Dansulfonamide-modified psma targeting compounds and preparation methods and applications thereof

Abstract

This invention provides a dansulphamide-modified PSMA-targeting compound with the structure shown in Formula A or Formula I; wherein R1 is an amino acid analog structure; R2 is a prostate-specific membrane antigen-targeting compound structure; R3 is a radionuclide chelating group; L is -(X)n-(CH2)m-(Y)q-, where X and Y are independently selected from lysine, glutamic acid, or derivatives containing lysine or glutamic acid, n is an integer from 0 to 10, m is an integer from 0 to 20, and q is an integer from 0 to 6, wherein each CH2 can be individually replaced by -OCH2-, -NH(CO)-, or -(CO)-NH-. This invention also provides a radionuclide label based on the structure of the compound. The compound and the radionuclide label exhibit high tumor uptake and retention times, making them suitable for radionuclide therapy and imaging of tumors with high PSMA expression.

Description

Technical Field

[0001] This invention relates to the field of cancer diagnosis and treatment technology, including but not limited to prostate cancer, specifically to a prostate-specific membrane antigen (PSMA) targeting compound with high tumor uptake and long retention properties, and a disease diagnosis and treatment probe labeled with a radionuclide. Prior Technology

[0002] Prostate-specific membrane antigen (PSMA) is expressed in only small amounts in normal tissues, but it is overexpressed in approximately 90% of prostate cancer lesions (100-1000 times higher than the reference value). Furthermore, the expression level of PSMA in lesions is closely related to tumor invasiveness and malignancy. Therefore, accurately evaluating PSMA expression in lesions is of great significance for the diagnosis, staging, efficacy evaluation, progression, and prognostic assessment of prostate cancer. Currently, PSMA imaging has become an indispensable imaging tool for the diagnosis and treatment of prostate cancer. In addition, PSMA can also be highly expressed in other tumors, such as lung cancer, liver cancer, gastric cancer, breast cancer, colorectal cancer, renal cell carcinoma, bladder cancer, brain tumors, thyroid cancer, and adenoid cystic carcinoma. Therefore, PSMA imaging also has great potential for application in the diagnosis and treatment of tumors other than prostate cancer.

[0003] Radioligand therapy (RLT) targeting PSMA is an important treatment for metastatic castration-resistant prostate cancer (mCRPC), and 177Lu-PSMA-617 is the most commonly used radionuclide therapy drug for RLT. In September 2021, Novartis announced the results of the Phase III VISION clinical trial of 177Lu-PSMA-617 in treating PSMA-positive mCRPC patients. The study showed that, compared with the best standard of care (SOC), 177Lu-PSMA-617 combined with SOC significantly improved the median radiological progression-free survival (8.7 months vs. 3.4 months) and median overall survival (15.3 months vs. 11.3 months) in mCRPC patients. On March 23, 2022, Pluvicto (lutetiumLu-177 vipivotide tetraxetan, 177Lu-PSMA-617) was approved by the FDA for the treatment of adult patients with PSMA-positive, previously treated androgen receptor inhibitors and taxane-based chemotherapy for mCRPC. However, it is noteworthy that despite the progress made by 177Lu-PSMA-617 in treating mCRPC patients, a significant proportion of prostate cancer patients still do not respond well to treatment. Furthermore, in terms of treatment, because PSMA-617 is a small molecule peptide, it is rapidly metabolized and eluted in vivo, resulting in a low effective dose at the tumor site and a short retention time. This necessitates the use of high-dose or more frequent dosing to meet treatment needs, increasing the likelihood of adverse reactions and creating a heavy economic and social burden. Therefore, optimizing the pharmacokinetics of molecular probes, improving the utilization rate of radionuclides such as 177Lu, and reducing the dose of radionuclides used are important directions for the development of integrated radionuclide diagnosis and therapy. Therefore, this study proposes PSMA-targeting compounds modified with dansulphamide compounds, which greatly improves the uptake and retention of PSMA probes at tumor sites, and has significant implications for the clinical application of PSMA-based integrated diagnosis and treatment. Summary of the Invention

[0004] Against this background, the primary objective of this invention is to develop a novel prostate-specific membrane antigen-targeting compound with dansulphamide as its core, which can enhance tumor uptake and prolong tumor retention. This invention overcomes the shortcomings of small-molecule PSMA-targeting probes, such as excessively rapid metabolism and short target organ retention time, thereby improving the efficacy of targeted PSMA radionuclide therapy and imaging, and has the potential for widespread clinical application.

[0005] Another object of the present invention is to provide a series of radiolabeled prostate-specific membrane antigen-targeting complexes with high tumor uptake and long retention properties.

[0006] Another object of the present invention is to provide a method for preparing the radiolabeled prostate-specific membrane antigen-targeting complex with high tumor uptake and long retention.

[0007] Another object of the present invention is to provide the application of the aforementioned complex in targeted radionuclide imaging and treatment of prostate cancer.

[0008] The technical solutions for achieving the primary objective of this invention include the following two aspects: ligand synthesis and radiolabeling.

[0009] In a first aspect, the present invention provides a dansulphamide-modified PSMA-targeting compound with high tumor uptake and long retention properties. Its molecular structure is formed by linking a dansulphamide-like compound and a PSMA-targeting compound together using a linking group L, as shown in formula A below: ; Alternatively, its molecular structure is formed by linking dansylamine compounds, PSMA-targeting compounds, and radionuclide chelating groups together with a linking group L, as shown in Formula I below; ; in: L is -(X)n-(CH2)m-(Y)q-, where n is an integer from 0 to 10 (preferably an integer from 0 to 5), X and Y are independently selected from lysine, glutamic acid, or derivatives containing lysine or glutamic acid, m is an integer from 0 to 20 (preferably an integer from 0 to 10), and q is an integer from 0 to 6 (preferably an integer from 0 to 3), wherein each CH2 can be individually replaced by -OCH2-, -NH(CO)-, or -(CO)-NH-; R1 refers to amino acid analogs, including L-amino acids and D-amino acids and their derivatives. R4 includes, but is not limited to: (CH2)r-aryl, (CH2)r-heteroaryl, (CH2)r-COOH, (CH2)r-NH2, (CH2)r-OH, (CH2)r-SH, (CH2)r-naphthylmethyl, (CH2)r-indolyl, (CH2)r-benzothiazolyl, where r is an integer from 0 to 10 (e.g., it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0010] In the preferred embodiment, R1 can specifically have the following structure: , , , , , , , , , .

[0011] R2 is derived from a prostate-specific membrane antigen-targeting compound, as shown in Formula II: in: a is an integer from 0 to 20 (preferably an integer from 0 to 10). The X1 structures at different positions may be the same or different, and are independently selected from O, N, S, or P; Y1 represents O, S, and NH; Z represents O, S, and NH; Q is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkylaryl, and cycloalkyl, preferably from substituted or unsubstituted C5-C14 aryl, substituted or unsubstituted C5-C14-alkylaryl, or substituted or unsubstituted C5-C14 cycloalkyl; W is selected from -(CH2)b-aryl or -(CH2)b-heteroaryl, where b is an integer from 0 to 10 (specifically, it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0012] R2 is derived from a prostate-specific membrane antigen-targeting compound, preferably but not limited to the following structures: , , , , , , .

[0013] R3 is a radionuclide chelating group, including but not limited to the following structures or derived from the following structures:

[0014] In a preferred embodiment of the present invention, the... R2 is R3 is or , That is, the structure of compound A is as shown in formula B: ; The structure of the compound of formula I is shown in formula III or formula III-1 as follows: , , At this time, L is selected from: Lys,‑Lys‑(CO)‑CH2‑(CO)‑NH‑CH2‑(CO),‑Lys‑(CO)‑CH2‑(OCH2CH2)‑O‑CH2(CO)‑NH‑CH2‑(CO),‑Lys‑(CO)‑CH2‑(OCH2CH2)3‑O ‑CH2(CO)‑NH‑CH2‑(CO), ‑(CO)‑CH2‑(CO)‑Lys‑, ‑(CO)‑(OCH2CH2)‑O‑CH2(CO)‑Lys‑ or ‑(CO)‑CH2‑(OCH2CH2)3‑O‑CH2(CO)‑Lys‑.

[0015] In a more preferred embodiment of the present invention, R2 is R3 is or L stands for Lys, meaning the structure of compound A is as shown in formula C; ; The structure of the compound of formula I is shown in formula IV or IV' below: ; ; R1 in formulas B, C, III, III-1, IV, and IV' above represents an amino acid analogue, including L-amino acids and D-amino acids and their derivatives. R4 includes, but is not limited to: (CH2)r-aryl, (CH2)r-heteroaryl, (CH2)r-COOH, (CH2)r-NH2, (CH2)r-OH, (CH2)r-SH, (CH2)r-naphthylmethyl, (CH2)r-indolyl, (CH2)r-benzothiazolyl, where r is an integer from 0 to 10 (e.g., it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0016] In the preferred embodiment, R1 can specifically have the following structure: , , , , , , , , , .

[0017] In a further preferred embodiment of the present invention, the structure of compound A can be any of the following: , , , , , , , , , , , .

[0018] The present invention further provides compounds with any structure shown in formulas A'-1 to A'-12: , , , , , , , , , , .

[0019] R5 is a radionuclide chelating group, selected from any of the following structures: .

[0020] The compound of formula I is any one of the compounds shown in formulas IV-1 to IV-24: , , , , , , , , , , , or, , , , , , , , , , , , , .

[0021] Based on this, the present invention also provides a method for preparing the compound shown in formula IV-1, comprising the following steps: The dansylamine derivative was obtained by reacting 5-(dimethylamino)naphthyl-1-sulfadiazine with the amino group of an amino acid. The exposed carboxyl group of the amino acid was then activated by NHS (N-hydroxybutyrylimide), and further reacted with Nε-Boc-L-lysine or (tert-butoxycarbonyl)-L-lysine by acetylamine condensation. Then, it was reacted with PSMA-617 in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and NHS. The Boc protecting group was then removed by TFA, followed by a reaction with PSMA-617 in the presence of EDC and NHS. The Fmoc protecting group was then removed by piracetam. Finally, it was reacted with DOTA-NHS (hydroxysuccinimide-tetraazacyclododecanetetraacetic acid) to obtain the compound with the structure shown in Formula IV-1.

[0022] The present invention also provides methods for preparing other compounds, which can be specifically prepared by referring to the preparation of the compound shown in Formula IV-1 above, with necessary structural substitutions.

[0023] On the other hand, the present invention further provides a radiolabeled complex, which is a complex obtained by labeling a radionuclide with compounds of formula A'-1 to A'-12 or formula I as ligands.

[0024] The radiolabeled complex can be used as a novel tumor radiodiagnostic probe, that is, as a radionuclide diagnostic probe or a radionuclide therapeutic probe.

[0025] The radionuclides mentioned are preferably any one of 177Lu, 18F, 43Sc, 44Sc, 47Sc, 55Co, 57Co, 68Ga, 67Ga, 62Cu, 64Cu, 67Cu, 89Zr, 86Y, 90Y, 99mTc, 99Tc, 90ln, 111ln, 89Sr, 152Gd, 153Gd, 153Sm, 149Tb, 151Tb, 161Tb, 166Ho, 166Dy, 186Re, 188Re, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 226Th, 227Th, or 211At.

[0026] In a preferred embodiment of the present invention, the structure of the radiolabeled complex is shown in formula V: Where L is -(X)n-(CH2)m-(Y)q-, n is an integer from 0 to 10 (preferably an integer from 0 to 5), X and Y are independently selected from lysine, glutamic acid, or derivatives containing lysine and glutamic acid, m is an integer from 0 to 20 (preferably an integer from 0 to 10), q is an integer from 0 to 6 (preferably an integer from 0 to 3), and each CH2 can be individually replaced by -OCH2-, -NH(CO)-, or -(CO)-NH-; R1 refers to amino acid analogs, including L-amino acids and D-amino acids and their derivatives. R4 includes, but is not limited to: (CH2)r-aryl, (CH2)r-heteroaryl, (CH2)r-COOH, (CH2)r-NH2, (CH2)r-OH, (CH2)r-SH, (CH2)r-naphthylmethyl, (CH2)r-indolyl, (CH2)r-benzothiazolyl, where r is an integer from 0 to 10 (e.g., it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

[0027] In the preferred embodiment, R1 can specifically have the following structure: , , , , , , , , , .

[0028] R2 is derived from a prostate-specific membrane antigen-targeting compound, as shown in Formula II: ; in: The X1 structures at different positions may be the same or different, and are independently selected from O, N, S, or P; Y1 represents O, S, and NH; Z represents O, S, and NH; a is an integer from 0 to 20; Q is selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkylaryl, or substituted or unsubstituted cycloalkyl. W is selected from -(CH2)b-aryl or -(CH2)b-heteroaryl, where b is an integer from 0 to 10 (e.g., it can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

[0029] In the preferred embodiment, R2 can be any of the following structures: , , , , , , M is a radioactive nuclide selected from any one of 177Lu, 18F, 43Sc, 44Sc, 47Sc, 55Co, 57Co, 68Ga, 67Ga, 62Cu, 64Cu, 67Cu, 89Zr, 86Y, 90Y, 99mTc, 99Tc, 90ln, 111ln, 89Sr, 152Gd, 153Gd, 153Sm, 149Tb, 151Tb, 161Tb, 166Ho, 166Dy, 186Re, 188Re, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 226Th, 227Th, or 211At.

[0030] The remaining limitations are as described above and will not be repeated.

[0031] In some preferred embodiments, the compound described in Formula V above can also be replaced with a chelating agent, such as replacing DOTA with NOA, with the other limitations as described above, and will not be repeated.

[0032] The radiolabeled complexes of this invention can be prepared by combining a compound containing a radionuclide with the compound of formula I of this invention using various existing labeling methods; the preferred labeling method of this invention is the following wet method or lyophilization method: The wet process is as follows: Dissolve an appropriate amount of the compound of formula I described in this invention in a buffer solution or deionized water, then add a solution containing a radionuclide, react at room temperature to 100°C for 5 to 30 minutes, then dilute with physiological saline or water for injection, and filter through a sterile filter membrane to generate a radionuclide-labeled complex injection solution. The lyophilization process is as follows: Dissolve an appropriate amount of the compound of formula I described in this invention in a buffer solution or deionized water, dispense it into a lyophilization container, freeze-dry it, and then seal it to form a lyophilized drug box. Excipients, antioxidants, or acid-base regulators can be added to the lyophilized drug box as needed. Add deionized water or buffer solution to the lyophilized drug box to dissolve the compound, then add a solution containing a radionuclide. React at room temperature to 100°C for 5-30 minutes. Dilute with physiological saline or water for injection, and filter through a sterile filter membrane to generate a radionuclide-labeled complex injection solution.

[0033] The present invention also provides the application of radiolabeled complexes of Formula V or other radiolabeled complexes (such as replacing DOTA in Formula V with NOA or other chelating agents) in radionuclide therapy and imaging of mammalian PSMA-overexpressing tumors.

[0034] In a preferred application of the present invention, the complex is prepared as an injection and administered intravenously to human patients or mammals with PSMA-overexpressing tumors.

[0035] This invention provides a dansulphamide-modified prostate-specific membrane antigen-targeting compound, its radionuclide-labeled complex, and provides methods for preparing and labeling this compound. Biological assays show that it exhibits a moderately prolonged circulating half-life, enhanced tumor uptake and accumulation, and retention time. These novel properties are not found in other PSMA imaging agents, making it suitable for radionuclide therapy and imaging of tumors with high PSMA expression. Simple Explanation of the Diagram

[0036] Figure 1 is a mass spectrometry chromatogram of the compound of formula IV-1 of this invention; Figure 2 is a mass spectrometry chromatogram of compound IV-5 of this invention; Figure 3 is a mass spectrometry chromatogram of compound IV-9 of this invention; Figure 4 is a mass spectrometry chromatogram of the compound of formula IV-13A of this invention; Figure 5 is a mass spectrometry chromatogram of compound IV-13 of this invention; Figure 6 shows the 68Ga labeling identification results of the compound of formula IV-1 of this invention; Figure 7 illustrates the saline and serum stability of the 68Ga-form IV-1 compound of this invention; Figure 8 illustrates the stability of the 68Ga-form IV-3 compound of this invention in physiological saline and serum; Figure 9 illustrates the saline and serum stability of the 68Ga-form IV-9 compound of this invention; Figure 10 shows the results of cellular uptake and saturation binding experiments of the 68Ga-form IV-1 compound and 68Ga-PSMA617 of this invention; Figure 11 shows the MicroPET imaging of the 68Ga-form IV-1 compound of the present invention in a PC3-PIP model mouse and the quantitative data of its region of interest; Figure 12 illustrates the region of interest quantification in MicroPET imaging of the 68Ga-form IV-9 compound of this invention in PC3-PIP model mice; Figure 13 illustrates the biodistribution of the 177Lu-form IV-1 compound of the present invention in normal mice; Figure 14 is a SPECT image of a PC3-PIP model mouse after administration of the compound 177Lu-form IV-1 of the present invention; Figure 15 illustrates the tissue uptake results in PC3-PIP model mice after administration of the 177Lu-form IV-1 compound of the present invention; Figure 16 is a PET image of a prostate cancer patient after administration of the 68Ga-form IV-1 compound of the present invention; Figure 17 illustrates the quantification of the region of interest in prostate cancer patients after administration of the 68Ga-form IV-1 compound of the present invention; Figure 18 shows PET imaging results of prostate cancer patients after administration of the 68Ga-form IV-9 compound of the present invention; and Figure 19 illustrates the quantification of the region of interest in prostate cancer patients after administration of the 68Ga-form IV-9 compound of this invention. Implementation

[0037] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0038] Example 1: Preparation of compound IV-1

[0039] The synthesis route is as follows:

[0040] The specific steps are as follows:

[0041] compound Synthesis of [2-1]: L-phenylalanine (1.69 g, 10 mmol) was dissolved in 20 mL of 10% sodium carbonate solution, and 5-(dimethylamino)naphthalene-1-sulfonyl chloride (2.69 g, 10 mmol) was dissolved in 20 mL of 1,4-dioxane. This solution was added dropwise to the L-phenylalanine solution under ice bath conditions, and the reaction was allowed to proceed at room temperature for 4 h. After the reaction was complete, 1 M dilute hydrochloric acid was added to adjust the pH to approximately 4, followed by extraction. The organic phase was then removed by vacuum distillation to remove the solvent, yielding a crude product. This crude product was purified by silica gel column chromatography to obtain a pale yellow solid compound 2-1, with a yield of 81%.

[0042] compound Synthesis of [3-1]: compound [2-1](400 mg, 1 mmol) and NHS(230 mg, 2 mmol) were dissolved in 10 mL of tetrahydrofuran. DCC (dicyclohexylcarbodiimide)(412 mg, 2 mmol) was added dropwise under ice bath conditions, and the reaction was carried out overnight at room temperature. After the reaction was completed, the filtrate was filtered, and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid compound. [3-1], yield 34%.

[0043] compound Synthesis of [4-1]: compound [3-1] (495 mg, 1 mmol) and (tert-butyloxycarbonyl)-L-lysine (246 mg, 1 mmol) were dissolved in 10 mL of DMSO (dimethyl sulfoxide), and then triethylamine (505 mg, 5 mmol) was added. The mixture was reacted overnight at room temperature. After the reaction was completed, the mixture was extracted three times with dilute hydrochloric acid. The organic phase filtrate was collected and the solvent was removed by vacuum distillation to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain a pale yellow solid compound. [4-1], yield 76%.

[0044] compound Synthesis of [5-1]: Compounds were added to 10 mL flasks respectively [4-1] [(]112 mg, 0.2 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (40 mg, 0.2 mmol), N-hydroxybutyric acid (23 mg, 0.2 mmol) and 5 mL of N,N-dimethylmethamide. After reacting for 4 h, N,N-diisopropylethylamine (60 mg, 0.5 mmol) and PSMA-617 (130 mg, 0.2 mmol) were added, and the resulting mixture was stirred and the reaction was monitored by HPLC. The solvent was removed by vacuum distillation to obtain the crude product. The crude product was subjected to reverse-phase column chromatography and freeze-dried to obtain the pure compound. [5-1], yield 59%.

[0045] compound Synthesis of [6-1]: 120 mg of the compound [5-1] Dissolved in 5 mL of 2 M hydrochloric acid-ethyl acetate solution, stirred at room temperature for 4 h. After the reaction was complete, the insoluble residue was filtered off, and washed three times with ethyl acetate to obtain the compound. [6-1] [,] Yield 92% [。]

[0046] Synthesis of compound IV-1: compound [6-1] (5.5 mg, 5 μmol), DOTA-NHS (hydroxysuccinimide-tetraazacyclododecanetetraacetic acid) (8 mg, 2 μmol), and N,N-diisopropylethylamine (3.2 mg, 25 μmol) were sequentially added to 1 ml of N,N-dimethylformamide. The reaction system was stirred at room temperature, and the reaction was monitored by HPLC until completion. The crude product was purified by HPLC and freeze-dried to obtain pure compound IV-1 in 52% yield; the structure is shown in Figure 1.

[0047] Example 2. Preparation of compound IV-3

[0048] The synthesis route in this embodiment is as follows:

[0049] This embodiment refers to Example 1, but replaces L-phenylalanine, which reacted with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in Example 1, with L-aspartic acid to obtain compound 2-3. Then, referring to the process of preparing compound 3-1 in Example 1, compound 2-1 is replaced with compound 2-3 to obtain compound 3-3. Further referring to the process of preparing compound 4-1 to compound IV-1 in Example 1, compound IV-3 is finally obtained.

[0050] Example 3. Preparation of Compound IV-5

[0051] The synthesis route in this embodiment is as follows:

[0052] The preparation steps described in this embodiment are based on Example 1. The L-phenylalanine that reacted with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in Example 1 was replaced with L-valine to obtain compound 2-5. Then, referring to the process of preparing compound 3-1 in Example 1, compound 2-1 was replaced with compound 2-5 to obtain compound 3-5. Further referring to the process of preparing compound 4-1 to compound IV-1 in Example 1, compound IV-5 was finally obtained, and its structural characterization is shown in Figure 2.

[0053] Example 4: Preparation of Compound IV-7

[0054] The synthesis route in this embodiment is as follows:

[0055] The preparation steps described in this embodiment are based on Example 1. The L-phenylalanine that reacted with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in Example 1 was replaced with L-lysine to obtain compound 2-7. Then, referring to the process of preparing compound 3-1 in Example 1, compound 2-1 was replaced with compound 2-7 to obtain compound 3-7. Further referring to the process of preparing compound 4-1 to compound IV-1 in Example 1, compound IV-7 was finally obtained.

[0056] Example 5. Preparation of compound IV-9

[0057] The synthesis route in this embodiment is as follows:

[0058] The preparation steps described in this embodiment are based on Example 1. The L-phenylalanine that reacted with 5-(dimethylamino)naphthalene-1-sulfonyl chloride in Example 1 was replaced with L-glycine to obtain compound 2-9. Then, referring to the process of preparing compound 3-1 in Example 1, compound 2-1 was replaced with compound 2-9 to obtain compound 3-9. Further referring to the process of preparing compound 4-1 to compound IV-1 in Example 1, compound IV-9 was finally obtained. The structural characterization is shown in Figure 3.

[0059] Example 6. Preparation of Compound IV-2 Referring to Example 1, by replacing the (tert-butoxycarbonyl)-L-lysine that reacted with compound 3-1 in Example 1 with Nε-Boc-L-lysine, compounds 4-2, 5-2, and 6-2 can be prepared sequentially, and finally compound IV-2 can be obtained.

[0060] The reaction route is as follows:

[0061] Example 7. Preparation of Compound IV-4 Referring to Example 2, by replacing the (tert-butoxycarbonyl)-L-lysine that reacted with compound 3-3 in Example 2 with Nε-Boc-L-lysine, compounds 4-4, 5-4, and 6-4 can be prepared sequentially, and finally compound IV-4 can be obtained.

[0062] The reaction route is as follows:

[0063] Example 8. Preparation of compound IV-6 Referring to Example 3, by replacing the (tert-butoxycarbonyl)-L-lysine that reacted with compounds 3-5 in Example 3 with Nε-Boc-L-lysine, compounds 4-6, 5-6, and 6-6 can be prepared sequentially, and finally, compound IV-6 can be obtained.

[0064] The reaction route is as follows:

[0065] Example 9. Preparation of compound IV-8 Referring to Example 4, by replacing the (tert-butoxycarbonyl)-L-lysine that reacted with compounds 3-7 in Example 4 with Nε-Boc-L-lysine, compounds 4-8, 5-8, and 6-8 can be prepared sequentially, and finally, compound IV-8 can be obtained.

[0066] The reaction route is as follows:

[0067] Example 10. Preparation of compound IV-10 Referring to Example 5, by replacing the (tert-butoxycarbonyl)-L-lysine that reacted with compounds 3-9 in Example 5 with Nε-Boc-L-lysine, compounds 4-10, 5-10, and 6-10 can be prepared sequentially, and finally, compound IV-10 can be obtained.

[0068] The reaction route is as follows:

[0069] Examples 11-12: Preparation of compounds of formula IV-11 and IV-12 Their preparation methods can all refer to Examples 1-10. For example, based on Example 1, the compound is prepared by... [3-1] Replacing Lys with Lys-PEG2-COOH or Lys-PEG4-COOH in the reaction yields the following corresponding structures:

[0070] Example 13. Preparation of compound IV-13

[0071] The synthesis route in this embodiment is as follows:

[0072] The specific steps are as follows: Nota-Bis(t-BuEster) was dissolved in DMF, DIPEA was added, the temperature was lowered to 10°C, and then HBTU was added. The reaction was maintained at this temperature for 30 min. Compound of formula 6-1 prepared in Example 1 was added to DIPEA to form a suspension, and the above reaction solution was added. After stirring for 1 h, MTBE was added dropwise to the reaction solution, and a solid precipitated. After standing, the supernatant was poured off to prepare and purify compound IV-13A. The structure characterization is shown in Figure 4. Compound IV-13A was dissolved in a TFA:water solution of 100:5 and reacted at 30°C for 3 h. Then, MTBE was added dropwise to the reaction solution, and a solid was precipitated. After filtration and purification, the compound was lyophilized to obtain the compound shown in formula IV-13. The structural characterization is shown in Figure 5.

[0073] In addition, other compounds provided by the present invention, such as IV-14, IV-15, IV-16, IV-17, IV-18, IV-19, IV-20, IV-21, IV-22, IV-23, and IV-24, can be prepared by referring to the methods provided in Examples 1-13.

[0074] Example 14. Preparation of radiolabeled complexes of the radionuclide 68Ga

[0075] The 68Ga nuclide labeling process is as follows: Wet method: Add approximately 37-3700 MBq of 68GaCl3 hydrochloric acid solution (rinsed from the germanium-gallium generator) to a centrifuge tube containing the compound solution to be labeled (the compound solution to be labeled is 0.5-2 mL of acetate-acetate solution containing 20-200 µg of each compound prepared in Examples 1-13), react at room temperature to 100°C for 20 min, cool to room temperature, dilute with physiological saline or water for injection, and sterile filter to obtain the labeled compound injection solution, denoted as "68Ga-Form IV". Lyophilization method: A certain amount of buffer solution and about 37~3700 MBq of 68GaCl3 hydrochloric acid solution (rinsed from the germanium gallium generator) were added to a lyophilization kit containing 20~200 µg of the compound to be labeled (the compound to be labeled is the compound prepared in Examples 1-13). After mixing and dissolving, the mixture was placed at room temperature to 100°C for 20 minutes and then cooled to room temperature. The solution was diluted with physiological saline or water for injection and then sterile filtered to obtain the labeled compound injection solution, denoted as "68Ga-Form IV".

[0076] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeling solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 68Ga ions, and then rinse with ethanol solution to obtain purified 68Ga-IV. Remove the organic solvent by nitrogen blowing, dilute with physiological saline, and sterilely filter to obtain the labeled compound injection solution.

[0077] The compound of formula IV-1 in Example 1 was labeled according to the above method to obtain "68Ga-formula IV-1". The identification results are shown in Figure 6. 68Ga-formula IV-1 has a high labeling rate and specific activity. The nuclide is readily available and the operation is simple, which is conducive to large-scale clinical application.

[0078] This invention also uses 68Ga to label other compounds, including but not limited to 68Ga-form IV-2, 68Ga-form IV-3, 68Ga-form IV-4, 68Ga-form IV-5, 68Ga-form IV-6, 68Ga-form IV-7, 68Ga-form IV-8, 68Ga-form IV-9, 68Ga-form IV-10, 68Ga-form IV-11, and 68Ga-form IV-12. Experiments have shown that the compounds provided by this invention have high labeling rates and specific activities, and the nuclides are readily available and the operation is simple, which is conducive to large-scale clinical application.

[0079] Example 15. In vitro stability test Following the labeling method of Example 14, compounds IV-1, IV-3, and IV-9 prepared in Examples 1, 3, and 9 were labeled, and the resulting 68Ga-labeled complexes were designated as "68Ga-form IV-1", "68Ga-form IV-3", and "68Ga-form IV-9", respectively. Solutions of 68Ga-form IV-1, 68Ga-form IV-3, and 68Ga-form IV-9 with radiochemical purity greater than 95% were dissolved in physiological saline and left at room temperature for different times. Samples were then analyzed by HPLC. At the tested time points, each probe still maintained a radiochemical purity >95%, indicating that it was stable in the specified solution and not easily decomposed. The HPLC results of the in vitro physiological saline stability of 68Ga-form IV-1, 68Ga-form IV-3, and 68Ga-form IV-9 solutions are shown in Part A of Figures 7-9, indicating that they maintained high stability (>95%) in the physiological saline system up to 2 hours. Solutions of 68Ga-IV-1, 68Ga-IV-3, and 68Ga-IV-9 with radiochemical purity greater than 95% were co-incubated with serum at room temperature for different times. Acetonitrile was added to remove proteins, and the supernatant was collected by centrifugation. Samples were analyzed by HPLC. At the tested time points, each probe still maintained a radiochemical purity >95%, indicating that it was stable and not easily decomposed in the specified solution. The HPLC results of the in vitro serum stability of 68Ga-IV-1, 68Ga-IV-3, and 68Ga-IV-9 are shown in part B of Figures 7-9, indicating that they maintained high stability (>95%) in physiological saline (i.e., serum) systems up to 2 hours.

[0080] Example 16. Preparation of radiolabeled complexes of radionuclide 177Lu Wet method: Add approximately 18.5~1850 MBq of 177LuCl3 sodium acetate solution to centrifuge tubes containing the solution of the compound to be labeled (the solution of the compound to be labeled is 0.5~2 mL of acetic acid-acetate solution containing 20-200 µg of each compound prepared in Examples 1-13), react at 90°C for 20 min, cool to room temperature, dilute with physiological saline or water for injection, and sterile filter to obtain the labeled compound injection solution, denoted as "177Lu-Form IV". Lyophilization method: A certain amount of buffer solution and about 37~3700 MBq of 177LuCl3 are added to a lyophilization kit containing 20~200 µg of the compound to be labeled (the compound to be labeled is the compound provided by the present invention, including but not limited to the compounds prepared in Examples 1-13). After mixing and dissolving, the mixture is placed at room temperature to 100°C for 20 minutes and then cooled to room temperature. It is then diluted with physiological saline or water for injection and sterile filtered to obtain the labeled compound injection solution, denoted as "177Lu-Form IV".

[0081] If the radiochemical purity is below 95%, purification is required. The purification steps are as follows: Take a Sep-Pak C18 separation column and activate and rinse it successively with 10 mL of anhydrous ethanol and 10 mL of water. Dilute the labeling solution with 10 mL of water and load it onto the separation column. Rinse the separation column with water to remove unreacted 177Lu ions, and then rinse with ethanol solution to obtain the purified 177Lu-labeled complex. Remove the organic solvent by nitrogen blowing, dilute with physiological saline, and sterilely filter to obtain the labeled compound injection solution.

[0082] This invention also uses 177Lu to label other compounds, including but not limited to 177Lu-form IV-1, 177Lu-form IV-2, 177Lu-form IV-3, 177Lu-form IV-4, 177Lu-form IV-5, 177Lu-form IV-6, 177Lu-form IV-7, 177Lu-form IV-8, 177Lu-form IV-9, 177Lu-form IV-10, 177Lu-form IV-11, 177Lu-form IV-12, etc.

[0083] The compounds provided by this invention can also be labeled using other labeling methods involved in the prior art, such as 18F-form IV-13, 18F-form IV-14, 18F-form IV-15, 18F-form IV-16, 18F-form IV-17, 18F-form IV-18, 18F-form IV-19, 18F-form IV-20, 18F-form IV-21, 18F-form IV-22, 18F-form IV-23, 18F-form IV-24, etc.

[0084] In addition, other labeling methods provided by the prior art (including but not limited to the method provided by this invention) can be used to label the radionuclide-labelable compounds provided by this invention. The radionuclides include, but are not limited to: 18F, 51Cr, 64Cu, 67Cu, 67Ga, 68Ga, 89Zr, 111In, 99mTc, 186Re, 188Re, 139La, 140La, 175Yb, 153Sm, 166Ho, 86Y, 90Y, 149Pm, 165 Dy, 169Er, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi, 213Bi, 72As, 72Se, 97Ru, 109Pd, 105Rh, 101mRh, 119Sb, 128Ba, 123I, 124I, 131I, 197Hg, 211At, 151Eu, 153Eu, 169Eu, 201Tl, 203Pb, 212Pb, 198Au, 225Ac, 227Th, or 199Ag, etc.

[0085] Example 17. Cellular uptake experiment

[0086] Tumor cell uptake assay: One day before the experiment, 2 × 10⁵ PC3-PIP cells were added to each well of a 24-well plate and incubated overnight. Cells were divided into three groups: total binding group, internalization group, and inhibition group, with four wells in each group. First, 100-fold excess PSMA617 was added to the wells of the inhibition group as an inhibitor. Then, 500 μL of serum-free medium diluted with 68Ga-form IV-1 solution (18.5 kBq / well) prepared according to the method in Example 14 (with a radiochemical purity greater than 95%) was added to each well of all wells. The plates were then capped and incubated at 37°C in a cell culture incubator. For the total binding group and the internalization group, cells were removed from the incubator at 10, 30, 60, and 120 min after incubation, respectively. The cells were washed three times with PBS, and the internalization group was washed once more with elution buffer. Finally, the cells were digested with 1 mL of digestion solution. For the inhibition group, after incubation at 37°C for 10, 30, 60, and 120 min, the cells were washed twice with ice-cold PBS (pH 7.4) and then digested with digestion solution. All solution and cells from the wells were aspirated into labeled tubes. Three 500 μL aliquots of the 68Ga-form IV-1 solution diluted in serum-free medium were taken, the total radioactive dose added to each well was calculated, the radioactivity count was measured using a gamma counter, and the cell binding rate and internalization rate were calculated at each time point.

[0087] Cellular receptor saturation competition binding assay: Based on the results of the cell uptake assay, cells with high binding rates were selected as cells for determining receptor affinity. This was then used to screen labeled probes for affinity and specificity, guiding and optimizing probe design. In short: Before the experiment, 50 μL of serum-free PC3-PIP (2 × 10⁶ / mL) was added to each well of a 96-well plate. Incremental concentrations of the radiolabeled substance to be tested (18 kBq / well) were added, adjusting the final reaction volume in all wells to 200 μL. The plate was then shaken at room temperature for 4 h, the reaction solution was dried, and the filter membrane was collected and the radioactivity count was measured using a gamma counter. The Kd value was calculated using Graph-Pad Prism software.

[0088] The experimental results are shown in Figure 10. The Kd value of 68Ga-IV-1 is comparable to that of 68Ga-PSMA617 (see curves in sections B and D), but the cell uptake of 68Ga-IV-1 within 2 hours is much higher than that of 68Ga-PSMA617 (see bar charts in sections A and C).

[0089] Example 18.68 Ga-type IV-1 tumor model mouse MicroPET imaging 68Ga-form IV-1 with a radiochemical purity greater than 95% was prepared according to the labeling method in Example 14. Mice bearing PC3-PIP tumors were randomly divided into experimental and control groups. 0.2 mL (approximately 7.4 MBq) of 68Ga-form IV-1 solution was injected into the experimental group mice via the tail vein, while the control group mice were simultaneously injected with 68Ga-PSMA 617. MicroPET imaging was performed at 1 h, 2 h, and 4 h post-injection. The imaging results are shown in Figure 11. The area indicated by the arrow in part A is the tumor location. The tumor uptake of the 68Ga-PSMA 617 injected into the control group was approximately 20% ID / g 4 h post-injection (see part B and the vertical bar graph in part C of Figure 11), while the tumor uptake of the 68Ga-form IV-1 of this invention was approximately 50% ID / g 4 h post-injection, significantly higher than that of the control group. This indicates that the 68Ga-form IV-1 complex of this invention has significantly enhanced tumor uptake and retention time (see part A and the grid bar graph in part C of Figure 11). Meanwhile, 68Ga-IV-1 can achieve a high target-to-non-target ratio (tumor / flesh ratio, tumor / heart ratio, and tumor / kidney ratio) within 1 hour, and the target-to-non-target ratio gradually increases over time (see the bar chart in section D of Figure 11). In summary, the 68Ga-type IV-1 of this invention facilitates longer imaging time, and with the guarantee of high imaging contrast, the diagnosis of primary micro-lesions will be more accurate, which is beneficial for target delineation. On the other hand, the high uptake and long retention of the radionuclide-labeled probe at the tumor site will lay the foundation for radionuclide targeted therapy.

[0090] Example 19.68: Ga-type IV-9 complex for MicroPET imaging in a mouse tumor model. A 68Ga-form IV-9 solution with a radiochemical purity greater than 95% was prepared according to the labeling method in Example 14. Mice bearing PC3-PIP tumors were randomly divided into experimental and control groups. 0.2 mL (approximately 7.4 MBq) of the 68Ga-form IV-9 solution was injected into the experimental group mice via the tail vein, while the control group mice were simultaneously injected with 68Ga-PSMA 617. MicroPET imaging was performed at 1 h, 2 h, and 4 h after injection. The imaging results are shown in Figure 12. The area indicated by the arrow in part A is the tumor location. The tumor uptake of the 68Ga-PSMA 617 injected into the control group was approximately 20% ID / g 4 h after injection (see parts B and C of Figure 11), while the tumor uptake of the 68Ga-form IV-9 complex of this invention was approximately 50% ID / g 4 h after injection, which was much higher than that of the control group. Meanwhile, 68Ga-IV-9 can achieve high target-to-non-target ratios (tumor / flesh ratio, tumor / heart ratio, and tumor / kidney ratio) within 1 hour, and the target-to-non-target ratio gradually increases over time (see the bar chart in part B of Figure 12).

[0091] Example 20.177 Biodistribution of Lu-labeled complexes in normal mice Compound IV-1 prepared in Example 1 was labeled using the labeling method of Example 16 to obtain 177Lu-form IV-1 with a radiochemical purity greater than 95%. 0.2 mL (approximately 1.85 MBq) was injected via the tail vein of normal mice. Mice were sacrificed at 4 h, 12 h, 24 h, and 48 h post-injection, and organs of interest were harvested and measured using a γ-counter. As shown in Figure 13, the blood uptake value of 177Lu-form IV-1 remained high at 4 hours, and the radioactivity was gradually cleared from the blood over time. This indicates that the 177Lu-labeled IV-1 of the present invention has the ability to moderately enhance blood circulation time, which is beneficial for enhancing tumor uptake while avoiding the problem of excessively long drug circulation in the body.

[0092] Example 21.177 Lu-type IV-1 tumor model mouse SPECT imaging A 177Lu-form IV-1 solution with a radiochemical purity greater than 95% was prepared according to Example 16. 0.2 mL (approximately 37 MBq) was injected via the tail vein of mice bearing PC3-PIP tumors. SPECT imaging was performed at 2, 4, 12, 24, 48, and 72 hours post-injection. The imaging results are shown in Figure 14. The arrows in Figure 14 indicate the tumor location. The imaging results show that the 177Lu-form IV-1 of this invention significantly enhances tumor uptake and retention time at the tumor site. The high uptake and long retention of the radionuclide-labeled probe at the tumor site will lay the foundation for radionuclide-targeted therapy. As shown in Figure 15, the tissue uptake results after administration indicate that a high target-to-non-target ratio (tumor / tumor ratio, tumor / heart ratio, and tumor / kidney ratio) can be obtained within 2 hours, and the target-to-non-target ratio gradually increases with time.

[0093] Example 22.68 Ga-labeled complexes for MicroPET imaging of prostate cancer patients Subject selection criteria: 1) Patients with a high suspicion of prostate cancer based on clinical symptoms, laboratory tests, and other imaging examinations (CT, MRI); 2) Age 18-80 years; surgical or biopsy is feasible to obtain a pathological diagnosis; 3) Exclude patients with renal failure (serum Cr > 3 mg / dl); exclude patients whose condition is too severe to accept further diagnosis and treatment.

[0094] 68Ga-form IV-1 and 68Ga-form IV-9 solutions with a radiochemical purity greater than 95% were prepared according to the labeling method in Example 14 and administered intravenously to the subjects. The intravenous radiotracer dose was 1.85-2.22 MBq / Kg. PET / CT imaging was performed using a Polestar m660 (Sino Union, China) integrated PET / CT scanner at 60, 120, 180, and 240 minutes after injection. Low-dose CT scans (120 kV, 35 mAs) from the tip of the skull to the mid-thigh were used for attenuation correction and anatomical reference. Whole-body PET / CT scans were performed at 5-6 beds (2 min / bed). The acquired images were reconstructed using an ordered subset expectation-maximization algorithm.

[0095] All images were transferred to MIM software (version 7.1.4, MIM Software, Cleveland, USA), and regions of interest (ROIs) were plotted on the CT images using PET edges. Positive lesions were defined as those with significantly increased tracer uptake compared to the surrounding normal prostate tissue. Radioactivity concentration was quantitatively assessed using standardized uptake values ​​(SUVs).

[0096] Figures 16-19 show PET / CT images taken at 1h, 2h, 3h, and 4h after injection of 68Ga-form IV-1 and 68Ga-form IV-9 solutions. The SUVmean of organs throughout the body and the SUVmax of tumors were measured. Imaging results for 68Ga-form IV-1 are shown in Figures 16 and 17, and for 68Ga-form IV-9 in Figures 18-19. The imaging results show that, with increasing time, the radioactivity concentration of the two complexes of this invention gradually decreases in the cardiac blood pool and in blood-rich organs such as the liver and spleen, while gradually increasing in tumors, reaching its highest value after 4h and exhibiting a high target-to-source ratio. This indicates that the 68Ga-labeled complexes of this invention not only possess high diagnostic efficacy but also offer advantages in applications involving therapeutic radionuclides with long half-lives.

Claims

1. A dansulphamide-modified PSMA-targeting compound, characterized in that its structure is shown in Formula I, wherein: L is lysine; R1 is an amino acid analog structure with the structure of , R4 is (CH2)r-aryl, where r is 1 and the aryl group is phenyl; or R1 is ; R2 is a structure targeting prostate-specific membrane antigen; and the structure of R2 is ; R3 is a radionuclide chelating group that can be linked to L.

2. The dansulphamide-modified PSMA-targeting compound as claimed in claim 1, characterized in that: R1 has the following structure: or.

3. A radionuclide-labeled PSMA-targeting compound, characterized in that: it is a complex obtained by labeling a radionuclide with a PSMA-targeting compound modified with dansulphamide as a ligand, as described in any one of claims 1 to 2.

4. The radiolabeled PSMA-targeting compound as claimed in claim 3, characterized in that: the radiolabeled PSMA-targeting compound is a radionuclide diagnostic probe or a radionuclide therapeutic probe.

5. The radiolabeled PSMA-targeting compound as claimed in claim 3, characterized in that: the radionuclide is selected from any one of 177Lu, 18F, 43Sc, 44Sc, 47Sc, 55Co, 57Co, 68Ga, 67Ga, 62Cu, 64Cu, 67Cu, 89Zr, 86Y, 90Y, 99mTc, 99Tc, 90ln, 111ln, 89Sr, 152Gd, 153Gd, 153Sm, 149Tb, 151Tb, 161Tb, 166Ho, 166Dy, 186Re, 188Re, 212Pb, 212Bi, 213Bi, 223Ra, 225Ac, 226Th, 227Th or 211At.

6. A pharmaceutical composition, characterized in that it comprises a PSMA-targeting compound modified with dansulphamide as described in any one of claims 1 to 2 or a PSMA-targeting compound labeled with a radionuclide as described in any one of claims 3 to 5, and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, characterized in that: the pharmaceutically acceptable carrier is selected from binders, buffers, colorants, diluents, disintegrants, emulsifiers, flavoring agents, flow aids, lubricants, preservatives, stabilizers, surfactants, tablets or wetting agents, or combinations thereof.

8. The use of a radionuclide-labeled PSMA-targeting compound, said use being the use of any one of claims 3 to 5 of the radionuclide-labeled PSMA-targeting compound in the preparation of a therapeutic or diagnostic medicament for cancers in which PSMA is highly expressed in mammals.

9. The application as described in claim 8, characterized in that: the radiolabeled PSMA-targeting compound having a diagnostic or therapeutic effect on the mammal is combined with one or more other active ingredients.

10. The use of a pharmaceutical composition, said use being the use of any one of claims 6 to 7 in the preparation of a therapeutic or diagnostic medicament for cancers in which PSMA is highly expressed in mammals.

11. The application as described in claim 10, characterized in that: the pharmaceutical composition of any one of claims 6 to 7, having a diagnostic or therapeutic effect on the mammal, is combined with one or more other active ingredients.

12. The application as claimed in any one of claims 8 to 11, characterized in that the PSMA-overexpressing tumor is prostate cancer.

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