Compound dota-mdla and pet probe for detecting ass1 protein and synthesis method and application thereof

Abstract

This invention relates to the compound DOTA-MDLA, a PET probe for detecting ASS1 protein, its synthesis method, and its applications. The compound DOTA-MDLA is shown in Formula IV. The compound DOTA-MDLA or its pharmaceutically acceptable salt provided by this invention is simple to prepare, has a short preparation time, high radiochemical yield, strong targeting, good specificity, and high uptake. It exhibits good stability in both physiological saline and serum, and can detect ASS1 expression levels at the cellular level and in mice bearing lung cancer, colon cancer, and ovarian cancer tumors. Furthermore, the invention provides… 68 Ga-labeled radiolabeled DOTA-MDLA has been used to produce a product that can be used for PET detection. 68 The Ga-DOTA-MDLA molecular probe, with its simple labeling method, short preparation time, and high yield, provides a simple and reliable molecular imaging tool for the in vivo detection, prediction, and monitoring of platinum-based drug resistance of ASS1 expression in clinical tumors, as well as for guiding the accurate administration of drugs targeting aspartate metabolism. This invention utilizes the compound DOTA-MDLA (Formula IV) or its pharmaceutically acceptable salt as a PET probe targeting the ASS1 protein.

Description

Technical Field

[0001] This invention relates to the fields of probe synthesis technology and radiolabeling, and in particular to a compound DOTA-MDLA and a PET probe capable of detecting ASS1 protein, as well as their synthesis methods and applications. Background Technology

[0002] Aspartate is a crucial source of nitrogen and carbon for nucleotide synthesis, and its metabolic changes are essential for cell proliferation and growth. Cellular uptake of aspartate is limited; therefore, restricting aspartate intake can inhibit tumor growth, making aspartate metabolism a cancer vulnerability that can be utilized for targeted therapy. Argininosuccinate synthase 1 (ASS1) is a key enzyme in aspartate metabolism and the arginine-citrulline cycle, regulated by transfer factors such as p53, SP4, and MYC, and closely related to tumorigenesis, development, and drug resistance. More importantly, the expression and function of ASS1 differ significantly across different types of tumors. On one hand, the tumor-promoting mechanisms of lung cancer, colon cancer, and ovarian cancer benefit from the upregulation of ASS1 activity. High levels of ASS1 can promote cell proliferation in colon cancer and migration and metastasis in gastric cancer cells. Simultaneously, it can trigger catabolic processes, restoring energy balance in the low-energy tumor microenvironment. Furthermore, our recent research has found that tumors with high ASS1 levels are resistant to platinum-based drugs such as cisplatin. ASS1-mediated aspartate metabolism reprogramming can lead to resistance to platinum-based chemotherapy drugs in tumor cells. Upregulation of ASS1 causes more aspartate to be diverted into the urea cycle in tumor cells, reducing nucleotide synthesis and decreasing the number of DNA sites that platinum-based drugs can target, thus leading to resistance. Therefore, ASS1 protein can serve as a target for recognizing alterations in tumor aspartate metabolism reprogramming and resistance to platinum-based drugs. ASS1 inhibitors are needed to achieve anti-tumor effects in this large class of tumors with high ASS1 levels. However, ASS1 expression is downregulated in various cancers. Decreased ASS1 is an independent prognostic biomarker in pancreatic cancer and sarcoma. Decreased ASS1 inhibits aspartate catabolism, allowing more aspartate to flow into the pyrimidine synthesis pathway to support rapid tumor cell proliferation. ASS1 activators have good anti-tumor effects in these low-ASS1 tumors. Furthermore, ASS1 silencing leads to cancer cells becoming dependent on exogenous arginine, making arginine an essential amino acid, thus giving rise to arginine deprivation therapy. Targeting ASS1 and aspartate metabolism provides new ideas and directions for the clinical diagnosis and treatment of cancer patients, and also provides new targets and a solid theoretical basis and experimental foundation for the subsequent development of novel molecular probes.

[0003] Given the significant heterogeneity and important dual effects of ASS1 in anti-tumor therapy, non-invasive, in vivo, and whole-body visualization detection methods targeting ASS1 and its mediated aspartate metabolism are particularly important for precision tumor treatment.

[0004] Positron emission tomography (PET) is a highly sensitive and non-invasive diagnostic technique that visualizes the accumulation of radioactive molecules / probes in tissues throughout the body. By combining with registered CT or MRI, it can precisely locate anatomical sites and detect molecular characteristics in tissues throughout the body. Tracer compounds synthesized from electron-emitting isotopes such as carbon, nitrogen, oxygen, and fluorine are similar to substances naturally present in the human body. These nuclide markers can participate in physiological, pathological, and biochemical metabolic processes, thus PET can be used for real-time monitoring during the treatment of diseases such as cancer. PET molecular imaging targeting ASS1 can not only non-invasively detect systemic tumor lesions and detect ASS1 expression in tumors, but also predict / monitor the early development and outcome of tumor resistance to platinum-based chemotherapy. Therefore, ASS1-targeted PET probes that are simple to prepare, stable, highly sensitive, and have a high target-to-probe ratio can help clinicians determine tumor ASS1 expression, predict / monitor the sensitivity of cancer patients to platinum-based chemotherapy drugs, and are crucial for the selection of cancer treatment strategies and the evaluation of treatment efficacy. Summary of the Invention

[0005] To further improve the specificity of PET probes targeting arginine succinate synthase 1 (ASS1), this invention provides a compound DOTA-MDLA, its synthesis method, and its applications.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a compound DOTA-MDLA or a pharmaceutically acceptable salt thereof, the compound DOTA-MDLA being shown in Formula IV.

[0008]

[0009] Secondly, the present invention provides a method for preparing the compound DOTA-MDLA.

[0010] A method for preparing the compound DOTA-MDLA, wherein the compound of formula (IV) is prepared from the compound of formula (III):

[0011]

[0012] In one embodiment of the present invention, in the preparation method of compound DOTA-MDLA, the compound shown in formula (III) is reacted in the presence of a first acid, and the compound shown in formula (IV) is obtained by a first post-treatment.

[0013] In one embodiment of the present invention, the first acid is selected from trifluoroacetic acid (TFA).

[0014] In one embodiment of the present invention, the first post-treatment includes pouring into diethyl ether, washing the precipitate with diethyl ether, and filtering to obtain the compound DOTA-MDLA represented by formula (IV).

[0015] In one embodiment of the present invention, the first acid is present in an aqueous solution, and the ratio of the first acid to the solvent water is 95% TFA: 5% H2O.

[0016] In one embodiment of the present invention, the reaction time of the compound shown in formula (III) with the first acid can be 1 hour, and the reaction conditions can be room temperature.

[0017] In one embodiment of the present invention, the compound represented by formula (III) is prepared from the compound represented by formula (II), and the synthetic route of the compound represented by formula (III) is shown below:

[0018]

[0019] In one embodiment of the present invention, in the method for preparing the compound of formula (III), the compound of formula (II) is reacted in a second solvent in the presence of a second base, and then condensed with the chelating agent DOTA through a second condensation reaction, and the compound of formula (III) is obtained by a second post-treatment.

[0020] In one embodiment of the present invention, the second base comprises piperazine (PIP);

[0021] In one embodiment of the present invention, the second solvent comprises DMF;

[0022] In one embodiment of the present invention, the second post-treatment includes adding methanol and DCM washing and drying.

[0023] In one embodiment of the present invention, the reaction time for preparing compound (III) can be 3 hours, and the reaction conditions can be room temperature.

[0024] In one embodiment of the present invention, the compound represented by formula (II) is prepared from the compound represented by formula (I), and the synthetic route of the compound represented by formula (II) is shown below:

[0025]

[0026] In one embodiment of the present invention, in the method for preparing the compound of formula (II), the compound of formula (I) is reacted in a third solvent in the presence of a third resin and a third alkaline environment, and the compound of formula (II) is obtained by a third post-treatment.

[0027] In one embodiment of the present invention, the third resin comprises 2-CTC Resin;

[0028] In one embodiment of the present invention, the third alkalinity includes DIPEA;

[0029] In one embodiment of the present invention, the third solvent includes DCM.

[0030] In one embodiment of the present invention, the third post-treatment includes DMF washing and drying, etc.

[0031] In one embodiment of the present invention, the reaction time of the compound of formula (I) with the resin can be 3.5 h, and the reaction conditions can be room temperature.

[0032] Thirdly, the present invention provides the use of the compound DOTA-MDLA or a pharmaceutically acceptable salt thereof. The use of the compound DOTA-MDLA or a pharmaceutically acceptable salt thereof in the preparation of products for detecting diseases related to aspartate metabolism or ASS1.

[0033] In one embodiment of the invention, the ASS1-related disease may include a tumor.

[0034] In one embodiment of the present invention, the tumor is a type of tumor with abnormal aspartate metabolism, including but not limited to tumors with abnormal changes in ASS1 protein or resistance to platinum-based therapy, such as lung cancer, colon cancer, ovarian cancer, etc.

[0035] In one embodiment of the invention, the product includes a diagnostic tracer.

[0036] The application of the compound DOTA-MDLA or its pharmaceutically acceptable salt in the preparation of products for detecting ASS1-related diseases provided by this invention can specifically be the application of drugs for clinical / preclinical diagnosis and / or efficacy evaluation, and can be used in research on radiochemical labeling, preclinical animal imaging and treatment, as well as its application in products for ASS1-related diseases.

[0037] The compound DOTA-MDLA or its pharmaceutically acceptable salt provided by this invention can serve as a small molecule precursor for a PET probe targeting the ASS1 protein. This application's research has found that PET visualization of ASS1 can not only detect ASS1 expression levels in vivo, but also reflect the sensitivity of tumors to platinum-based drugs in vivo.

[0038] Fourthly, the present invention further modifies the compound DOTA-MDLA or its pharmaceutically acceptable salt, including but not limited to replacing it with a different radiolabeled nuclide (e.g., using a different radiolabeled nuclide for DOTA-MDLA). 177 Lu、 64 Cu、 90 Y、 225 Ac marker), and conventional modifications to DOTA-MDLA. Preferably, the present invention provides 68 The Ga-labeled compound DOTA-MDLA, denoted as 68 Ga-DOTA-MDLA.

[0039] Based on this, a series of radionuclide-labeled radiolabeled PET probes are provided, specifically radionuclide-labeled compounds DOTA-MDLA or pharmaceutically acceptable salts thereof; the radionuclides include 68 Ga, Tc-99 177 Lu、 64 Cu、 90 Y、 225 Ac.

[0040] Fifthly, the present invention provides 68 A radiolabeling method for Ga-labeled tracers.

[0041] A sort of 68 The Ga-labeled tracer radiolabeling method includes the following steps:

[0042] 1) Rinse the germanium-gallium generator with 0.1M HCl to obtain... 68 GaCl3 solution;

[0043] 2) Adjust with 0.1M NaAC 68 GaCl3 is adjusted to pH 2.3-3.5 to form a labeling system;

[0044] 3) Add the precursor solution to the labeling system in 2) above, and react at 100°C for 14 minutes to obtain... 68 Ga-labeled tracers;

[0045] The precursor is the compound DOTA-MDLA of Formula IV or a pharmaceutically acceptable salt thereof.

[0046] In one embodiment of the present invention, the concentration of the precursor solution used is 1 mg / mL.

[0047] In one embodiment of the present invention, the solvent in the precursor solution may be pure water.

[0048] Sixthly, utilizing the provided basis 68 A radiolabeling method for Ga-labeled tracers, producing reagents suitable for PET detection. 68 Ga-DOTA-MDLA molecular probe. 68 Ga-DOTA-MDLA molecular probe is a type of 68 Ga-labeled radiolabeled PET probes provide 68 The application of Ga-labeled radiolabeled PET probes in the preparation of PET / CT detection kits can be used for PET / CT detection.

[0049] This invention modifies the ASS1 substrate, aspartic acid analogue N-methylaspartic acid (α-Methyl-DL-aspartic acid, MDLA), and develops a novel PET probe for detecting ASS1 protein, DOTA-MDLA, which is the world's first PET probe for targeted detection of ASS1.

[0050] The compound DOTA-MDLA of Formula IV provided by this invention or its pharmaceutically acceptable salt is simple to prepare, has a short preparation time, high radiochemical yield, strong targeting, good specificity, and high uptake. It has good stability in both physiological saline and serum, and can detect the expression level of ASS1 at the cellular level and in mice bearing lung cancer, colon cancer, and ovarian cancer.

[0051] Furthermore, the present invention provides 68 Ga-labeled radiolabeled DOTA-MDLA has been used to produce a product suitable for PET detection. 68 The Ga-DOTA-MDLA molecular probe uses a labeling method that is simple to operate, has a short preparation time, and a high yield.

[0052] The compound DOTA-MDLA of Formula IV or its pharmaceutically acceptable salt provided by this invention can be used as a PET probe targeting the ASS1 protein. The PET probe has the specificity to target and bind to the ASS1 protein and can be used to detect the expression and changes of the ASS1 protein in tumor tissue in vivo to monitor aspartate catabolism, assess the resistance of tumors to platinum drugs, and guide the precise use of ASS1 drugs.

[0053] Given the significant heterogeneity and dual effects of ASS1 in anti-tumor therapy, non-invasive, in vivo, and whole-body visualization methods targeting ASS1 are crucial for precision tumor treatment. Therefore, the compound DOTA-MDLA (Formula IV) or its pharmaceutically acceptable salt, as a PET probe targeting the ASS1 protein, provided in this invention, offers an important and reliable molecular imaging tool for in vivo detection of ASS1 expression in clinical tumors, prediction and monitoring of platinum resistance, and guidance for accurate administration of drugs targeting aspartate metabolism. Furthermore, the PET probe targeting the ASS1 protein provides a non-invasive and effective imaging method for predicting and monitoring disease progression in potential beneficiaries of platinum-based therapy or aspartate metabolism therapy. This invention also provides new targets and a solid theoretical and experimental basis for the subsequent development of novel molecular probes.

[0054] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0055] (1) The preparation of the present invention 68 Ga-DOTA-MDLA exhibits high sensitivity and specificity for ASS1.

[0056] (2) The preparation of the present invention 68 Ga-DOTA-MDLA exhibits good stability.

[0057] (3) High uptake of platinum-resistant tumors mediated by ASS1 protein upregulation 68 Ga-DOTA-MDLA.

[0058] (4)Use 68 PET / CT imaging of the Ga-DOTA-MDLA probe can effectively reflect the tumor's response to platinum-based chemotherapy drugs, as well as the expression of ASS1 protein in tumor cells and changes in aspartate metabolism and / or urea cycle pathways. Attached Figure Description

[0059] Figure 1 The mass spectrum and proton spectrum of the compound DOTA-MDLA prepared in Example 1 are shown.

[0060] Figure 2 The radioactive iTLC spectrum of the compound DOTA-MDLA prepared in Example 2 is shown.

[0061] Figure 3 Example 2 illustrates the analysis of DOTA-MDLA and... by HPLC. 68 Ga-DOTA-MDLA.

[0062] Figure 4 As shown in Example 3 68Stability of Ga-DOTA-MDLA in PBS, fetal bovine serum and human serum.

[0063] Figure 5 As shown in Example 4 68 Blood clearance rate of Ga-DOTA-MDLA in mice.

[0064] Figure 6 The effects of ASS1 high- and low-expression cells in Example 5 are shown. 68 Results of Ga-DOTA-MDLA uptake.

[0065] Figure 7 The example shown in Example 6 68 Biodistribution of Ga-DOTA-MDLA in tumor-bearing mouse models at 0.5, 1, and 2 hours.

[0066] Figure 8 The images shown are PET / CT images of cisplatin-resistant tumor-bearing mouse models with high and low ASS1 expression from Example 7.

[0067] Figure 9 The images shown are PET / CT images of ovarian cancer tumor-bearing animal models with different ASS1 expression levels in Example 8 and Western blotting images of ASS1 protein level detection. Detailed Implementation

[0068] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0069] Example 1: Preparation of compound DOTA-MDLA

[0070]

[0071] The specific steps are as follows:

[0072] 1. Weigh 0.2 g of resin (as shown in Formula I), i.e., 2-CTC Resin (degree of substitution 1.0 mmol / g) and Fmoc-αMe-Asp(OtBu)-OH (425 mg), and place them in a solid-phase synthesis reactor. Add 10 ml of DCM, add 0.2 ml of DIPEA, and react under nitrogen for 3 hours. Add 0.2 ml of methanol and continue the reaction for 30 minutes. Wash three times with 10 ml of DMF to obtain intermediate resin II, i.e., Fmoc-αMe-Asp(OtBu)-2-CTC Resin.

[0073] 2. Removal of Fmoc: Add 10 ml of 20% PIP / DMF, react under nitrogen for 30 minutes, wash 5 times with 10 ml of DMF to obtain the intermediate resin H-αMe-Asp(OtBu)-2-CTC Resin.

[0074] 3. Condensation of DOTA(OtBu)3: Weigh 0.572 g of DOTA(OtBu)3 and 0.38 g of HATU, add them to a solid-phase synthesis reactor, add 10 ml of DMF, and purge with nitrogen to completely dissolve them. Add 0.9 ml of NMM and purge with nitrogen for 3 hours. Wash three times with 10 ml of DMF, three times with 10 ml of methanol, three times with 10 ml of DCM, and three times with 10 ml of methanol. Dry the intermediate resin to obtain 0.36 g of DOTA(OtBu)3-αMe-Asp(OtBu)-2-CTC Resin peptide resin.

[0075] 4. Resin lysis: 0.36g peptide resin was added to 10ml (95% TFA / H2O), reacted at room temperature for 1 hour, filtered and the filtrate was collected. The filtrate was precipitated with 50ml of ice-cold ether, centrifuged and the supernatant was discarded. The precipitate was washed 3 times with ether and dried to obtain product IV, namely DOTA-αMe-Asp-OH, 65mg, also known as DOTA-MDLA.

[0076] The mass spectrum and proton spectrum of the compound DOTA-MDLA prepared in this embodiment are as follows: Figure 1 As shown,

[0077] Example 2: Preparation of tracer

[0078] The preparation of a tracer using the compound DOTA-MDLA as a precursor includes the following steps:

[0079] 1) Dissolve DOTA-MDLA as a labeling precursor in sterile water for injection to prepare a precursor solution with a concentration of 1 mg / mL.

[0080] 2) The germanium-gallium generator was rinsed with 0.1M hydrochloric acid solution to obtain... 68 GaCl3 solution;

[0081] 3) Adjust with 0.1M NaAC 68 The pH of the GaCl3 solution was adjusted to 2.3-3.5 to form a reaction system;

[0082] 4) Add the precursor solution from 1) to the reaction system in 3) above, react at 100°C for 14 minutes, and obtain 68 Ga-labeled tracers 68 Ga-DOTA-MDLA.

[0083] Its radiochemical purity was determined by Radio-TLC and Radio-HPLC, and the relevant analytical spectra are shown below. Figure 2 and Figure 3 As shown.

[0084] Results: After preparing the precursor solution, the following were obtained from the elution. 68 Starting with GaCl3 solution, the tracer was successfully prepared within 20 minutes. 68 Ga-DOTA-MDLA has a radiochemical purity of over 95%.

[0085] The tracer prepared in Example 2 was prepared in this embodiment. 68 The radioactive iTLC spectrum of Ga-DOTA-MDLA is as follows: Figure 2 As shown.

[0086] DOTA-MDLA and [other components] in this embodiment were analyzed by HPLC. 68 Ga-DOTA-MDLA, results as follows Figure 3 As shown.

[0087] Example 3: Stability Experiment

[0088] Collect and prepare human serum for later use. Add 200 μL of PBS, fetal bovine serum (FBS), or 200 μL of human serum to several EP tubes, respectively. Add 1 mCi of freshly prepared human serum to each of the above EP tubes. 68 Ga-DOTA-MDLA was incubated in a water bath at room temperature (25℃) or 37℃ for different times (1, 2, 3 h). After incubation, 300 μL of ice-cold acetonitrile was added to FBS and human serum EP tubes, respectively, and vortexed to precipitate plasma proteins. Then, 300 μL of water for injection was added and vortexed. After centrifugation at room temperature (12000 g / min, 5 min), the plasma proteins were separated, and the supernatant was used to determine the radiochemical purity by Radio-TLC. The mixture in the PBS tube was directly determined to have radiochemical purity by Radio-TLC after incubation. 68 The stability of Ga-DOTA-MDLA in PBS, FBS, or human serum is as follows: Figure 4 As shown.

[0089] result: 68 The Ga-labeled DOTA-MDLA tracer remained intact and undegraded in vitro in PBS (room temperature 25°C), fetal bovine serum, and human serum (37°C); indicating that the tracer... 68 Ga-DOTA-MDLA exhibits high stability in PBS and serum.

[0090] Example 4: Blood Clearance Experiment

[0091] Prepare several BALB / c mice and take 3.7 MBq of pre-labeled... 68 Ga-DOTA-MDLA solution was injected into mice via the tail vein. The contralateral tail vein was punctured, and blood samples were collected at 1, 3, 5, 10, 15, 30, 60, 90, and 120 minutes post-injection using a capillary tube. The Cpm (radioactivity count) values ​​of the collected blood samples were immediately measured using a gamma counter, with each time point independently repeated five times. The processed Cpm measurements were then subjected to biphasic attenuation fitting using GraphPad Prism 9.0 software. The fitting formula is as follows:

[0092] Y=Plateau+SpanFast*exp(-KFast*X)+SpanSlow*exp(-KSlow*X)

[0093] 68 The blood clearance rate of Ga-DOTA-MDLA in BALB / c mice is as follows: Figure 5 As shown.

[0094] Results: Bidirectional attenuation analysis yielded... 68 The distribution phase half-life (t1 / 2) of Ga-DOTA-MDLA in BALB / c mice was 2.0 min, and the blood clearance phase half-life (t1 / 2) was 56.2 min.

[0095] Example 5: Cell Uptake Experiment

[0096] Experimental studies have revealed that the cisplatin-sensitive cell line A549... P A2780 P HCT-8 P The cell line was ASS1-low expression, while the cisplatin-resistant cell line was A549. CisR A2780 CisR HCT-8 CisR The cell line highly expresses ASS1.

[0097] The cells were seeded into 6-well plates, with approximately 2 x 10 cells per well. 5 After the cells adhere, the labeled 68Ga-DOTA-MDLA (185 kBq) was added to each well, and the cells were incubated at 37°C for 30 min. After incubation, the culture medium was discarded, and the tumor cells were gently washed three times with 4°C PBS (0.5 mL / well). The tumor cells were then digested with 0.25% trypsin (1 mL / well) to detach them from the 6-well plate, and transferred to the corresponding test tubes. Each well was then rinsed three times with 4°C PBS (0.5 mL / well), and the PBS was transferred to the corresponding cell tubes. The process was monitored using an optical microscope to ensure complete cell separation and collection. The radioactivity of the cell suspension in each tube was measured using a gamma counter. The experiment was repeated three times.

[0098] Cisplatin-sensitive (low ASS1 expression) and cisplatin-resistant (high ASS1 expression) cell lines 68 Ga-DOTA-MDLA uptake results as follows Figure 6 As shown.

[0099] Example 6: In vivo biodistribution experiment

[0100] Prepare several BALB / c nude mice and take 7.4 MBq of pre-labeled... 68 Ga-DOTA-MDLA solution was injected into mice via the tail vein. Mice were sacrificed at 0.5, 1, and 2 hours, and dissected to remove tissues and organs including the heart, liver, spleen, lungs, kidneys, brain, duodenum, stomach, bladder, bones, blood, muscles, and tumors. The radioactivity of these tissues and organs was immediately measured using a gamma counter, and the radioactivity per gram of tissue or organ was calculated. The uptake value within the tissues and organs is expressed as the percentage of the injected dose taken up per gram of tissue (%ID / g).

[0101] 68 Ga-DOTA-MDLA in A549 CisR In the tumor-bearing BALB / c nude mouse model, at 0.5, 1, and 2 hours... Figure 7 As shown. 68 Ga-DOTA-MDLA exhibits significantly high biodistribution in tumors with high ASS1 expression.

[0102] result: 68 Ga-DOTA-MDLA is highly expressed in A549 on ASS1. CisR It is highly uptaken in tumor tissues. In addition, organs and tissues such as the heart, brain, duodenum, stomach, bones, and muscles also absorb it. 68 Ga-DOTA-MDLA low uptake. 68 Ga-DOTA-MDLA is primarily excreted through the kidneys and urinary system. This result indicates... 68The Ga-DOTA-MDLA probe is specific for detecting tumor ASS1 expression levels in in vivo PET imaging, and also indicates high background uptake in the kidneys and urinary system.

[0103] Example 7: PET / CT Imaging Experiment of Cisplatin-Resistant Tumor-Bearing Animal Models with High and Low ASS1 Expression

[0104] Build A549 P A2780 P A549 CisR A2780 CisR A BALB / c nude mouse subcutaneous xenograft model was established, and small animal PET / CT imaging was performed when the tumor reached a long diameter of 1 cm. The labeled... 68 The Ga-DOTA-MDLA probe was injected into mice via the tail vein, with each tumor-bearing mouse receiving a dose of 7.4-11.1 MBq. Imaging was performed 30 minutes after injection. The uptake of the probe by each tumor tissue was analyzed using Inveon Research Workplace image analysis software.

[0105] PET / CT images of tumor-bearing animal models with high and low ASS1 expression are shown below. Figure 8 As shown.

[0106] Results: A549 cisplatin-resistant tumor cells with high ASS1 expression. CisR A2780 CisR The constructed subcutaneous xenograft model for 68 A549, a cisplatin-sensitive tumor cell line with high Ga-DOTA-MDLA uptake and low ASS1 expression. P A2780 P right 68 Low Ga-DOTA-MDLA uptake. These experimental results demonstrate that cisplatin-resistant tumors induced by ASS1 overexpression... 68 The Ga-DOTA-MDLA probe exhibits high specificity and uptake.

[0107] Example 8: PET / CT Imaging Experiment of Ovarian Cancer Tumor-Bearing Animal Models with Different ASS1 Expression Levels

[0108] Subcutaneous xenograft models of OVCA433, HeyA8, ES2, A2780, OVCAR5, SKOV3, and OVCAR8 BALB / cnude mice were constructed. Small animal PET / CT imaging was performed when the tumors reached a long diameter of 1 cm. The labeled tumors were then... 68The Ga-DOTA-MDLA probe was injected into mice via the tail vein, with each tumor-bearing mouse receiving a dose of 7.4-11.1 MBq. Imaging was performed 30 minutes after injection. The uptake of the probe by each tumor tissue was analyzed using Inveon Research Workplace image analysis software.

[0109] PET / CT images of ovarian cancer-bearing animal models with different ASS1 expression levels are as follows: Figure 9 As shown, Figure A is 68 PET / CT images of Ga-DOTA-MDLA in subcutaneous xenograft tumor models of OVCA433, HeyA8, ES2, A2780, OVCAR5, SKOV3, and OVCAR8 mice; Figure B shows the expression of ASS1 in tumor tissues detected by Western Blot experiment.

[0110] Results: Subcutaneous xenograft models constructed from ovarian cancer cells expressing different ASS1 types (OVCA433, HeyA8, ES2, A2780, OVCAR5, SKOV3, and OVCAR8) showed efficacy against [the disease / problem]. 68 Ga-DOTA-MDLA exhibits varying degrees of uptake. Tumors with higher ASS1 expression are more susceptible to... 68 Higher Ga-DOTA-MDLA uptake was positively correlated with ovarian cancer. These experimental results demonstrate that ovarian cancers with different ASS1 expression levels... 68 Ga-DOTA-MDLA probes specifically take up the protein.

[0111] Examples 3-8 lead to the following conclusions:

[0112] (1) Stability experiments show 68 Ga-DOTA-MDLA exhibits excellent stability in PBS and serum.

[0113] (2) Blood clearance experiment showed 68 After intravenous injection, Ga-DOTA-MDLA can be rapidly distributed throughout the body's tissues and organs via circulation. It also has a short half-life in the blood, making it suitable as an imaging probe.

[0114] (3) Cell uptake experiments confirmed that cells... 68 Ga-DOTA-MDLA uptake is positively correlated with ASS1 expression in cells.

[0115] (4) In vivo biodistribution experiments showed that tumor tissues with high ASS1 expression were effective against... 68 Ga-DOTA-MDLA high uptake 68 Ga-DOTA-MDLA is mainly excreted in the body through the kidneys and urinary system. Other tissues and organs in the body have low uptake of this probe, i.e., low background, and good tumor specificity.

[0116] (5) Animal PET / CT imaging experiments proved 68 Ga-DOTA-MDLA exhibits good specificity and sensitivity against tumor tissues with high ASS1 expression, with a high target-to-species ratio and strong specificity; it is effective against platinum-resistant tumors with high ASS1 expression. 68 High uptake of Ga-DOTA-MDLA.

[0117] (6) In conclusion, 68 Ga-DOTA-MDLA can be used as a tumor imaging probe to image tumor ASS1 and identify the occurrence of platinum resistance in tumors associated with upregulated ASS1 levels.

[0118] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention. For example, Tc-99, 177 Lu、 64 Cu、 90 Y、 225 MDLA labeled with radionuclides such as Ac are all within the scope of protection of this patent.

Claims

1. The compound DOTA-MDLA or a pharmaceutically acceptable salt thereof, characterized in that, The compound DOTA-MDLA is shown in Formula IV.

2. A method for preparing the compound DOTA-MDLA, characterized in that, The compound shown in formula (III) was reacted in the presence of a first acid, and then subjected to a first post-treatment to obtain the compound shown in formula (IV), DOTA-MDLA:

3. The method for preparing the compound DOTA-MDLA according to claim 2, characterized in that, The compound shown in formula (III) is prepared from the compound shown in formula (II). The compound shown in formula (II) was deprotected from Fmoc by 20% PIP / DMF, and further synthesized by solid-state synthesis and condensation with a DOTA chelating agent to obtain the compound shown in formula (III).

4. The method for preparing the compound DOTA-MDLA according to claim 3, characterized in that, The compound shown in formula (II) is prepared from the compound shown in formula (I). The synthetic route for the compound represented by formula (II) is shown below: The compound shown in formula (I) was reacted in DCM in the presence of 2-CTC Resin and DIPEA, and then post-treated to give the compound shown in formula (II).

5. The use of the compound DOTA-MDLA of claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The use of compound DOTA-MDLA or a pharmaceutically acceptable salt thereof in the preparation of products for the detection of diseases associated with abnormal aspartate metabolism or ASS1; The diseases associated with abnormal aspartate metabolism are tumors or metabolic diseases caused by abnormal changes in the aspartate metabolic pathway. The diseases associated with ASS1 are tumors or metabolic diseases caused by abnormal changes in the urea cycle related to ASS1. The diseases include tumors with high ASS1 expression and the types of platinum-resistant tumors mediated by them, including but not limited to lung cancer, colon cancer, and ovarian cancer.

6. The application according to claim 5, characterized in that, The products include diagnostic tracers.

7. A radiolabeled PET probe, characterized in that, The compound DOTA-MDLA of claim 1, or a pharmaceutically acceptable salt thereof, is labeled with a radionuclide. The nuclides include 68 Ga, Tc-99 177 Lu、 64 Cu、 90 Y、 225 Ac.

8. A kind 68 Ga-labeled radiolabeled PET probe, characterized in that... for 68 The Ga-labeled compound of claim 1, DOTA-MDLA, or a pharmaceutically acceptable salt thereof.

9. A kind 68 A Ga-labeled tracer radiolabeling method, characterized in that... Includes the following steps: 1) Rinse the germanium-gallium generator with 0.1M HCl to obtain... 68 GaCl3 solution; 2) Adjust with 0.1M NaAC 68 GaCl3 is adjusted to pH 2.3-3.5 to form a labeling system; 3) Add the precursor solution to the labeling system in 2) above, and react at 100°C for 14 minutes to obtain... 68 Ga-labeled tracers; The precursor is the compound DOTA-MDLA of Formula IV or a pharmaceutically acceptable salt thereof.

10. The claim 8 68 Application of Ga-labeled radiolabeled PET probes in the preparation of PET / CT detection kits.

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