[0031] This application introduces a quinoline or naphthalene structure and a linker R2 between the targeting group and the coordination structure R3. This 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. 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 PSMA-targeted small molecule compounds or radionuclide-labeled probes in non-target tissues, increasing the uptake of target molecules and / or probes in PSMA-high expression lesions, and increasing the target / non-target ratio. Compared with existing PSMA-targeted radionuclide-labeled probes, the radionuclide-labeled probe provided in this application has excellent metabolic kinetic properties, high lesion uptake and retention time, and a high target / non-target ratio. It exhibits excellent diagnostic and therapeutic effects for PSMA protein-mediated diseases and has the potential to become a diagnostic and therapeutic reagent and / or drug with clinical application and commercial promotion value. As shown in the test results of the examples, the probe provided in this application… 68 The absolute uptake of Ga-labeled radionuclide-labeled probes in tumors is 68 Ga-PSMA-617 is twice as potent as this radionuclide diagnostic probe, making it a highly promising candidate for applications. Furthermore, by adjusting the appropriate specific activity or drug combination, it is possible to obtain a better target / non-target ratio, thereby enhancing the uptake of the radionuclide-labeled probe in tumors.

[0032] The PSMA-targeting small molecule compound provided in this application can chelate with radionuclides through the R3 group to form PSMA-targeting radionuclide-labeled probes with high affinity and high specificity. These probes feature convenient labeling, strong labeling ability, short labeling time, and high labeling yield. The prepared radionuclide-labeled probes exhibit good stability, high sensitivity, excellent pharmacokinetic properties, high lesion uptake, and a high target / non-target ratio. They are suitable for labeling various diagnostic and therapeutic radionuclides and can also be used to construct diagnostic and therapeutic platforms based on therapeutic radionuclide pairs. They show great promise in the preparation of therapeutic and / or drug agents, diagnostic reagents, and / or drugs for PSMA protein-mediated diseases. This is beneficial for the application and clinical promotion of radionuclide-labeled probes.

[0033] The method for preparing PSMA-targeting small molecule compounds provided in this application is simple to synthesize, easy to operate, and has low production cost, making it suitable for industrial production.

[0034] The nuclide labeling probes provided in this application have the characteristics of convenient labeling, strong labeling ability, short labeling time, and high labeling yield. Attached Figure Description

[0035] Figure 1 shows the mass spectrometry identification of compound DNB8P1;

[0036] Figure 2 shows the mass spectrometry identification of compound DNA8P1;

[0037] Figure 3 shows the mass spectrometry identification of compound DNC8P1;

[0038] Figure 4 shows the mass spectrometry identification of compound DND8P1;

[0039] Figure 5 shows the mass spectrometry identification of compound DNA6P1;

[0040] Figure 6 shows the mass spectrometry identification of compound DNB6P1;

[0041] Figure 7 shows the mass spectrometry identification of compound DCA8P1;

[0042] Figure 8 shows the mass spectrometry identification of compound DCB8P1;

[0043] Figure 9 shows the mass spectrometry identification of compound DNB8P2;

[0044] Figure 10 shows the mass spectrometry identification of compound DCB8P2;

[0045] Figure 11 shows the mass spectrometry identification of compound NNA8P1;

[0046] Figure 12 shows the mass spectrometry identification of compound NCB8P1;

[0047] Figure 13 shows the mass spectrometry identification of compound NNB8P1;

[0048] Figure 14 shows the mass spectrometry identification of compound DGNB8P1;

[0049] Figure 15 shows the mass spectrometry identification of compound DSNB8P1;

[0050] Figure 16 shows the mass spectrometry identification of compound DGNE8P1;

[0051] Figure 17 shows the mass spectrometry identification of compound DGNF8P1;

[0052] Figure 18 shows the HPLC chromatograms of compounds DNA8P1 and DNB8P1;

[0053] Figure 19 shows the HPLC chromatograms of compounds DNC8P1 and DND8P1;

[0054] Figure 20 shows the HPLC chromatograms of compounds DNA6P1 and DNB6P1;

[0055] Figure 21 shows the HPLC chromatograms of compounds DCA8P1 and DCB8P1;

[0056] Figure 22 shows the HPLC chromatograms of compounds DNB8P2 and DCB8P2;

[0057] Figure 23 shows the HPLC chromatograms of compounds NNA8P1, NNB8P1 and NCB8P1;

[0058] Figure 24 shows the HPLC chromatograms of compounds DGNB8P1 and DSNB8P1;

[0059] Figure 25 shows the HPLC chromatograms of compounds DGNE8P1 and DGNF8P1;

[0060] Figure 26 shows the probe. 68 Ga]Ga-DNA8P1 and [ 68 HPLC chromatogram of Ga]Ga-DNB8P1;

[0061] Figure 27 shows the probe. 68 Ga]Ga-DNC8P1 and [ 68 HPLC chromatogram of Ga]Ga-DND8P1;

[0062] Figure 28 shows the probe. 68 Ga]Ga-DCA8P1 and [ 68 HPLC chromatogram of Ga]Ga-DCB8P1;

[0063] Figure 29 shows the probe. 68 Ga]Ga-DNA6P1 and [ 68 HPLC chromatogram of Ga]Ga-DNB6P1;

[0064] Figure 30 shows the probe. 68 Ga]Ga-DNB8P2 and [ 68 HPLC chromatogram of Ga]Ga-DCB8P2;

[0065] Figure 31 shows the probe. 68 Ga]Ga-DGNB8P1 and [ 68 HPLC chromatogram of Ga]Ga-DSNB8P1;

[0066] Figure 32 shows the probe. 68 Ga]Ga-DGNE8P1 and [ 68 HPLC chromatogram of Ga]Ga-DGNF8P1;

[0067] Figure 33 shows the probe. 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 HPLC chromatogram of F]AlF-NCB8P1;

[0068] Figure 34 shows the probe. 177 Lu]Lu-DGNB8P1、[ 177 Lu]Lu-DSNB8P1、[ 177 Lu]Lu-DGNE8P1 and [ 177HPLC chromatogram of Lu]Lu-DGNF8P1;

[0069] Figure 35 shows the probe. 44 Sc]Sc-DNB8P1 and [ 44 HPLC chromatogram of Sc]Sc-DGNB8P1;

[0070] Figure 36 shows the probe. 131 HPLC chromatogram of IPB-NB8P1;

[0071] Figure 37 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DNA8P1 in physiological saline and serum;

[0072] Figure 38 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DNB8P1 in physiological saline and serum;

[0073] Figure 39 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DNA6P1 in physiological saline and serum;

[0074] Figure 40 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DNB6P1 in physiological saline and serum;

[0075] Figure 41 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DNC8P1 in physiological saline and serum;

[0076] Figure 42 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DND8P1 in physiological saline and serum;

[0077] Figure 43 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DCA8P1 in physiological saline and serum;

[0078] Figure 44 shows the probe. 68 HPLC chromatogram of the stability of Ga]Ga-DCB8P1 in physiological saline and serum;

[0079] Figure 45 shows the probe. 18 HPLC chromatogram of the stability of F]AlF-NNA8P1 in physiological saline and serum;

[0080] Figure 46 shows the probe. 18 HPLC chromatogram of the stability of F]AlF-NNB8P1 in physiological saline and serum;

[0081] Figure 47 shows the probe. 18HPLC chromatogram of the stability of F]AlF-NCB8P1 in physiological saline and serum;

[0082] Figure 48 shows the probe. 177 HPLC chromatogram of the stability of Lu]Lu-DGNB8P1 in physiological saline and serum;

[0083] Figure 49 shows the probe. 177 HPLC chromatogram of the stability of Lu]Lu-DGNF8P1 in physiological saline and serum;

[0084] Figure 50 shows the probe. 44 HPLC chromatogram of the stability of Sc]Sc-DNB8P1 in physiological saline and serum;

[0085] Figure 51 shows the probe. 44 HPLC chromatogram of the stability of Sc]Sc-DGNB8P1 in physiological saline and serum;

[0086] Figure 52 shows the probe. 68 Ga]Ga-DNA8P1 and [ 68 Ga]Ga-DNB8P1 in PSMA-positive PC3-PIP cells K d Test curve;

[0087] Figure 53 shows the probe. 68 Ga]Ga-DNC8P1 and [ 68 Ga]Ga-DND8P1 in PSMA-positive PC3-PIP cells K d Test curve;

[0088] Figure 54 shows the probe. 68 Ga]Ga-DNA6P1 and [ 68 Ga]Ga-DNB6P1 in PSMA-positive PC3-PIP cells K d Test curve;

[0089] Figure 55 shows the probe. 68 Ga]Ga-DCA8P1 and [ 68 Ga]Ga-DCB8P1 in PSMA-positive PC3-PIP cells K d Test curve;

[0090] Figure 56 shows the probe. 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 F]AlF-NCB8P1 in PSMA-positive PC3-PIP cells K d Test curve;

[0091] Figure 57 shows the probe. 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 The cell uptake and inhibition results of F]AlF-NCB8P1 in PSMA-positive PC3-PIP cells at different time points (60 min time point) were compared with the cell uptake results of the probe in PSMA-negative PC3 cells at 60 min.

[0092] Figure 58 shows the probe. 177 Lu]Lu-DGNB8P1 and [ 177 The cell uptake and inhibition results of Lu-DSNB8P1 in PSMA-positive PC3-PIP cells at different time points (2h and 4h) were compared with the cell uptake results of the probe in negative PC3 cells at 2h and 4h.

[0093] Figure 59 shows the probe. 68 PET imaging results of Ga]Ga-DNA8P1 (A) and uptake values ​​and target / non-target ratios of major tissues (B);

[0094] Figure 60 shows the probe. 68 PET imaging results of Ga-DNB8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0095] Figure 61 shows the probe. 68 PET imaging results of Ga]Ga-DNC8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0096] Figure 62 shows the probe. 68 PET imaging results of Ga]Ga-DND8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0097] Figure 63 shows the probe. 68 PET imaging results of Ga]Ga-DNA6P1 (A) and uptake values ​​and target / non-target ratios of major tissues (B);

[0098] Figure 64 shows the probe. 68 PET imaging results of Ga-DNB6P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0099] Figure 65 shows the probe. 68 PET imaging results of Ga-DCA8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0100] Figure 66 shows the probe.68 PET imaging results of Ga-DCB8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0101] Figure 67 shows the probe. 68 PET imaging results of Ga-DNB8P2 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0102] Figure 68 shows the probe. 68 PET imaging results of Ga-DCB8P2 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0103] Figure 69 shows the probe. 68 PET imaging results of Ga]Ga-DGNB8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0104] Figure 70 shows the probe. 68 PET imaging results of Ga-DSNB8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0105] Figure 71 shows the probe. 68 PET imaging results of Ga]Ga-DGNE8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0106] Figure 72 shows the probe. 68 PET imaging results of Ga]Ga-DGNF8P1 (A) and uptake values ​​of major tissues and target / non-target ratio (B);

[0107] Figure 73 shows the results of competition binding agents (DNB8P1, ...) with different contents. nat Ga-DNB8P1, PSMA-617) have a lower probe [ 68 Comparison of PET imaging results (A) and uptake values ​​(B) of Ga-DNB8P1 in major tissues;

[0108] Figure 74 shows the probe. 68 Ga]Ga-DNB8P1 and classic probes[ 68 PET imaging results of Ga-PSMA-617 (A), comparison of uptake values ​​of major tissues (B), and comparison of target / non-target ratio (C);

[0109] Figure 75 shows the probe. 18 PET imaging results of F]AlF-NNA8P1 (A) and uptake values ​​of major tissues (B);

[0110] Figure 76 shows the probe. 18PET imaging results of F]AlF-NNB8P1 (A) and uptake values ​​of major tissues (B);

[0111] Figure 77 shows the probe. 18 PET imaging results of F]AlF-NCB8P1 (A) and uptake values ​​of major tissues (B);

[0112] Figure 78 shows the probe. 44 PET imaging results of Sc]Sc-DGNB8P1 (A) and comparison of uptake values ​​and target / non-target ratios of major tissues (B);

[0113] Figure 79 shows the probe. 177 SPECT imaging results of Lu-DGNB8P1 (A) and target / non-target ratio (B);

[0114] Figure 80 shows the probe. 177 SPECT imaging results of Lu-DSNB8P1 (A) and target / non-target ratio (B);

[0115] Figure 81 shows the probe. 177 SPECT imaging results of Lu-DGNF8P1 (A) and target / non-target ratio (B);

[0116] Figure 82 shows the probe. 177 SPECT imaging results of Lu]Lu-PSMA-617;

[0117] Figure 83 shows the probe in the presence of 20 μg of the competing binder DGNB8P1. 177 SPECT imaging results of Lu-DGNB8P1 (A) and target / non-target ratio (B);

[0118] Figure 84 shows the probe. 18 Biodistribution of F]AlF-NNA8P1 in tumor-bearing mice;

[0119] Figure 85 shows the probe. 18 Biodistribution of F]AlF-NCB8P1 in tumor-bearing mice;

[0120] Figure 86 shows the probe. 177 Biodistribution of Lu]Lu-DGNB8P1 in tumor-bearing mice;

[0121] Figure 87 shows the probe. 44 Biodistribution of Sc]Sc-DGNB8P1 in tumor-bearing mice;

[0122] Figure 88 is [ 177Lu-PSMA-617 probe (18.5 MBq) and different injection doses 177 Tumor inhibition results of Lu]Lu-DGNB8P1 probe in tumor-bearing mice. Detailed Implementation

[0123] This application provides a PSMA-targeting small molecule compound having the structure shown in Formula I:

[0124] In this application, n in Formula I is an integer from 0 to 5. In specific embodiments, n can be 0, 1, 2, 3, 4 or 5.

[0125] In this application, R1 in Formula I includes any one of the following structures:

[0126] In this application, R2 may or may not exist in Formula I. When R2 exists, it includes any of the following structures:

[0127] Wherein, a, b, and t are independently integers from 0 to 5. In a specific embodiment, a, b, and t can be 0, 1, 2, 3, 4, or 5.

[0128] In this application, R3 in Formula I is the group to be labeled, including any one of the following structures:

[0129] In this application, the -CO- end of R1 is connected to the -NH- of the lysine residue in Formula I to form an amide bond; when R2 is absent, the -NH- end of R1 is directly connected to R3; when R2 is present, the -NH- end of R1 is connected to the -CO- of R2, and the -NH- end of R2 is then connected to R3.

[0130] In this application, the PSMA-targeting small molecule compound preferably has any one of the structures shown in Formula I-1 to Formula I-10. In Formula I-1 to Formula I-10, n is independently 1 or 2, Q is independently N or CH, and R2 is independently absent or a different linking group. PSMA-targeting small molecule compounds with the structures shown in Formula I-1 to Formula I-10 and their names are shown in Tables 1 to 10.

[0131] Table 1. PSMA-targeting small molecule compounds with the structure shown in Formula I-1 and their names.

[0132] Table 2. PSMA-targeting small molecule compounds with Formula I-2 and their names.

[0133] Table 3. Names of PSMA-targeting small molecule compounds with Formula I-3

[0134] Table 4 lists the PSMA-targeting small molecule compounds with structures shown in Formulas I-4 and their names.

[0135] Table 5. Names of PSMA-targeting small molecule compounds with structures of formulas I-5

[0136] Table 6 lists the names of PSMA-targeting small molecule compounds with structures shown in Formulas I-6.

[0137] Table 7 lists PSMA-targeting small molecule compounds with structures shown in Formulas I-7 and their names.

[0138] Table 8 lists the PSMA-targeting small molecule compounds with structures shown in Formulas I-8 and their names.

[0139] Table 9 lists PSMA-targeting small molecule compounds with structures shown in Formulas I-9 and their names.

[0140] Table 10 lists PSMA-targeting small molecule compounds with structures shown in Formula I-10 and their names.

[0141] Clinical and preclinical studies have shown that existing PSMA-targeting radiopharmaceuticals suffer from some non-target organ retention, particularly in the kidneys, liver, and normal glands, which to some extent affects treatment efficacy. Furthermore, existing drugs have limited lesion detection capabilities, potentially missing some micrometastases, necessitating the development of drugs capable of detecting a wider range of lesions. Existing therapeutic radiopharmaceuticals also have limited lesion retention time and require relatively high doses. Therefore, developing radionuclide-targeted drugs with higher lesion uptake levels, better therapeutic effects, and a superior target / non-target ratio can improve treatment outcomes while reducing patient dosage or frequency and minimizing the possibility of adverse reactions. The PSMA-targeting small molecule compound provided in this application is convenient to label, exhibits good stability, high sensitivity, excellent pharmacokinetic properties, high lesion uptake, and a high target / non-target ratio, making it of significant value as a radiopharmaceutical.

[0142] This application provides a method for preparing the PSMA-targeted small molecule compound described in the above technical solution, including method 1, method 2 or method 3.

[0143] In this application, method 1 includes the following steps: reacting compound 1 with an R3 active compound bearing an activated ester group or an anhydride group to generate an amide bond, thereby obtaining the PSMA-targeting small molecule compound; the R3 active compound bearing an activated ester group or an anhydride group includes any one of the following structures:

[0144] The definitions of R1, R2, and n in compound 1 are the same as those in formula I.

[0145] Unless otherwise specified, all materials and equipment used in this application are commercially available products in this field.

[0146] In this application, the molar ratio of compound 1 to the active compound R3 with an activated ester or anhydride group is preferably 1:1 to 10, and in specific embodiments, it can be 1:1, 1:1.2, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In this application, the solvent used for the substitution reaction in method 1 preferably includes one or more of organic solvents, water, and PBS (phosphate buffered saline). In this application, the organic solvent preferably includes one or more of N-methylpyrrolidone (NMP), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). The pH value of the PBS is preferably 7 to 12, and in specific embodiments, it can be 7, 8, 9, 10, 11, or 12. This application does not have a specific limitation on the amount of solvent used, as long as it ensures the smooth progress of the substitution reaction.

[0147] In this application, the substitution reaction is preferably carried out in the presence of a basic reagent; the basic reagent is preferably an organic base, more preferably triethylamine (TEA) and / or N,N-diisopropylethylamine (DIPEA). In this application, the molar ratio of compound 1 to the basic reagent is preferably 1:0.1 to 1000, and in specific embodiments it can be 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.

[0148] In this application, the temperature of the substitution reaction is preferably 15-60°C, and in specific embodiments it can be 15°C, 20°C, 30°C, 40°C, 50°C or 60°C; the time of the substitution reaction is preferably 0.5-48h, and in specific embodiments it can be 0.5h, 1h, 2h, 5h, 10h, 15h, 20h, 25h, 30h, 35h, 40h, 45h or 48h.

[0149] After completing the substitution reaction, this application preferably further includes: purifying the obtained substitution reaction solution by high-performance liquid chromatography (HPLC) followed by freeze-drying to obtain the PSMA-targeted small molecule compound. This application does not have specific limitations on the temperature and time of the freeze-drying; freeze-drying to constant weight (i.e., lyophilization) is sufficient. This application also does not have specific limitations on the HPLC purification conditions; any HPLC purification conditions that achieve the required compound purity are acceptable.

[0150] In this application, method 2 includes the following steps: reacting an R3 active compound with an isothiocyanate group to form a thiourea bond with compound 1 to obtain the PSMA-targeting small molecule compound; the R3 active compound with the isothiocyanate group is shown below:

[0151] In this application, the molar ratio of compound 1 to the isothiocyanate-based R3 active compound is preferably 1:1 to 10, and in specific embodiments it can be 1:1, 1:1.2, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In this application, the substitution reaction conditions and post-treatment methods of method 2 are the same as those of method 1, and will not be repeated here.

[0152] In this application, method 3 includes the following steps: using an R3 chelating reagent with a carboxyl group to perform an amidation reaction with compound 1 under condensation reagent conditions to generate an amide bond, and then removing the protecting group to obtain the PSMA-targeting small molecule compound; the R3 chelating reagent with a carboxyl group includes any one of the following structures:

[0153] Wherein, A is a carboxyl protecting group, preferably including methyl ester, ethyl ester or tert-butyl ester; the definitions of R1, R2 and n in compound 1 are the same as the definitions of R1, R2 and n in formula I.

[0154] In this application, the molar ratio of compound 1 to the R3 chelating agent with a carboxyl group is preferably 1:1 to 10, and in specific embodiments it can be 1:1, 1:1.2, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0155] In this application, the amidation reaction is preferably carried out in the presence of a condensing reagent, an organic amine, and a solvent. In this application, the condensing reagent preferably includes one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), dicyclohexylcarbodiimide (DCC), N,N-diisopropylcarbodiimide (DIC), carbodiimide hydrochloride (EDC), oxaloyl chloride, thionyl chloride, phosphorus oxychloride, carbon disulfide, and activated succinimide ester; the molar ratio of compound 1 to the condensing reagent is preferably 1:0.5 to 10, and in specific embodiments it can be 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In this application, the organic amine preferably includes N,N-diisopropylethylamine (DIPEA) and / or triethylamine (Et3N); the molar ratio of compound 1 to the organic amine is preferably 1:0.5 to 10, and in specific embodiments it can be 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10. In this application, the solvent preferably includes one or more of organic solvents, water and PBS (phosphate buffered solution). In this application, the organic solvent preferably includes one or more of N-methylpyrrolidone (NMP), dichloromethane (DCM), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF); this application does not have a special limitation on the amount of the organic solvent used, as long as it is sufficient to ensure the smooth progress of the amidation reaction. In this application, the temperature of the amidation reaction is preferably room temperature, and the time is preferably 0.5 to 48 hours. In specific embodiments, it can be 0.5 hours, 1 hour, 2 hours, 5 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, or 48 hours.

[0156] In this application, the deprotecting agent used to remove the protecting group preferably includes a strong acid or a strong base, more preferably trifluoroacetic acid (TFA) or piperidine; this application does not have a special limitation on the concentration of the deprotecting agent solution, as long as it can ensure that the deprotecting group removal proceeds smoothly. In this application, the temperature for deprotecting the group is preferably 0 to 100°C, and in specific embodiments it can be 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C or 100°C; the time for deprotecting the group is preferably 1 min to 24 h, and in specific embodiments it can be 1 min, 5 min, 30 min, 1 h, 5 h, 10 h, 15 h, 20 h or 24 h.

[0157] After the deprotection of the protecting group is completed, this application preferably further includes: purifying the obtained deprotected reaction solution by high-performance liquid chromatography (HPLC) followed by freeze-drying to obtain the PSMA-targeted small molecule compound. This application does not have specific limitations on the temperature and time of the freeze-drying; freeze-drying to constant weight (i.e., lyophilization) is sufficient. This application also does not have specific limitations on the HPLC purification conditions; any HPLC purification conditions that achieve the required compound purity are acceptable.

[0158] This application provides a radionuclide-labeled probe having the structure shown in Formula II or Formula III:

[0159] The definitions of R1, R2, and n in Formulas II and III are the same as those in Formula I; R4 is a radionuclide labeling group, obtained by coordinating the R3 labeling group in the PSMA-targeted small molecule compound described in the above technical solution with a radionuclide; R5 includes any one of the following structures:

[0160] Where y and z are independent integers from 0 to 5, and in specific embodiments can be 0, 1, 2, 3, 4 or 5; X is carbon or hydrogen or nitrogen; R6 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At.

[0161] In this application, 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, 86 Y、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 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 At least one of Ac.

[0162] In this application, Formula III preferably has any one of the structures shown in Formula III-1 to Formula III-6. In Formula III-1 to Formula III-6, Q is independently N or CH, and R2 is independently absent or has different linking groups. The nuclide-labeled probes with the structures shown in Formula III-1 to Formula III-6 and their names are shown in Tables 11 to 16. In Tables 11 to 16, / indicates or, for example, 123 / 124 / 125 / 131I indicates 123 I, 124 I, 125 I or 131 I.

[0163] Table 11. Nuclide-labeled probes with the structure shown in Formula III-1 and their names.

[0164] Table 12 shows the nuclide-labeled probes with the structure shown in Formula III-2 and their names.

[0165] Table 13 lists the nuclide-labeled probes with the structure shown in Formula III-3 and their names.

[0166] Table 14 lists the nuclide-labeled probes with the structure shown in Formula III-4 and their names.

[0167] Table 15 lists the nuclide-labeled probes with the structure shown in Formula III-5 and their names.

[0168] Table 16 lists the nuclide-labeled probes with the structure shown in Formula III-6 and their names.

[0169] This application provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: coordinating the PSMA-targeting small molecule compound with a radionuclide to obtain a radionuclide-labeled probe having the structure shown in Formula II.

[0170] In this application, the nuclide-labeled probe having the structure shown in Formula II is preferably prepared by wet labeling or lyophilization labeling.

[0171] In this application, the preparation of a radionuclide-labeled probe having the structure shown in Formula II using a wet labeling method preferably includes the following steps: mixing a solution containing a precursor compound with a radionuclide solution, performing a coordination reaction, and then diluting to obtain a radionuclide-labeled probe solution containing the structure shown in Formula II; wherein the precursor compound is a PSMA-targeting small molecule compound as described in the above technical solution or a pharmaceutically acceptable form of the PSMA-targeting small molecule compound as described in the above technical solution.

[0172] This application does not specifically limit the solvent in the solution of the PSMA-targeting small molecule compound; any solvent well-known to those skilled in the art can be used, such as a buffer solution, water, or one or more organic solvents. The buffer solution preferably includes PBS, acetate-acetate solution, or aluminum chloride-acetate solution; the organic solvent preferably includes one or more of acetonitrile, ethanol, DMSO, and DMF. In this application, when the nuclide is... 18 When F, the solvent preferably includes an acetic acid-acetate solution or an aluminum chloride-acetate solution; when the nuclide is 68 Ga or 177In this application, the solvent preferably includes an acetic acid-acetate solution. The concentration of the PSMA-targeting small molecule compound solution is preferably 0.001–1000 mg / mL, more preferably 0.01–1 mg / mL. The ratio of the mass of the PSMA-targeting compound to the radioactivity of the radionuclide in the radionuclide 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 application does not specifically limit the radionuclide solution; any radionuclide solution well known to those skilled in the art can be used, such as… 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.

[0173] In this application, 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, this application preferably further includes cooling the obtained coordination reaction system to room temperature after the coordination reaction. This can be done by dilution as needed, followed by sterile membrane filtration to obtain a radionuclide-labeled probe solution. This application does not have specific limitations on the cooling method; any cooling method well known to those skilled in the art can be used, such as natural cooling. In this application, the dilution is preferably done using physiological saline or water for injection. In this application, the concentration of the radionuclide-labeled probe solution is preferably 0.037–3700 MBq / mL.

[0174] In this application, 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; the organic solvent is preferably acetonitrile.

[0175] In this application, when the nuclide is 99mAt Tc, the coordination reaction is preferably carried out in the presence of a reducing agent and a cooperating ligand. The reducing agent preferably includes SnCl2 or sodium triphenylphosphine tris(m-sulfonate) (TPPTS), and the SnCl2 is preferably used in the form of a hydrochloric acid solution of SnCl2; the concentration of the hydrochloric acid solution is preferably 0.00001–12 mol / L. Each time the label is applied, the amount of SnCl2 used is preferably 0.001–100 mg, more preferably 0.01–0.2 mg. In this application, the synergistic ligand preferably includes at least one of N-tris(hydroxymethyl)methylglycine (Tricine), sodium triphenylphosphine tri-m-sulfonate (TPPTS), ethylenediamine-N,N'-diacetic acid (EDDA), ethylenediaminetetraacetic acid (EDTA), and sodium 3-diphenylphosphine benzenesulfonate (TPPMS); the mass ratio of the PSMA-targeting small molecule compound to the synergistic ligand is preferably 0.01-0.5:0.1-50, more preferably 0.02-0.1:1-10.

[0176] In this application, the preferred method for preparing a radionuclide-labeled probe with the structure shown in Formula II using a lyophilization labeling method includes the following steps: lyophilizing a solution containing a PSMA-targeting small molecule compound and then sealing it to obtain a lyophilized kit; adding a solvent to the lyophilized kit for dissolution, then adding a radionuclide solution for coordination reaction and dilution to obtain a radionuclide-labeled probe solution with the structure shown in Formula II. In this application, the lyophilization preferably includes dispensing the solution containing the PSMA-targeting small molecule compound into a lyophilization container before lyophilization; this application does not specifically limit the lyophilization conditions, and lyophilization conditions well known to those skilled in the art can be used.

[0177] This application preferably adds pharmaceutically acceptable excipients to the radionuclide-labeled probe solution containing the structure shown in Formula II obtained by the wet labeling method or the lyophilized drug box obtained by the lyophilization labeling method, as needed. The pharmaceutically acceptable excipients preferably include at least one of excipients, antioxidants, binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, flow aids, lubricants, preservatives, stabilizers, surfactants, tableting agents, and wetting agents. This application does not have a special limitation on the types of the above-mentioned pharmaceutically acceptable excipients, and pharmaceutically acceptable excipients well known to those skilled in the art can be used.

[0178] In this application, when the radiochemical purity of the radionuclide-labeled probe solution with the structure shown in Formula II 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 with the structure shown in Formula II. 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 application, 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 application, the dilution is preferably performed using physiological saline or water for injection. In this application, the radioactivity concentration of the high-purity radionuclide-labeled probe solution is preferably 0.037–3700 MBq / mL.

[0179] This application provides a method for preparing the radionuclide-labeled probe described in the above technical solution, comprising the following steps: subjecting compound 1 to an amidation reaction with an active compound to obtain a radionuclide-labeled probe having the structure shown in Formula III; wherein the active compound includes compound 2 or compound 3;

[0180] The definitions of y, z, X and R6 are the same as those of y, z, X and R6 in R5.

[0181] In this application, the molar ratio of compound 1 to the active compound is preferably 1:0.0000001 to 10, and in specific embodiments it can be 1:0.0000001, 1:0.000001, 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. In this application, the organic solvent used in the amidation reaction preferably includes one or more of organic solvents, water, and PBS (phosphate buffered saline). In this application, the organic solvent preferably includes one or more of N-methylpyrrolidone (NMP), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF). This application does not have a special limitation on the amount of the organic solvent used, as long as it can ensure that the amidation reaction proceeds smoothly.

[0182] In this application, the temperature of the amidation reaction is preferably 25 to 100°C, and the time is preferably 10 min to 24 h.

[0183] The preparation method provided in this application 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.

[0184] This application provides a pharmaceutically acceptable form of the compound, including the pharmaceutically acceptable form of the PSMA-targeting small molecule compound described in the above-described technical solutions and / or the pharmaceutically acceptable form of the radionuclide-labeled probe described in the above-described technical solutions. The pharmaceutically acceptable form includes one or more of salts, stereoisomers, racemates, hydrates, and solvates. In this application, the salt preferably includes trifluoroacetate, phosphate, formate, acetate, potassium salt, or sodium salt. This application does not have any particular limitation on the tautomers, racemates, hydrates, or solvates; any tautomers, racemates, hydrates, or solvates known to those skilled in the art can be used. This application does not have any particular limitation on the preparation method of the tautomers, racemates, hydrates, solvates, or salts; any preparation method known to those skilled in the art can be used.

[0185] This application also provides a pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable excipients. The active ingredient comprises one or more of the following: the PSMA-targeting small molecule compound described in the above-described technical solutions, a pharmaceutically acceptable form of the PSMA-targeting small molecule compound described in the above-described technical solutions, a radionuclide-labeled probe described in the above-described technical solutions, and a pharmaceutically acceptable form of the radionuclide-labeled probe described in the above-described technical solutions. In this application, the optional types of pharmaceutically acceptable excipients are the same as those described above and will not be repeated here. In this application, when the active ingredient 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 intravenously. When the active ingredient does not contain a radionuclide-labeled probe and / or a pharmaceutically acceptable form of the radionuclide-labeled probe, this application does not have any special limitations on the dosage form and administration method of the pharmaceutical composition; dosage forms and administration methods well known to those skilled in the art can be used.

[0186] This application also provides a lyophilized kit comprising a precursor compound and pharmaceutically acceptable excipients, wherein the precursor compound comprises one or more of the PSMA-targeting small molecule compounds described in the above-described technical solutions and pharmaceutically acceptable forms of the PSMA-targeting small molecule compounds described in the above-described technical solutions.

[0187] This application provides the use of the PSMA-targeting small molecule compounds, the radionuclide-labeled probes, and / or the pharmaceutically acceptable forms described in the above-described technical solutions, the pharmaceutical compositions described in the above-described technical solutions, or the lyophilized kits described in the above-described technical solutions in the preparation of therapeutic agents, therapeutic drugs, diagnostic reagents, or diagnostic drugs for PSMA protein-mediated diseases. In this application, the PSMA protein-mediated diseases preferably include tumors and / or their metastases; the tumors preferably include one or more of 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 application, the diagnostic methods of the diagnostic reagents and diagnostic drugs independently preferably include one or more of single-photon emission computed tomography (SPECT), positron emission tomography (PET), and magnetic resonance imaging (MRI); the treatment methods of the therapeutic reagents and therapeutic drugs independently preferably include radionuclide-targeted therapy and / or chemotherapy.

[0188] The PSMA-targeting small molecule compound, the PSMA-targeting small molecule compound and its pharmaceutically acceptable form, and the radionuclide-labeled probe and its pharmaceutically acceptable form provided in this application all possess 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, making them 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.

[0189] To further illustrate this application, the technical solutions of this application will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of this application.

[0190] Example 1

[0191] Synthesis of DNB8P1

[0192] The synthesis process is as follows: 3.18 μmol of precursor NB8P1, 7.95 μmol of hydroxysuccinimide-tetraazacyclododecanetetraacetic acid (DOTA-NHS), and 15.91 μmol of N,N-diisopropylethylamine (DIPEA) were dissolved in 500 μL of dimethyl sulfoxide (DMSO). After stirring at room temperature for 12 h, the product was separated and purified by semi-preparative HPLC. The target product peak was collected and lyophilized to obtain DNB8P1.

[0193] Semi-preparative HPLC separation and purification conditions: reversed-phase C18 semi-preparative column (10 mm × 250 mm); mobile phase A: water + 0.1 v / v% trifluoroacetic acid (TFA); mobile phase B: acetonitrile + 0.1 v / v% TFA; gradient elution conditions: 0–10 min, mobile phase B volume fraction increased from 5% to 50%; 10–15 min: mobile phase fraction remained constant; 15–17 min: mobile phase B volume fraction decreased to 5% and maintained until 30 min. Mobile phase flow rate was 3 mL / min.

[0194] HPLC purity analysis conditions: Reversed-phase C18 column (4.6 × 250 mm); Mobile phase A: Water + 0.1 v / v% TFA; Mobile phase B: Acetonitrile + 0.1 v / v% TFA; Gradient elution program: 0–10 min, mobile phase B volume fraction increased from 5% to 50%; 10–15 min: mobile phase B volume fraction 50%; 15–17 min: mobile phase B volume fraction decreased from 50% to 5%; 17–30 min: mobile phase B volume fraction 5%; Mobile phase flow rate: 1 mL / min; Radioactive signal was detected by a radioactive detector.

[0195] Examples 2-17

[0196] Following the preparation method of DNB8P1, by replacing the structures of n, R1, R2, and R3 in compound II, the corresponding PSMA-targeting small molecule compounds DNA8P1, DNC8P1, DND8P1, DNA6P1, DNB6P1, DCA8P1, DCB8P1, DNB8P2, DCB8P2, NNA8P1, NCB8P1, NNB8P1, DGNB8P1, DSNB8P1, DGNE8P1, and DGNF8P1 were obtained, respectively. n, R1, R2, and R3 are shown in Table 17.

[0197] The mass spectra of the PSMA-targeting small molecule compounds prepared in Examples 1-17 are shown in Figures 1-17, indicating that the target PSMA-targeting small molecule compounds were successfully prepared in this application.

[0198] The HPLC purity analysis chromatograms of the PSMA-targeting small molecule compounds prepared in Examples 1-17 are shown in Figures 18-25. It can be seen that the HPLC purity of each PSMA-targeting small molecule compound is greater than 95%.

[0199] Table 17. n, R1, R2, and R3 in the PSMA-targeting small molecule compounds prepared in Examples 1-17

[0200] Example 18

[0201] 68Ga-labeled nuclide-labeled probes[ 68 Synthesis of Ga-DNB8P1

[0202] The labeling method is as follows: 50 μg of compound DNB8P1 prepared in Example 1 was dissolved in 500 μL of 0.3 M acetate-sodium acetate buffer solution, and then added to a solution containing approximately 185–555 MBq of eluted germanium-gallium generator (2 mL). 68 The GaCl3 hydrochloric acid solution was placed in a vial, incubated at 95°C with shaking for 20 min, then cooled to room temperature, diluted with physiological saline or water for injection, and filtered through a sterile membrane to obtain the radionuclide. 68 Ga-marked [ 68 Ga]Ga-DNB8P1 solution.

[0203] 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: 0–10 min, mobile phase B volume fraction increased from 5% to 50%; 10–15 min, mobile phase B volume fraction 50%; 15–17 min, mobile phase B volume fraction decreased from 50% to 5%; 17–30 min, mobile phase B volume fraction 5%; mobile phase flow rate was 1 mL / min; radioactivity was detected by a radiodetector.

[0204] Examples 19-31

[0205] according to[ 68 The Ga-DNB8P1 was prepared by replacing the PSMA-targeting small molecule compound DNB8P1 with DNA8P1, DNC8P1, DND8P1, DCA8P1, DCB8P1, DNA6P1, DNB6P1, DNB8P2, DCB8P2, DGNB8P1, DSNB8P1, DGNE8P1, and DGNF8P1, respectively, to obtain the radionuclide-labeled probes. 68 Ga]Ga-DNA8P1, [ 68 Ga]Ga-DNC8P1, [ 68 Ga]Ga-DND8P1, [ 68 Ga]Ga-DCA8P1, [ 68 Ga]Ga-DCB8P1, [ 68 Ga]Ga-DNA6P1, [ 68 Ga]Ga-DNB6P1, [ 68 Ga]Ga-DNB8P2, [ 68Ga]Ga-DCB8P2, [ 68 Ga]Ga-DGNB8P1, [ 68 Ga]Ga-DSNB8P1, [ 68 Ga]Ga-DGNE8P1 and [ 68 Ga]Ga-DGNF8P1. The radio-HPLC results of the various nuclide-labeled probes prepared in Examples 18-31 are shown in Figures 26-32. It can be seen that the probes synthesized in this application... 68 The radiochemical purity of Ga-labeled probes is greater than 95%.

[0206] Example 32

[0207] 18 F-labeled nuclide-labeled probes [ 18 Synthesis of F]AlF-NNB8P1

[0208] 50 μg of the labeled precursor NNB8P1 dissolved in 20 μL of dimethyl sulfoxide was mixed with 100 μL of 0.1 M acetate-sodium acetate buffer (pH=4), 200 μL of acetonitrile, and 3 μL of 2 mM AlCl3 solution (prepared from 0.1 M acetate-sodium acetate buffer at pH=4), and then 100 μL of approximately 740 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 The F]AlF-NNB8P1 solution was analyzed and identified by radio-HPLC according to the conditions of Example 18.

[0209] Examples 33-34

[0210] according to[ 18 The method for preparing F]AlF-NNB8P1 involves replacing NNB8P1 with NNA8P1 and NCB8P1, respectively, to obtain probes. 18 F]AlF-NNA8P1 and [ 18 F]AlF-NCB8P1.

[0211] The probes prepared in Examples 32-34 were sampled and analyzed by HPLC. The HPLC results are shown in Figure 33. It can be seen that the probes prepared in this application... 18 The radiochemical purity of the F-labeled nuclide-labeled probes was greater than 95%.

[0212] Example 35

[0213] 177 Lu-labeled nuclide-labeled probes[ 177 Synthesis of Lu]Lu-DGNB8P1

[0214] 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 DGNB8P1. 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-DGNB8P1 solution was analyzed and identified by radio-HPLC according to the conditions of Example 18.

[0215] Examples 36-38

[0216] according to[ 177 The Lu-DGNB8P1 probe was prepared by replacing DGNB8P1 with DSNB8P1, DGNE8P1, and DGNF8P1, respectively, to obtain probes. 177 Lu]Lu-DSNB8P1、[ 177 Lu]Lu-DGNE8P1 and [ 177 Lu]Lu-DGNF8P1.

[0217] The radionuclide-labeled probes were sampled and identified by radio-HPLC according to the conditions of Example 18. The radio-HPLC results of the probes prepared in Examples 35-38 are shown in Figure 34. It can be seen that the probes prepared in this application... 177 The radiochemical purity of Lu-labeled nuclide-labeled probes is greater than 95%.

[0218] Example 39

[0219] 44 Sc-labeled nuclide-labeled probes[ 44 Synthesis of Sc]Sc-DGNB8P1

[0220] 50 μg of compound DGNB8P1 prepared in Example 2 was mixed with a radionuclide prepared by an accelerator at approximately 5.55 MBq. 44 Sc- The solution (2.6 mL, adjusted to pH 4 with ammonium acetate) was mixed in a vial and heated at 100 °C for 20 min. The volume was then reduced to approximately 300 μL by nitrogen blowing. After cooling to room temperature, it was diluted with physiological saline or water for injection and filtered through a sterile membrane to obtain the radionuclide. 44 Sc-marked [ 44 The Sc]Sc-DGNB8P1 solution was subjected to radio-HPLC purity analysis and identification according to the conditions of Example 18.

[0221] Example 40

[0222] according to[ 44 The probe [Sc]Sc-DGNB8P1 was prepared by replacing DGNB8P1 with DNB8P1. 44 Sc]Sc-DNB8P1.

[0223] The radionuclide-labeled probe samples were analyzed and identified by radio-HPLC according to the conditions of Example 18.

[0224] Figure 35 shows the probe. 44 Sc]Sc-DNB8P1 and [ 44 The HPLC chromatogram of Sc]Sc-DGNB8P1 shows that the material prepared in this application... 44 The radiochemical purity of the Sc-labeled nuclide-labeled probes is greater than 95%.

[0225] Example 41

[0226] 131 I-labeled nuclide-labeled probe 131 Synthesis of IPB-NB8P1

[0227] Dissolve 500 μg of NB8P1 in 200 μL of anhydrous DMSO. 131 After mixing IPBA-NHS with NB8P1 solution, 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-high performance liquid chromatography (Radio-HPLC), and its radiochemical purity was further analyzed.

[0228] The radionuclide-labeled probe was identified by radio-HPLC purity analysis. 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, mobile phase B volume fraction increased from 5% to 95%; 20–22 min: mobile phase B volume fraction 95%; 22–25 min: mobile phase B volume fraction decreased from 95% to 5%; mobile phase flow rate was 1 mL / min. The radioactive signal was detected by a radiodetector. Figure 36 shows the probe. 131 The HPLC chromatogram of IPB-NB8P1 shows that it is radioactive. 131 IPB-NB8P1 has a radiochemical purity greater than 95%.

[0229] Test Example 1

[0230] Evaluation of the stability and lipid-water distribution properties of radionuclide-labeled probes

[0231] 1. Stability test of injection solution: The radionuclide-labeled probe was placed in physiological saline or in vitro serum at room temperature for different time periods, and samples were analyzed by HPLC. 68 Ga、 18 F, 177 Lu and 44 The HPLC analysis conditions for the Sc nuclide-labeled probes were the same as those for the corresponding probes in Examples 18, 32, 35, and 39.

[0232] Figures 37-51 show the probes [ 68 Ga]Ga-DNA8P1, [ 68 Ga]Ga-DNB8P1, [ 68 Ga]Ga-DNA6P1, [ 68 Ga]Ga-DNB6P1, [ 68 Ga]Ga-DNC8P1, [ 68 Ga]Ga-DND8P1, [ 68 Ga]Ga-DCA8P1, [ 68 Ga]Ga-DCB8P1, [ 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1, [ 18 F]AlF-NCB8P1, [ 177 Lu]Lu-DGNB8P1、[ 177 Lu]Lu-DGNF8P1、[ 44 Sc]Sc-DNB8P1 and [ 44The HPLC chromatograms of the stability of Sc-DGNB8P1 in physiological saline or in vitro serum show that, at the tested time points, the stability of each... 68 Ga、 18 F, 177 Lu and 44 The radiochemical purity of the Sc-labeled nuclide-labeled probes is greater than 95%, indicating that they are stable in the specified medium.

[0233] 2. Determination of the lipid-water distribution coefficient (logP):

[0234] 100 μL of the radionuclide-labeled probe solution (3.7 MBq / mL) 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. After vortexing for 3 min, the tube was centrifuged at 10000 r / min 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. The formula for calculating logP is shown in equation (1):

[0235] P = I 有机相 / I 水相 Equation (1);

[0236] 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. The lipid-water distribution coefficients of the labeled probes for each radiolabeled element are shown in Table 18.

[0237] Table 18 Lipid-water distribution coefficient of radionuclide labeled probes

[0238] As can be seen from Table 18, 68 Ga、 18 F, 117 Lu and 44 Sc-labeled nuclide-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.

[0239] Test Example 2

[0240] Assay for radionuclide-labeled probe receptor affinity and cellular uptake

[0241] 1. Receptor affinity test of probe in PSMA-positive PC3-PIP cells

[0242] 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 the 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 the other plate. 100 μL of different concentrations (0.156-400 nM) of [a specific ingredient] was added to each plate. 68 Ga-labeled radioligands were applied in triplicate for each concentration. 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.

[0243] Figures 52-55 show the probes [ 68 Ga]Ga-DNA8P1 and [ 68 Ga]Ga-DNB8P1, [ 68 Ga]Ga-DNC8P1 and [ 68 Ga]Ga-DND8P1, [ 68 Ga]Ga-DNA6P1 and [ 68 Ga]Ga-DNB6P1, [ 68 Ga]Ga-DCA8P1 and [ 68 Ga]Ga-DCB8P1 in PSMA-positive PC3-PIP cells K d Test curves. Figure 56 shows [ 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 F]AlF-NCB8P1 in PSMA-positive PC3-PIP cells K d Test curves. KA of each of the above nuclide-labeled probes. d The values ​​are shown in Table 19. All the probes tested have good affinity for PSMA.

[0244] Table 19 K of the radionuclide-labeled probes d value

[0245] 2. Cellular uptake and inhibition assay of radionuclide-labeled probes

[0246] PC3-PIP cells with high PSMA expression and PC3 cells with negative expression were seeded in 24-well plates containing culture medium (containing fetal bovine serum and antibiotics) and cultured for 48 h (cell count using a cell counting chamber, approximately 200,000 cells / well). At the start of the uptake experiment, the original culture medium was aspirated, and the cells were washed with PBS (500 μL, pH 7.4, containing 0.2% BSA). An equal volume of the target radionuclide-labeled probe, diluted in culture medium without fetal bovine serum and antibiotics, was added to each well, and 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 sodium hydroxide solution was added to each well to lyse the cells. After 5 min, the lysed cells were transferred to disposable centrifuge tubes to measure the radioactivity count. The cell uptake percentage was obtained by dividing the count by the total amount of radioactivity added.

[0247] To investigate the PSMA targeting specificity of the radionuclide-labeled probe, an inhibition group was also set up. Specifically, an appropriate amount of the inhibitor PSMA-617 was added to each well of cells before the addition of the radionuclide-labeled probe, and the cells were co-incubated at 37°C for a certain period of time. 18 F-labeled probes were co-incubated for 1 hour; 177 Lu probe was incubated for 2 h and 4 h. After incubation, the radioactive culture medium was aspirated, and sodium hydroxide solution (500 μL, 1 M) was added to each well to lyse the cells. After 5 min, the lysed cells were placed in a disposable centrifuge tube to measure the radioactivity count. The cell uptake percentage was obtained by dividing the count by the total amount of radioactivity added.

[0248] Figure 57 shows the radionuclide-labeled probe. 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 The cell uptake and inhibition results of [F]AlF-NCB8P1 in PSMA-positive PC3-PIP cells at different time points (60 min) were compared with the cell uptake results of the probe in PSMA-negative PC3 cells at 60 min. It can be seen that, taking the results at 60 min as an example, all radionuclide-labeled probes showed significant uptake in PSMA-positive cells, while uptake was significantly reduced in negative cells. The uptake of the probes in PSMA-positive cells was inhibited by PSMA-617, indicating that the [F]AlF-NCB8P1 in this application... 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 The F]AlF-NCB8P1 probe is specific for targeting the PSMA protein.

[0249] Figure 58 shows the radionuclide-labeled probe. 177 Lu]Lu-DGNB8P1 and [ 177The cell uptake and inhibition results of Lu-DSNB8P1 in PSMA-positive PC3-PIP cells at different time points (2h and 4h) were compared with the cell uptake results of radionuclide-labeled probes in PC3-negative cells at 2h and 4h. It can be seen that, taking the results at the 4h time point as an example, [ 177 Lu]Lu-DGNB8P1 and [ 177 Lu-DSNB8P1 showed significant uptake in PSMA-positive cells, while uptake was significantly reduced in PSMA-negative cells. The uptake of the radionuclide-labeled probe in PSMA-positive cells was inhibited by PSMA-617, indicating that the […] provided in this application… 177 Lu]Lu-DGNB8P1 and [ 177 The Lu]Lu-DSNB8P1 radionuclide-labeled probe is specific for targeting the PSMA protein.

[0250] Test Example 3

[0251] PET Imaging Experiment with Radionuclide-Labeled Probes

[0252] The radiochemical purity prepared in the examples is greater than 95%. 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 PC3-PIP model 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 probe were calculated.

[0253] Figures 59-72 show the probes [ 68 Ga]Ga-DNA8P1, [ 68 Ga]Ga-DNB8P1, [ 68 Ga]Ga-DNC8P1, [ 68 Ga]Ga-DND8P1, [ 68 Ga]Ga-DNA6P1, [ 68 Ga]Ga-DNB6P1, [ 68 Ga]Ga-DCA8P1, [ 68 Ga]Ga-DCB8P1, [ 68 Ga]Ga-DNB8P2, [ 68 Ga]Ga-DCB8P2, [ 68 Ga]Ga-DGNB8P1, [ 68 Ga]Ga-DSNB8P1, [ 68 Ga]Ga-DGNE8P1 and [ 68PET imaging of Ga-DGNF8P1 (A) and uptake values ​​and target / non-target ratios in major tissues (B). It is evident that the radionuclide-labeled probe exhibits high uptake at the tumor site. A slight radioactive signal is observed in the kidney at early time points, but this signal rapidly diminishes over time. The background values ​​of the radionuclide-labeled probe in other non-target organs are very low, and the absolute uptake and target / non-target ratio of the tumor significantly increase over time.

[0254] Figure 73 shows the competing binders [DNB8P1, ...] with different contents. nat In the presence of Ga-DNB8P1 (non-radioactive gallium), PSMA-617, nuclide-labeled probes [ 68 Comparison of PET imaging results (A) and uptake values ​​(B) of Ga-DNB8P1 in major tissues. It can be seen that when 80 μg of Ga-DNB8P1 is present in the injection solution... nat When using Ga-DNB8P1 or PSMA-617, the probe [ 68 The uptake of Ga-DNB8P1 at the tumor site was significantly reduced. However, the presence of a certain dose of DNB8P1 did not affect the probe's uptake. 68 Ga]Ga-DNB8P1 significantly affects PET imaging quality and tumor uptake.

[0255] Figure 74 shows the radionuclide-labeled probe. 68 Ga]Ga-DNB8P1 and classic probes[ 68 A comparison of PET imaging (A), uptake values ​​(B), and target / non-target ratios (C) of Ga-PSMA-617 (self-made, prepared according to the procedure in Example 18) is shown. It can be seen that compared to [ 68 Ga]Ga-PSMA-617, [ 68 The Ga-DNB8P1 significantly improves PET imaging quality, absolute tumor uptake, and target / non-target ratio.

[0256] Figures 75-77 show the nuclide-labeled probes. 18 F]AlF-NNA8P1, [ 18 F]AlF-NNB8P1 and [ 18 PET imaging (A) and uptake values ​​of major tissues for AlF-NCB8P1 (B) are shown in Figures 75-77. The 1-hour inhibition group used PSMA-617 as an inhibitor. 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, which contributes to achieving better efficacy.

[0257] Take 0.2 mL (0.37 MBq) of the radiochemically pure [prepared in the example] with a purity greater than 95%. 44 Sc]Sc-DGNB8P1 injection solution was administered via tail vein injection to PC3-PIP model mice. MicroPET imaging was performed at different time points, and regions of interest (ROIs) were delineated on the images. The probe distribution values ​​were then calculated. Figure 78 shows the radionuclide-labeled probes. 44 PET imaging of Sc-DGNB8P1 (A), comparison of uptake values ​​in major tissues and target / non-target ratio (B). The radionuclide-labeled probe showed significant uptake at the tumor site as early as 15 minutes after injection. The probe is primarily metabolized via the kidneys. Over time, the radionuclide-labeled probe was significantly metabolized in non-target organs, while uptake in the tumor remained stable, demonstrating an improved target / non-target ratio.

[0258] Test Example 4

[0259] SPECT imaging experiment with radionuclide-labeled probes

[0260] Tumor-bearing mice were injected with 37 MBq via the tail vein. 177 Lu-labeled nuclide-labeled probes ([ 177 Lu]Lu-DGNB8P1、[ 177 Lu]Lu-DSNB8P1、[ 177 Lu]Lu-DGNF8P1 and [ 177 Lu-PSMA-617 (one of the gold standards, self-made, prepared according to the procedure in Example 35) was used for static SPECT imaging at different time points after injection. After imaging, the images were reconstructed and the regions of interest (ROIs) in the mouse images were delineated to obtain the radioactivity count values. The target / non-target ratio of the nuclide-labeled probe distribution was calculated.

[0261] Nucleotide-labeled probes[ 177 Lu]Lu-DGNB8P1、[ 177 Lu]Lu-DSNB8P1、[ 177 The SPECT imaging results of Lu-DGNF8P1 are shown in Figures 79-81. [Control group] 177 Figure 82 shows the SPECT imaging results of Lu-PSMA-617. It can be seen that within the monitoring time range, [ 177 Lu]Lu-DGNB8P1、[ 177 Lu]Lu-DSNB8P1、[ 177 The enrichment of the Lu-DGNF8P1 radionuclide-labeled probe at the tumor site was higher than that in the control group. 177The high contrast and clear lesion outline of the Lu-PSMA-617 radionuclide probe provided in this application demonstrate that it has good tumor uptake effect and has the potential for PSMA radionuclide targeted therapy.

[0262] Figure 83 shows the nuclide-labeled probe in the presence of 20 μg of the competitive binder DGNB8P1. 177 SPECT imaging results and target / non-target ratio of Lu-DGNB8P1. It can be seen that when 20 μg of DGNB8P1 is present in the injection solution, the radionuclide-labeled probe […]. 177 The SPECT imaging quality of the Lu]Lu-DGNB8P1 was not affected, and it still has good tumor uptake and a high tumor / kidney ratio.

[0263] Test Example 5

[0264] Biodistribution experiment of radionuclide labeled probes

[0265] Radionuclide-labeled probes prepared by tail vein injection in tumor-bearing mice ([) 18 F]AlF-NNA8P1, [ 18 F]AlF-NCB8P1, [ 177 Lu]Lu-DGNB8P1 or [ 44 [Sc]Sc-DGNB8P1) 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).

[0266] Figures 84-85 show the radionuclide-labeled probes. 18 F]AlF-NNA8P1 and [ 18 Biodistribution results of [F]AlF-NCB8P1 in tumor-bearing mice. It is shown that both radionuclide-labeled probes exhibit high uptake at the tumor site, slight retention in the kidneys, and rapid clearance. In normal tissues, both radionuclide-labeled probes exhibit low background signal characteristics, which is highly advantageous for PSMA-targeted PET imaging.

[0267] Figure 86 shows the radionuclide-labeled probe. 177 The biodistribution of Lu-DGNB8P1 in tumor-bearing mice was as follows. It was found that 4 hours after injection, […]. 177 The tumor uptake of Lu-DGNB8P1 was greater than 30% ID / g. 24 hours after injection, the tumor uptake value remained above 20% ID / g and maintained a high level for 96 hours. Therefore, the […] provided in this application… 177Lu]Lu-DGNB8P1 exhibits excellent tumor uptake and prolonged retention time, making it highly advantageous for PSMA-targeted radionuclide therapy.

[0268] Figure 87 shows the radionuclide-labeled probe. 44 Uptake values ​​of Sc-DGNB8P1 in various organs at different time points in tumor-bearing mice. The radionuclide-labeled probe rapidly accumulates at the tumor site and maintains a long retention time, while uptake values ​​in most other organs are low and decay over time. The probe is mainly metabolized via the kidneys. Injection of 100 μg of the competitive binder PSMA-617 in the inhibition group significantly inhibited […]. 44 Tumor uptake of Sc]Sc-DGNB8P1.

[0269] Test Example 6

[0270] PSMA-targeted radionuclide therapy experiment using radionuclide-labeled probes

[0271] Tumor-bearing mice were divided into experimental groups ([ 177 Lu]Lu-DGNB8P1 group), [ 177 Lu]Lu-PSMA-617 control group and saline group. Each mouse in the experimental group was injected with a different dose of [Lu-PSMA-617 via tail vein] 177 Lu]Lu-DGNB8P1 (37MBq, 18.5MBq and 9.25MBq respectively); [ 177 The Lu-PSMA-617 control group received 18.5 MBq via tail vein injection per mouse. 177 Lu]Lu-PSMA-617; In the saline group, each mouse received the same volume of saline via the tail vein, and tumor size and body weight were monitored daily. The treatment results are shown in Figure 88. Compared to the saline group, the tumor volume in the experimental group decreased significantly over time, and the […] of each dose… 177 Lu-DGNB8P1 both showed significant therapeutic effects on tumors. 177 In the Lu-PSMA-617 control group, tumor growth was somewhat suppressed in the early stages of treatment, but a significant rebound occurred in the later stages. It is worth noting that the 18.5 MBq and 9.25 MBq doses... 177 Lu-DGNB8P1 showed significantly better tumor-suppressive effects than the 18.5 MBq dose. 177 Lu]Lu-PSMA-617, describing the […] provided in this application 177 The Lu-DGNB8P1 probe shows promising potential for therapeutic applications.

[0272] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A PSMA-targeting small molecule compound having the structure shown in Formula I: In Equation I, n is an integer from 0 to 5; R1 includes any of the following structures: R2 may or may not exist, and when R2 exists, it includes any of the following structures: In R2, a, b, and t are independent integers from 0 to 5; R3 is the group to be labeled, including any one of the following chelate structures:

2. The PSMA-targeting small molecule compound according to claim 1, characterized in that, The PSMA-targeting small molecule compound has any one of the structures shown in Formula I-1 to Formula I-10: Wherein, n is independently 1 or 2, Q is independently N or CH, and R2 is independently absent or a different linking group.

3. The method for preparing the PSMA-targeted small molecule compound according to claim 1 or 2, comprising method 1, method 2 or method 3; Method 1 includes the following steps: The PSMA-targeting small molecule compound is obtained by using an active compound R3 with an activated ester or anhydride group to undergo a substitution reaction with compound 1 to generate an amide bond. The R3 active compound with an activated ester group or an anhydride group includes any one of the following structures: Method 2 includes the following steps: reacting an R3 active compound with an isothiocyanate group to form a thiourea bond via a substitution reaction, thereby obtaining the PSMA-targeting small molecule compound; the R3 active compound with the isothiocyanate group includes any one of the following structures: Method 3 includes the following steps: using an R3 chelating reagent with a carboxyl group to perform an amidation reaction with compound 1 under condensation reagent conditions to generate an amide bond, followed by removal of the protecting group to obtain the PSMA-targeting small molecule compound; the R3 chelating reagent with a carboxyl group includes any one of the following structures, wherein A is a carboxyl protecting group: R1, R2, and n in compound 1 are the same as R1, R2, and n in formula I.

4. The preparation method according to claim 3, characterized in that, In Method 1, the molar ratio of compound 1 to the active compound R3 with an activated ester group or an anhydride group is 1:1 to 10.

5. The preparation method according to claim 3, characterized in that, In Method 1, the solvent used in the substitution reaction includes one or more of organic solvents, water, and phosphate buffer solutions; the organic solvent includes one or more of N-methylpyrrolidone, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide.

6. The preparation method according to any one of claims 3 to 5, characterized in that, In Method 1, the substitution reaction is carried out in the presence of a basic reagent.

7. The preparation method according to claim 6, characterized in that, In Method 1, the temperature of the substitution reaction is 15–60°C and the time is 0.5–48 h.

8. The preparation method according to claim 3, characterized in that, In Method 2, the molar ratio of compound 1 to the isothiocyanate-based R3 active compound is 1:1 to 10.

9. The preparation method according to claim 3, characterized in that, In Method 2, the solvent used in the substitution reaction includes one or more of organic solvents, water, and phosphate buffer solutions; the organic solvent includes one or more of N-methylpyrrolidone, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide.

10. The preparation method according to claim 3, 7, 8 or 9, characterized in that, In Method 2, the substitution reaction is carried out in the presence of a basic reagent.

11. The preparation method according to claim 10, characterized in that, In Method 2, the temperature of the substitution reaction is 15–60°C and the time is 0.5–48 h.

12. The preparation method according to claim 3, characterized in that, In method 3, the molar ratio of compound 1 to the R3 chelating reagent with a carboxyl group is 1:1 to 10.

13. The preparation method according to claim 3, characterized in that, In Method 3, the amidation reaction is carried out in the presence of a condensation reagent, an organic amine, and a solvent.

14. The preparation method according to claim 13, characterized in that, The condensing agent comprises one or more of the following: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate, dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, carbodiimide hydrochloride, oxalyl chloride, thionyl chloride, phosphorus oxychloride, carbon disulfide, and activated succinimide ester. The molar ratio of compound 1 to the condensing agent is 1:0.5 to 10.

15. The preparation method according to claim 13, characterized in that, The organic amine includes one or both of N,N-diisopropylethylamine and triethylamine; The molar ratio of compound 1 to organic amine is 1:0.5 to 10.

16. The preparation method according to claim 13, characterized in that, The solvent includes one or more of organic solvents, water, and phosphate buffer solutions; the organic solvent includes one or more of N-methylpyrrolidone, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide.

17. The preparation method according to any one of claims 3 and 11-16, characterized in that, In Method 3, the amidation reaction takes 0.5 to 48 hours.

18. The preparation method according to claim 3, characterized in that, In Method 3, the deprotecting reagent used to remove the protecting group includes strong acids or strong bases.

19. The preparation method according to claim 3 or 18, characterized in that, In method 3, the temperature for removing the protecting group is 0 to 100°C, and the time is 1 min to 24 h.

20. A radionuclide-labeled probe, characterized in that, It has the structure shown in Formula II or Formula III: Wherein, R1, R2, and n are defined in the same way as R1, R2, and n in Formula I; R4 is a radionuclide labeling group, obtained by coordinating the R3 labeling group in the PSMA-targeted small molecule compound of claim 1 or 2 with a radionuclide; R5 includes any one of the following structures: Where y and z are independent integers from 0 to 5; X is carbon, hydrogen, or nitrogen; R6 is... 18 F, 123 I, 124 I, 125 I, 131 I or 211 At.

21. The radionuclide-labeled probe according to claim 20, 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, 86 Y、 90 Y、 97 Ru、 99m Tc, 105 Rh、 101m Rh、 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 At least one of Ac.

22. The radionuclide-labeled probe according to claim 20 or 21, characterized in that, Formula III has any one of the structures shown in Formula III-1 to Formula III-6: Where Q is independently N or CH, and R2 is independently absent or a different linking group.

23. The method for preparing the radionuclide-labeled probe according to any one of claims 20 to 22, The method for preparing the nuclide-labeled probe having the structure shown in Formula II includes the following steps: The PSMA-targeting small molecule compound of claim 1 or 2 is coordinated with a nuclide to obtain a nuclide-labeled probe having the structure shown in Formula II. The method for preparing the nuclide-labeled probe having the structure shown in Formula III includes the following steps: subjecting compound 1 to an amidation reaction with an active compound to obtain a nuclide-labeled probe having the structure shown in Formula III; the active compound includes compound 2 or compound 3; 24. The preparation method according to claim 23, characterized in that, The nuclide-labeled probe with the structure shown in Formula II is prepared by wet labeling or freeze-drying labeling.

25. The preparation method according to claim 24, characterized in that, The preparation of a nuclide-labeled probe with the structure shown in Formula II using a wet labeling method includes the following steps: mixing a solution containing a precursor compound with a nuclide solution, performing a coordination reaction, and then diluting to obtain a nuclide-labeled probe solution containing the structure shown in Formula II.

26. The preparation method according to claim 25, characterized in that, The ratio of the mass of the PSMA-targeting compound to the radioactivity of the radionuclide in the radionuclide solution is 20–400 μg: 1 kBq–1000 GBq.

27. The preparation method according to any one of claims 24 to 26, characterized in that, The coordination reaction is carried out at a temperature of 25–100°C for a time of 10–60 min.

28. The preparation method according to claim 24, characterized in that, The preparation of the radionuclide-labeled probe with the structure shown in Formula II using the lyophilization labeling method includes the following steps: freezing-drying a solution containing a PSMA-targeting small molecule compound and then sealing it to obtain a lyophilized kit; adding a solvent to the lyophilized kit to dissolve the compound, then adding a radionuclide solution to perform a coordination reaction and diluting the solution to obtain a radionuclide-labeled probe solution with the structure shown in Formula II.

29. The preparation method according to claim 23, characterized in that, The molar ratio of compound 1 to the active compound is 1:0.0000001 to 10.

30. The preparation method according to claim 23, characterized in that, The organic solvent used in the amidation reaction includes one or more of organic solvents, water, and phosphate buffer solutions; the organic solvent includes one or more of N-methylpyrrolidone, dichloromethane, dimethyl sulfoxide, and N,N-dimethylformamide.

31. A pharmaceutically acceptable form of existence, characterized in that, The pharmaceutically acceptable forms include the PSMA-targeting small molecule compounds of any one of claims 1 to 2 and / or the radionuclide-labeled probes of any one of claims 20 to 22, wherein the pharmaceutically acceptable forms include one or more of salts, stereoisomers, racemates, hydrates and solvates.

32. A pharmaceutical composition, characterized in that, It includes an active ingredient and pharmaceutically acceptable excipients; the active ingredient includes one or more of the following: the PSMA-targeting small molecule compound according to any one of claims 1 to 2, the radionuclide-labeled probe according to any one of claims 20 to 22, and the pharmaceutically acceptable form according to claim 31.

33. A lyophilized reagent kit, characterized in that, It includes a precursor compound and a pharmaceutically acceptable excipient, wherein the precursor compound comprises one or more of the pharmaceutically acceptable forms of the PSMA-targeting small molecule compound of any one of claims 1 to 2 and the PSMA-targeting small molecule compound of claim 31.

34. The use of the PSMA-targeting small molecule compound of claim 1, the radionuclide-labeled probe of any one of claims 20 to 22, the pharmaceutically acceptable form of the radionuclide-labeled probe of claim 31, the pharmaceutical composition of claim 32, or the lyophilized kit of claim 33 in the preparation of therapeutic agents, therapeutic drugs, diagnostic agents, or diagnostic drugs for PSMA protein-mediated diseases.

35. The application according to claim 34, characterized in that, The diseases mediated by the PSMA protein include tumors and / or their metastases; The tumors 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.

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