Compound targeting CAIX and PSMA as well as nuclide marker, preparation method and application thereof

Abstract

The invention belongs to the technical field of nuclear medicine, and particularly relates to a compound targeting CAIX and PSMA as well as a nuclide marker, a preparation method and application of the compound. One purpose of the present invention is to provide an application of a compound targeting CAIX and PSMA or a salt thereof in preparation of a product for diagnosis or / and treatment of prostate cancer, prostate cancer before metastasis, colon cancer, breast cancer, kidney cancer or bladder cancer. The compound and the nuclide marker thereof have multiple advantages in diagnosis and treatment of tumors expressing PSMA and CAIX, including high affinity to two receptors, high target / non-target ratio (T / NT) and good in-vivo distribution characteristics, so that a more reliable imaging tool is provided for early detection and precise treatment of tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nuclear medicine, and particularly relates to a compound targeting CAIX and PSMA, a radionuclide label thereof, a preparation method and application. BACKGROUND

[0002] Prostate cancer is a heterogeneous disease that forms in a highly complex microenvironment. Most patients have entered the middle and late stages at the time of diagnosis, at which time androgen deprivation therapy (ADT) is often used as the first choice of treatment. However, the vast majority of patients receiving this therapy will eventually develop metastatic castration-resistant prostate cancer (mCRPC), resulting in a significant increase in treatment difficulty.

[0003] Prostate-specific membrane antigen (PSMA) is expressed in more than 80% of prostate adenocarcinoma cells, and its expression level is positively correlated with tumor grade and clinical stage, thus becoming an important target in the diagnosis and treatment of prostate cancer. It is worth noting that in addition to prostate cancer, PSMA is also highly expressed in the neovasculature of a variety of solid malignancies, one of which is renal cancer. This expression characteristic provides a molecular basis for developing a PSMA-targeted radiotracer for the detection of primary and metastatic lesions of renal cancer. In clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, the expression level of PSMA is particularly prominent, and studies have shown that its expression rate can reach 76.2%, which is significantly higher than that of other renal cancer subtypes.

[0004] Carbonic anhydrase IX (CAIX), a transmembrane protein, plays a crucial role in tumorigenesis and development. Composed of 459 amino acids, its structure comprises a signal peptide, an extracellular mucin-like domain, a catalytic domain, a transmembrane region, and a short cytoplasmic tail. Recent studies have shown that CAIX is a key downstream target protein of hypoxia-inducible factor-1α (HIF-1α), exhibiting highly specific expression on the surface of various hypoxia-associated malignancies (such as breast cancer, lung cancer, ovarian cancer, head and neck cancer, bladder cancer, colon cancer, cervical cancer, and renal cell carcinoma), while its expression level is extremely low in normal tissues. Therefore, it is considered a highly promising target for tumor diagnosis and treatment. Particularly in ccRCC (cytokine-mediated renal cell carcinoma), approximately 90% of tumor cells exhibit hypoxia or VHL gene deletion, leading to upregulation of CAIX expression. This upregulation not only results in extracellular pH acidification and decreased cell adhesion but also promotes tumor invasion and metastasis. Therefore, CAIX is also considered a key molecular marker in the diagnosis and treatment of renal cell carcinoma.

[0005] Based on the high specific expression of PSMA and CAIX in various tumors such as renal cell carcinoma, constructing a dual-targeting molecular probe that can simultaneously target these two molecules is of great research significance for improving the early diagnostic sensitivity of primary and metastatic tumors and shows broad clinical application prospects.

[0006] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] This invention belongs to the field of nuclear medicine technology, specifically relating to a compound targeting CAIX and PSMA, its radionuclide label, preparation method, and application.

[0008] To address the aforementioned technical problems, one objective of this invention is to provide a compound targeting CAIX and PSMA, having the structure shown in Formula I or a pharmaceutically acceptable salt thereof; Formula I is shown below: ..Formula I.

[0009] One object of the present invention is also to provide radiolabeled compounds targeting CAIX and PSMA, having the structure shown in Formula II or a pharmaceutically acceptable salt thereof; Formula II is shown below: Formula II, Where M is a radioactive nuclide.

[0010] According to a preferred embodiment, the radionuclide M is selected from... 68 Ga、 177 Lu、 99 mTc, 111 In、 67 Ga、 86 Y、 90 Y、 161 Tb, 186 Re、 188 Re、 64 Cu、 67 Cu、 211 At、 225 Ac、 18 F 、123 I, 124 I, 125 One of I. Preferably, the radionuclide M is selected from... 68 Ga、 177 Lu、 99 mTc, 111 In、 64 Cu、 18 F 、123 I, 124 I, 225 One of Ac. More preferably, the radionuclide M is selected from... 68 Ga、 177 Lu、 225 One of Ac.

[0011] According to a preferred embodiment, a radiolabeled compound or its salt targeting CAIX and PSMA has the structure shown in Formula III: …Form III.

[0012] According to a preferred embodiment, a radiolabeled compound or its salt targeting CAIX and PSMA has the structure shown in Formula IV: …Form IV.

[0013] One of the objectives of this invention is to provide a method for preparing compounds targeting CAIX and PSMA, comprising the following steps: Compound 1 reacts with compound 2 to give compound 3; The reaction of compounds 3 and 4 yields the compound shown in Formula I that targets CAIX and PSMA.

[0014] The structure of compound 1 is shown in Formula V: …Form V.

[0015] The structure of compound 1 is shown in Formula VI below: …Style VI.

[0016] The structure of compound 4 is shown in Formula VII: …Form VII.

[0017] According to a preferred embodiment, 1 eq of compound 2 is dissolved in N,N-dimethylformamide, then 1.5 eq of HATU and 3 eq of DIEA are added, and after stirring evenly, 1.2 eq of compound 1 is added. After the reaction is complete, add trifluoroacetic acid and allow the reaction to proceed for at least 1 hour. Add 50 mL of diethyl ether, centrifuge and dry, dissolve together with compound 4 in N,N-dimethylformamide, and add 3 eq of N,N-diisopropylethylamine.

[0018] One of the objectives of this invention is to provide a method for preparing radiolabeled compounds targeting CAIX and PSMA, comprising the following steps: The compound of Formula I is reacted with a radioactive nuclide salt to generate the radioactive nuclide label shown in Formula II.

[0019] One of the objectives of this invention is to provide a pharmaceutical composition comprising the above-mentioned compound targeting CAIX and PSMA or a salt thereof; or a radionuclide label comprising the above-mentioned compound targeting CAIX and PSMA or a salt thereof.

[0020] One of the objectives of this invention is to provide a kit comprising the above-mentioned compounds targeting CAIX and PSMA or their salts; or a radionuclide label comprising the above-mentioned compounds targeting CAIX and PSMA or their salts.

[0021] One of the objectives of this invention is to provide the use of the above-mentioned compounds targeting CAIX and PSMA or their salts, radionuclide markers of the above-mentioned compounds targeting CAIX and PSMA or their salts, the above-mentioned pharmaceutical compositions, and the above-mentioned kits in the preparation of products for detecting and / or detecting cells and / or tissues expressing PSMA receptors and CAIX receptors.

[0022] According to a preferred embodiment, the product is a diagnostic tracer or a therapeutic agent.

[0023] According to a preferred embodiment, the product is used in positron emission tomography (PET), computed tomography (CT), positron emission tomography (PET), single-photon emission tomography (SEP) or single-electron emission tomography (SEP).

[0024] According to a preferred embodiment, the product is used for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, colon cancer, breast cancer, kidney cancer, or bladder cancer.

[0025] One of the objectives of this invention is to provide the above-mentioned compounds targeting CAIX and PSMA or their salts, radionuclide labels of the above-mentioned compounds targeting CAIX and PSMA or their salts, the above-mentioned pharmaceutical compositions, and the above-mentioned kits for use in the preparation of products for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, colon cancer, breast cancer, kidney cancer, or bladder cancer.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The compound designed in this technical solution can simultaneously target both PSMA and CAIX receptors, effectively overcoming the shortcomings of existing tumor imaging agents in terms of diagnostic accuracy and sensitivity. Experimental results show that the radiolabeled compound has a more significant uptake effect at the tumor site, exhibiting higher sensitivity and better targeting performance, thus providing a more reliable imaging tool for early detection and precise treatment of tumors.

[0027] The compounds and their radionuclide markers of this invention exhibit multiple advantages in the diagnosis and treatment of tumors expressing both PSMA and CAIX, including high affinity for both receptors, a high target / non-target ratio (T / NT), and good in vivo distribution characteristics. This dual-targeting strategy not only enhances the enrichment of probes at lesions but also helps reduce non-specific uptake of normal tissues, thereby improving the signal-to-noise ratio and imaging contrast. Furthermore, these compounds have shown potential application value in various solid tumor models, particularly suitable for malignant tumor types such as prostate cancer and clear cell renal cell carcinoma, which co-express or separately highly express PSMA and CAIX, laying an important foundation for the development of next-generation multi-targeted therapies. Attached Figure Description

[0028] Figure 1 This is a synthetic route diagram of compound DOTA-CAIX-PSMA from Example 1 of the present invention; Figure 2 The mass spectrum of DOTA-CAIX-PSMA; Figure 3 The UV spectrum of DOTA-CAIX-PSMA; Figure 4 for 68 Radio-HPLC chromatogram of Ga-DOTA-CAIX-PSMA; Figure 5 for 68 Radio-HPLC chromatogram of Ga-DOTA-CAIX; Figure 6 for 68 PET / CT images of the OS-RC-2 model using Ga-CAIX-PSMA; Figure 7 for 68 PET / CT images of the OS-RC-2 model using Ga-DOTA-CAIX; Figure 8 for 68 Uptake of Ga-CAIX-PSMA in OS-RC-2 cells; Figure 9 for 68 Figure showing the in vitro stability of Ga-CAIX-PSMA in PBS and 10% FBS; Figure 10 for 68 Figure showing the albumin binding rate of Ga-DOTA-CAIX. Detailed Implementation

[0029] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0030] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0031] The Chinese name corresponding to the English abbreviation of this invention is: Sodium ascorbate: Ascorbic acid; DMF: N,N-dimethylformamide; HATU: 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; DIEA: N,N-diisopropylethylamine; TFA: Trifluoroacetic acid; Piperidine: Piperidine; EDCI: Carbodiimide; HOOBt: 3-hydroxy-1,2,3-benzotriazine-4(3H)-one; NMM: N-methylmorpholine; Compd: compound.

[0032] Example 1 This embodiment discloses the synthesis of compound DOTA-CAIX-PSMA of Formula I of the present invention, and the synthetic route is shown in the appendix. Figure 1 As shown.

[0033] The synthesis steps are as follows: Dissolve Compd 2 (1 eq) in 10 mL DMF, add HATU (1.5 eq) and DIEA (3 eq), stir for 30 minutes, and then add Compd 1 (1.2 eq); The reaction was carried out overnight at room temperature. LC-MS was used to monitor the completeness of the reaction. The reaction solvent DMF was removed by vacuum distillation (50°C, 0.1 MPa). 5 mL of TFA was added, and the reaction was carried out at room temperature for 60 minutes. 50 mL of diethyl ether was added, and the mixture was centrifuged at 5000 rpm for 10 minutes. The supernatant was removed, and the mixture was dried under vacuum. The product was purified by reverse-phase preparative HPLC to obtain Compd 3 (yield: 46%). Compd 3 (1 eq) and Cmpd 4 (1.2 eq) were dissolved in 5 mL of DMF, and DIEA (3 eq) was added. The reaction was carried out at room temperature for 12 hours, concentrated, and purified by reverse-phase preparative liquid chromatography to obtain compound I, DOTA-CAIX-PSMA (yield: 65%).

[0034] Figure 2 The mass spectrum of the compound of formula I is shown; Figure 3 The HPLC chromatogram of the compound of formula I is shown. Figure 2 , 3 The results showed that the compound of formula I was successfully synthesized based on the above steps.

[0035] Example 2 This embodiment discloses the nuclide markers for compounds of formula I. 68 Preparation method of Ga-DOTA-CAIX-PSMA.

[0036] The synthesis steps are as follows: Using DOTA-CAIX-PSMA as a precursor, DOTA-CAIX-PSMA was prepared into a 1 μg / μL aqueous precursor solution for later use. Take 30 μL of the above precursor solution and add it to 370 MBq. 68 In a GaCl3 solution, the pH was adjusted to 4-5 with 0.5 M sodium acetate buffer and reacted at 90℃ for 15 min. Add 5 mL of physiological saline to the reaction solution and pass it through a C18 light column. Rinse the C18 column with 20 mL of physiological saline to remove any residue.68 GaCl3; The product in the C18 column was eluted with 1 mL of 60% ethanol solution, passed through a 0.22 μm sterile filter membrane, and finally diluted to a mass fraction of less than 10%.

[0037] like Figure 4 As shown, the results 68 The radiochemical purity of Ga-DOTA-CAIX-PSMA is >98%.

[0038] Comparative Example 1 This comparative example is publicly available. 68 Preparation method of Ga-DOTA-CAIX.

[0039] The DOTA-CAIX used in this comparative example was provided by Ganzhou Tanzhen Biomedical Co., Ltd.

[0040] The synthesis steps are as follows: Using DOTA-CAIX compound as a precursor, DOTA-CAIX was prepared into a 1 μg / μL aqueous precursor solution for later use; Take 50 μL of the above precursor solution and add it to 370 MBq. 68 In a GaCl3 solution, the pH was adjusted to 4-5 with 0.5 M sodium acetate buffer and reacted at 90℃ for 15 min. Add 5 mL of physiological saline to the reaction solution and pass it through a C18 light column. Rinse the C18 column with 20 mL of physiological saline to remove any residue. 68 GaCl3; The product in the C18 column was eluted with 1 mL of 50% ethanol solution, passed through a 0.22 μm sterile filter membrane, and finally diluted with ethanol to a concentration of less than 10% by mass. like Figure 5 As shown, the results 68 The radiochemical purity of Ga-DOTA-CAIX is >98%.

[0041] Experimental Example 1 This experimental example discloses 68 Ga-DOTA-CAIX-PSMA 68 PET / CT imaging experiments of Ga-DOTA-CAIX.

[0042] (I) Experimental Procedure (1) Establishment of OS-RC-2 animal model: OS-RC-2 cells expressing CAIX receptor were cultured in 1640 medium containing 10% FBS. The culture conditions were 37℃, 5% CO2, and saturated humidity. After the cells grew to 70-80%, they were digested with trypsin and centrifuged before being inoculated.

[0043] After anesthetizing BALB / c-nu nude mice, subcutaneous inoculation was performed under their armpits, with each BALB / c-nu nude mouse receiving 5*10 cells. 6 0.1 mL / cell. Wait until the tumor size reaches 300-400 mm. 3 It can be used for imaging experiments.

[0044] In this experimental example 68 Ga-DOTA-CAIX-PSMA was prepared according to the method in Example 1; 68 Ga-DOTA-CAIX was prepared according to the method of Comparative Example 1. 100 μCi of... 68 Ga-DOTA-CAIX-PSMA was injected via tail vein into OS-RC-2 tumor-bearing mice with high CAIX receptor expression. PET / CT imaging was performed at 0.5 h, 1 h, 1.5 h, and 3 h post-injection. The results are as follows: Figure 6 As shown.

[0045] Take 100 μci 68 Ga-DOTA-CAIX was injected via tail vein into OS-RC-2 tumor-bearing mice with high CAIX receptor expression. PET / CT imaging was performed at 0.5 h, 1 h, 1.5 h, and 3 h post-injection. The results are as follows: Figure 7 As shown.

[0046] (2) 68 In vitro stability assay of Ga-DOTA-CAIX-PSMA: Incubation in PBS solution for 30 min, 60 min, 90 min, and 120 min yielded the following results: Figure 9 As shown.

[0047] (3) 68 Experimental study on the lipid-water partition coefficient of Ga-DOTA-CAIX-PSMA: 68 Ga-DOTA-CAIX-PSMA (100 μL, 0.74 Mbq) was added to a mixture of n-octanol and PBS, and the volume ratio of the organic phase to the aqueous phase was kept at 1:1 (n=5). The mixture was vortexed for 3 minutes and centrifuged at 3000 rpm for 5 minutes. Then, three samples (100 μL) were taken from each phase and the radioactivity count was determined using a gamma counter (HIDEX). The partition coefficient was calculated by dividing the count in n-octanol by the count in phosphate buffered saline.

[0048] (4) 68Ga-DOTA-CAIX-PSMA and 68Ga-DOTA-CAIX cell uptake assay: OS-RC-2 cells expressing CAIX were cultured in 1640 medium containing 10% FBS. After the cells grew to 70-80% confluence, they were digested with trypsin and seeded into 24-well plates (approximately 1.5*10⁻⁶ cells per well). 5 Cells were cultured overnight at 37°C with 5% CO2. The next day, the cells were starved with pure 1640 medium, followed by the addition of approximately 50 μL / 1 μCi / well of radiolabeled product. The cells were then incubated for 30 min, 60 min, and 120 min, and the cell uptake was measured at each time point using a gamma counter (n≥3).

[0049] (II) Experimental Results (1) Animal experiments according to Figure 6 As shown, the radionuclide labeling of the compound of formula I of the present invention 68 Ga-DOTA-CAIX-PSMA is specifically taken up at the OS-RC-2 tumor site.

[0050] according to Figure 7 As shown, in Comparative Example 1 68 Ga-DOTA-CAIX showed low uptake in the OS-RC-2 tumor model at 0.5 h, 1 h, and 1.5 h.

[0051] according to Figure 8 As shown, by comparing the uptake values ​​of major organs, SUVs MAX , 68 Ga-DOTA-CAIX-PSMA and 68Ga-DOTA-CAIX show significant differences in tumor uptake.

[0052] Based on the results of the above experimental examples, it can be seen that the radionuclide labeling of the present invention is superior to... 68 Ga-DOTA-CAIX-PSMA exhibits better uptake and higher sensitivity at tumor sites, with a longer uptake time at tumor sites, which is more beneficial for tumor diagnosis and treatment, and it is also rapidly cleared by the kidneys.

[0053] (2) In vitro stability according to Figure 9 As shown, via radio HPLC analysis of radiochemical purity showed that the radiochemical purity was greater than 95% within 2 hours after incubation in PBS.

[0054] (3) Lipid-water partition coefficient 68The lipid-water partition coefficient of Ga-DOTA-CAIX-PSMA is logD7.4 = -2.24. This result indicates that... 68 Ga-DOTA-CAIX-PSMA has excellent hydrophilicity.

[0055] (4) 68 Ga-DOTA-CAIX-PSMA and 68 Ga-DOTA-CAIX cellular uptake according to Figure 10 As shown, with 68 Compared to Ga-DOTA-CAIX, 68 Ga-DOTA-CAIX-PSMA showed higher uptake in CAIX-positive OS-RC-2 cells (>15% ID / 10). 6 Cell), at three time points of 30 min, 60 min, and 120 min, the cells responded to 68 The uptake of Ga-DOTA-CAIX-PSMA did not decrease and instead showed a slow upward trend. This result indicates 68 Ga-DOTA-CAIX-PSMA exhibits stable binding to the CAIX target and is less prone to off-target effects.

[0056] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A compound targeting CAIX and PSMA, characterized in that, It has the structure shown in Formula I or a pharmaceutically acceptable salt thereof; Formula I is shown below: ..Formula I.

2. A radionuclide label for compounds targeting CAIX and PSMA, characterized in that, It has the structure shown in Formula II or a pharmaceutically acceptable salt thereof; Formula II is shown below: Formula II, Where M is a radioactive nuclide.

3. The radionuclide marker according to claim 2, characterized in that, The radionuclide M is selected from... 68 Ga、 177 Lu、 99 mTc, 111 In、 67 Ga、 86 Y、 90 Y、 161 Tb, 186 Re、 188 Re、 64 Cu、 67 Cu、 211 At、 225 Ac、 18 F 、123 I, 124 I, 125 One of them is I.

4. The radionuclide marker according to claim 3, characterized in that, The radionuclide label has the structure shown in Formula III: …Form III.

5. The radionuclide marker according to claim 3, characterized in that, The radionuclide label has the structure shown in Formula IV: …Form IV.

6. A method for preparing a compound targeting CAIX and PSMA, characterized in that, Includes the following steps: Compound 1 reacts with compound 2 to give compound 3; The reaction of compounds 3 and 4 yields the compound shown in Formula I that targets CAIX and PSMA. The structure of compound 1 is shown in Figure V below: …Form V; The structure of compound 1 is shown in Figure VI below: …form VI; The structure of compound 4 is shown in the following diagram VII: …Form VII.

7. A method for preparing a radionuclide label of a compound targeting CAIX and PSMA, characterized in that, Includes the following steps: The compound of Formula I is reacted with a radioactive nuclide salt to generate a radioactive nuclide label as shown in Formula II, wherein Formula I is as follows: ..Formula I.

8. The use of the compound of claim 1, the radionuclide label of any one of claims 2 to 5, or the compound targeting CAIX and PSMA prepared based on the preparation method of any one of claims 6 to 7 in the preparation of products for the diagnosis and / or treatment of prostate cancer, pre-metastatic prostate cancer, colon cancer, breast cancer, kidney cancer, or bladder cancer.

9. The application according to claim 8, characterized in that, The product is used for positron emission tomography (PET), computed tomography (CT), single-photon emission tomography (SPECT), or single-electron emission tomography (SEP).

10. The use of the compound of claim 1, the radionuclide marker of any one of claims 2 to 5, or the compound targeting CAIX and PSMA prepared based on the preparation method of any one of claims 6 to 7 in the preparation of products for detecting and / or detecting cells and / or tissues expressing PSMA and CAIX.

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