A radioactive metal chelate and a preparation method and application thereof

Abstract

The application provides a radioactive metal chelate and a preparation method and application thereof, and belongs to the technical field of biological medicines.The radioactive metal chelate contains a dye derivative which can be efficiently chelated with a radioactive metal, and the dye derivative is obtained by coupling a desferrioxamine chelating group to a heptamethyl carbon cyanine dye as a mother nucleus; the structure can be rapidly and efficiently coordinated with the radioactive metal, and thus the radioactive metal chelate with high labeling rate and good radiochemical stability in a protein-containing environment is obtained, the radioactive metal chelate is used for long-term tumor PET imaging, the significant effect that the probe can realize long-time and high-contrast tumor imaging is realized, continuous visual monitoring and delayed diagnosis of malignant tumors can be realized, and a new technical path is provided for long-acting molecular imaging.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a radioactive metal chelate, its preparation method, and its application. Background Technology

[0002] Radiomold imaging is an important tool for achieving precise tumor diagnosis. By conjugating radionuclides with small molecule ligands, peptides, or antibodies, the metabolic and receptor expression status of tumors can be reflected at the molecular level. Currently, commonly used clinical imaging agents include metalloid imaging agents (such as...) 68 Ga、 64 Cu、 89 Zr and other radioactive probes primarily rely on chelating agents to form complexes with target molecules to achieve specific recognition. However, traditional small-molecule radioactive probes generally suffer from drawbacks such as rapid in vivo clearance, short tumor retention time, and limited imaging windows. Especially in solid tumors requiring long-term monitoring, their pharmacokinetic properties often do not match the half-life of the radionuclide, resulting in rapid signal decay and low imaging contrast.

[0003] Heptamethine carbocyanine dye (HMCD) is a class of organic dyes with near-infrared fluorescence properties and natural tumor-targeting capabilities. It can actively accumulate in tumor cell mitochondria via the HIF-1α / OATP pathway and remain in vivo for extended periods. In recent years, derivatives based on the HMCD backbone (such as IR-780, DZ-1, and MHI-148) have been widely used in optical imaging and tumor-targeted drug delivery research, demonstrating good biocompatibility and specificity. However, the original HMCD molecule suffers from poor water solubility, low stability, and a lack of metal chelating sites, limiting its application in the field of radiographic imaging.

[0004] To address these issues, researchers have attempted to introduce hydrophilic groups or chelating agents such as DOTA onto the HMCD backbone. For example, existing technologies... 68 The Ga-DOTA-DZ-1 probe achieved short-term PET imaging in a liver cancer model, but due to... 68 Ga has a short half-life (approximately 68 minutes), resulting in a limited imaging window; 64 While Cu-labeled HMCD derivatives have prolonged imaging time to some extent, they are prone to liver and kidney redistribution and increased background signal. Therefore, developing novel radionuclide-dye composite probes that are pharmacokineticly compatible and can achieve high stability and long-term imaging has become an important direction in the field of molecular imaging.

[0005] Existing technologies include those using DOTA as a chelating agent. 68Ga-labeled DZ derivatives. These probes typically chelate metal ions through a DOTA-DZ backbone to form a relatively stable radioactive complex, enabling early tumor imaging. However, their imaging window is generally less than 3 hours, and the signal decays rapidly, making delayed quantification and dynamic lesion tracking difficult. Especially for tumors with slow pharmacokinetic metabolism or abundant stroma, short-lived radionuclides cannot cover their optimal imaging time, leading to decreased image contrast and diagnostic accuracy. Summary of the Invention

[0006] The purpose of this invention is to provide a radioactive metal chelate, its preparation method and application, which has significant advantages in terms of extended imaging time, enhanced stability and improved imaging resolution. It can realize continuous visual monitoring and delayed diagnosis of malignant tumors, and provide a new technical path for long-term molecular imaging.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a radioactive metal chelate, which is a complex formed by chelating a compound of Formula I with a radioactive metal ion through a deferoxamine chelating group; Formula I;

[0009] The radioactive metal ions are selected from zirconium-89;

[0010] The method for preparing the radioactive metal chelate includes reacting the compound of Formula I with radioactive metal ions in a buffer solution with a pH of 6.0 to 7.0.

[0011] The present invention also provides the application of the above-mentioned radioactive metal chelate in the preparation of tumor imaging diagnostic agents.

[0012] Preferably, the tumor is cervical cancer or lung cancer.

[0013] The present invention also provides a pharmaceutical composition comprising the above-mentioned radioactive metal chelate and a pharmaceutically acceptable carrier.

[0014] The present invention also provides a method for preparing the above-mentioned radioactive metal chelate, comprising reacting the compound shown in Formula I with radioactive metal ions in a buffer solution with a pH of 6.0 to 7.0;

[0015] The preparation method of the compound shown in Formula I includes the following steps:

[0016] Compound DZ-1 is reacted with an activating reagent under alkaline conditions and in a first organic solvent to generate an activated intermediate compound;

[0017] The activated intermediate compound was coupled with deferoxamine in the presence of a second organic solvent and an organic base to obtain the compound.

[0018] The compound DZ-1 has the structure shown in formula a: Formula a.

[0019] Preferably, the activating agent is N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate, the first organic solvent is dichloromethane, and the alkaline conditions are provided by triethylamine.

[0020] Preferably, the second organic solvent is dimethyl sulfoxide, and the organic base in the coupling reaction is N,N-diisopropylethylamine.

[0021] The beneficial effects of this invention are:

[0022] The radioactive metal chelate provided by this invention contains zirconium-89 ( 89 The highly efficient Zr-chelated dye derivative DZ-HX1 is obtained by chemically coupling a desferrioxamine (DFO) chelating group with heptamethylcarbocyanine dye (DZ-1) as the parent core; this structure can react with Zr under near-neutral to slightly acidic conditions. 89 Zr provides rapid and efficient coordination, resulting in high labeling rates and good radiochemical stability in protein-containing environments. 89 Zr-DZ-HX1 is used for long-term tumor PET imaging.

[0023] The present invention provides 89 The Zr-DZ-HX1 complex represents a key breakthrough in both structure and performance, effectively preventing in vivo metal dechelation or transfer and significantly improving radiochemical purity and biological stability. 89 The pharmacokinetic properties of Zr and DZ-HX1 are highly compatible, enabling the probe to achieve significant effects such as long-duration, high-contrast tumor imaging. Furthermore, the optimized structure of HX1 significantly improves its water solubility and blood circulation characteristics, resulting in higher uptake rates and lower off-target background in tumor tissue. This invention… 89 Zr-DZ-HX1 has significant advantages in terms of extended imaging time, enhanced stability, and improved imaging resolution, enabling continuous visual monitoring and delayed diagnosis of malignant tumors, and providing a new technical approach for long-term molecular imaging. Attached Figure Description

[0024] Figure 1 For HPLC detection 89 The labeling rate of Zr-DZ-HX1, where: (A) pH 6~7, 89 In the Zr-chloride system 89 (B) Labeling rate analysis of Zr-DZ-HX1; pH 6–7, 89 In the Zr-oxalate system89 Labeling rate analysis of Zr-DZ-HX1; (C) pH 7-8, 89 In the Zr-chloride system 89 Labeling rate analysis of Zr-DZ-HX1; (D) pH 7–8, 89 In the Zr-oxalate system 89 Labeling rate analysis of Zr-DZ-HX1;

[0025] Figure 2 Radio-TLC detection 89 Figure showing the in vitro stability of Zr-DZ-HX1 under room temperature, PBS, and FBS conditions;

[0026] Figure 3 Tail vein injection for HeLa subcutaneous xenograft model 89 PET / CT images at different time points after Zr-DZ-HX1, with dashed circles indicating subcutaneous tumors in the images;

[0027] Figure 4 For micro-PET quantitative analysis of HeLa tumor-bearing animal models, tail vein injection 89 Radioactive uptake of tumors and vital organs at different time points after Zr-DZ-HX1 (A) and tumor / organ uptake ratio (B).

[0028] Figure 5 Injection into an animal model of lung carcinoma in situ 89 PET / CT images at different time points after Zr-DZ-HX1, with arrows indicating lung carcinoma in situ;

[0029] Figure 6 for 89 The distribution of Zr-DZ-HX1 in the HeLa tumor-bearing animal model over 168 h is shown in the figure, where: (A) the change of the percentage of radioactive injection dose in each tissue over time; and (B) the change of the ratio of radioactive uptake by the tumor to that of key organs over time. Detailed Implementation

[0030] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0031] Example

[0032] First, compound 5 was synthesized. The synthetic route is as follows: DZ-1 (1.0 equiv.) (purchased from Kairui Biotechnology Co., Ltd.) was dissolved in dichloromethane (DCM, 2 mL), and triethylamine (TEA, 2.0 equiv., 0.286 mmol) and N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate (TSTU, 1.5 equiv., 0.173 mmol) were added. After stirring at room temperature for 5 minutes, the reaction was monitored by LC-MS, which showed complete conversion of the starting material DZ-1. Water was then added, and the mixture was extracted with dichloromethane (6 mL × 3). The organic phases were combined, dried over anhydrous Na2SO4, and then distilled under reduced pressure. The crude reaction solution was purified by silica gel column chromatography (SiO2, DCM: MeOH = 10:1) to give 136 mg of green crude solid product 5 (C 44 H 52 ClN3O7S), with a yield of 76%. LC-MS analysis showed that the molecular weight was consistent with the theoretical molecular weight of the target compound (802.42), and the measured value was [M+H]. + : 802.30. 1 H NMR (400 MHz, MeOD) δ 8.44(t, J = 14.6 Hz, 2H), 7.55 – 7.49 (m, 2H), 7.48 – 7.38 (m, 3H), 7.29 (ddd, J= 17.5, 9.1, 6.5 Hz, 3H), 6.38 (d, J = 14.1 Hz, 1H), 6.28 (d, J = 14.0 Hz,1H), 4.21 (dt, J = 24.1, 7.3 Hz, 4H), 2.89 (t, J = 7.1 Hz, 2H), 2.84 (s, 3H),2.79 – 2.64 (m, 6H), 2.09 – 1.78 (m, 11H), 1.74 (d, J = 2.5 Hz, 12H), 1.61 (tt, J = 11.6, 5.9 Hz, 2H).

[0033] The synthetic route for compound 6 (DZ-HX1) is as follows:

[0034] The synthetic route for compound 6 (DZ-HX1) is as follows: Compound 5 (1.0 equiv.) was dissolved in dimethyl sulfoxide (DMSO, 4 mL), followed by the addition of deferoxamine (1.0 equiv., 0.171 mmol). Next, N,N-diisopropylethylamine (DIPEA, 2.0 equiv., 0.325 mmol) dissolved in DMSO (1 mL) was added dropwise to the reaction mixture over 1 minute at room temperature. After stirring at room temperature for 5 minutes, LC-MS monitoring showed that reactant 5 had reacted completely. Since heptamethylcarbocyanine dye derivatives are highly sensitive to alkaline systems, trifluoroacetic acid was added to adjust the pH of the reaction system to 6.0–7.0. After the reaction was complete, the mixture was washed with water and extracted with dichloromethane (6 mL × 3). The combined organic phases were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by preparative HPLC (Luna C18(2) 100A, 10 μm, 10 × 250 mm preparative column) to obtain 11.0 mg of green solid compound 6. (C 65 H 95 ClN8O 12 S), yield 7%. LC-MS analysis showed that the molecular weight was consistent with the theoretical molecular weight of the target compound, 1248.03, and the measured value was [M+2H]. 2+ : 624.50. 1 H NMR (400 MHz, DMSO- d 6 ) δ 9.61-9.60 (m, 3H), 8.29-8.21 (m, 2H),7.78 (t, J = 4.8 Hz, 2H), 7.72 (t, J = 5.2 Hz, 1H), 7.65-7.60 (m, 2H), 7.52(d, J = 8.4 Hz, 1H), 7.46-7.40 (m, 3H), 7.32-7.26 (m, 2H), 6.42 (d, J = 14.0Hz, 1H), 6.28 (d, J = 14.0 Hz, 1H), 4.24-4.17 (m, 4H), 3.46-3.42 (m, 6H), 3.02-2.94 (m, 6H), 2.73-2.67 (m, 4H), 2.58-2.55 (m, 4H), 2.52-2.51 (m, 2H),2.26 (t, J = 7.2 Hz, 4H), 2.04 (t, J= 7.2 Hz, 2H), 1.96 (s, 3H), 1.85-1.80(m, 4H), 1.77-1.70 (m, 4H), 1.67 (d, J = 3.2 Hz, 12H), 1.56-1.48 (m, 8H), 1.37-1.30 (m, 8H), 1.23-1.19 (m, 6H).

[0035] DZ-HX1 89 Zr labeling and detection

[0036] pH 89 Effect of Zr-DZ-HX1 labeling rate

[0037] To clarify 89 The optimal reaction conditions for Zr-labeled DZ-HX1 were investigated in this invention, and different forms of zirconium-89 were systematically examined. 89 Zr-chloride / 89 Zr-oxalate and the effect of pH on labeling efficiency.

[0038] like Figure 1 As shown, in zirconium chloride ( 89 Zr-chloride) is 89 In the Zr-based labeling system, different pH conditions significantly affected the labeling efficiency. When using pH 8–9 (Na₂CO₃, 2 M, 50 μL), the labeling system changed color from green to red with precipitation, and the labeling rate was 0%. Under pH 7–8 (Na₂CO₃, 2 M, 20 μL), the reaction system also showed color change and precipitation, with the labeling rate remaining 0%. However, in a buffer system at pH 6–7 (CH₃COONa, 1 M, 100 μL), the reaction solution remained clear green without precipitation, and the labeling rate increased to 90%. The experiments show that… 89 Zr-chloride The reaction labeled DZ-HX1 is extremely sensitive to alkaline conditions, but exhibits better coupling ability under acidic conditions at pH 6–7.

[0039] In zirconium oxalate ( 89 Zr-oxalate) is 89In the labeling experiments using Zr source, pH showed a similar effect on the labeling rate. At pH 8–9 (Na₂CO₃, 2 M, 50 μL) and pH 7–8 (Na₂CO₃, 2 M, 30 μL), the reaction of oxalate ions with sodium carbonate both resulted in a change in solution color from green to red and precipitation, with a labeling rate of 0%. When using a combined buffer system at pH 6–7 (Na₂CO₃ + HEPES, 2 M, 20 μL), the labeling efficiency reached 90%. These results suggest that in a weakly acidic environment (pH 6–7), 89 Zr-oxalate and DZ-HX1 can be efficiently coupled, and the labeling system is extremely sensitive under high pH conditions (7-9).

[0040] Therefore, different forms 89 Zr ( 89 Zr-chloride / 89 The labeling efficiency of Zr-oxalate for DZ-HX1 is affected by the pH value of the reaction system. At higher pH conditions (7–9), the Na2CO3 buffer system causes a color change in the reaction solution accompanied by precipitation, resulting in low or zero labeling efficiency. However, at lower pH conditions (6–7), the CH3COONa and HEPES buffer system improves the reaction state, stabilizing the system and producing a clear green reaction solution with a labeling rate reaching 90%. Different forms of... 89 Zr exhibits a consistent labeling trend under similar conditions, indicating that optimizing the pH of the reaction system is crucial for improving labeling efficiency.

[0041] 89 In vitro stability of Zr-DZ-HX1

[0042] For evaluation 89 The in vitro stability of Zr-DZ-HX1 was investigated in this invention, specifically examining the change in its labeling rate over time under PBS, FBS, and room temperature conditions. The results showed that at 100 μg DZ-HX1 substrate and 37 MBq... 89 Zr-oxalate was reacted in a buffer system (containing Na2CO3 and HEPES) with a pH of 6.0–7.0 at room temperature for 30 min. 89 The initial labeling rate of Zr-DZ-HX1 reached 98.42 ± 0.27%.

[0043] 89 The stability of Zr-DZ-HX1 in different media is as follows: Figure 2 As shown, 89 The labeling rate of Zr-DZ-HX1 after incubation with PBS for 4 h dropped sharply to 74.81 ± 11.88%. 89The labeling rate of Zr-DZ-HX1 after incubation with FBS for 24 h remained at 94.99 ± 2.36%, decreasing to 79.62 ± 4.85% after 168 h of incubation. Under room temperature conditions, 89 The labeling rate of Zr-DZ-HX1 remained at 96.53 ± 2.60% after 24 h, and decreased to 87.08 ± 5.50% after 168 h.

[0044] therefore, 89 Zr-DZ-HX1 exhibits the best stability when placed alone at room temperature, maintaining a high labeling rate for 168 h; however, it shows the worst stability in the PBS system, with significant dissociation occurring in just 4 h; its stability in the FBS system is relatively good but still shows a certain downward trend.

[0045] 89 The LogP of Zr-DZ-HX1 is -0.57 ± 0.04, indicating that the probe is hydrophilic.

[0046] 89 Micro-PET / CT Imaging Study of Zr-labeled DZ-HX1

[0047] 89 Zr-DZ-HX1 exhibited significant long-term circulating characteristics and tumor targeting in vivo distribution and metabolism in HeLa cervical cancer-bearing mice. For example... Figure 3 As shown, 89 At 12 h after tail vein injection of Zr-DZ-HX1, the radioactive uptake at the tumor site was 2.75 ± 0.58%ID / g, which increased to 3.01 ± 0.46%ID / g at 24 h, peaked at 48 h (3.52 ± 0.64%ID / g), and then gradually decreased to 3.08 ± 0.85%ID / g at 72 h and decreased to 1.90 ± 0.50%ID / g at 168 h. 89 Zr-DZ-HX1 clearly visualized the tumor site during the imaging cycle, with a distinct boundary between the tumor and surrounding tissues, demonstrating good tumor uptake and retention characteristics.

[0048] 89Zr-DZ-HX1 is primarily metabolized by the liver. Hepatic radioactive uptake is high, with significant radioactive retention. The peak hepatic uptake occurs at 72 h (7.71 ± 3.93% ID / g) and remains at 4.90 ± 2.54% ID / g at 168 h. Renal uptake is 2.38 ± 0.31% ID / g at 12 h, then gradually decreases, indicating partial renal metabolism. Cardiac and muscle uptake are low, consistently below 2.30% ID / g and 0.40% ID / g throughout the imaging cycle, with low background signal. Bone tissue uptake gradually increases over time, reaching 1.19 ± 0.31% ID / g at 168 h, suggesting a possible presence of some radioactive residues in the body. 89 Zr de-standardization phenomenon ( Figure 4 ).

[0049] A lung carcinoma in situ model was constructed using cells with a Kras-mutated background, and the results were examined. 89 Metabolic characteristics and targeting of Zr-DZ-HX1 in an orthotopic lung cancer model. Results showed... 89 Zr-DZ-HX1 also showed good targeting in orthotopic lung tumor models. 12 hours after injection, visible radioactive signals appeared in the tumor area (indicated by the arrow). Figure 5 The tumor uptake value begins to accumulate. At 24 hours, the signal at the tumor site further increases, reaching a peak at 96 hours, showing a high tumor uptake rate and significant target specificity. Subsequently, the radioactive signal gradually weakens, and a clear tumor imaging signal can still be observed at 120 hours. At 168 hours, the tumor signal further weakens, but the background signal remains at a low level, and the imaging contrast is good.

[0050] 89 In vivo distribution experiment of Zr-labeled DZ-HX1

[0051] Injection in tumor-bearing animal models 89 Following Zr-DZ-HX1, biodistribution was assessed and quantitatively evaluated at 4, 24, 48, 96, and 168 h. 89 Metabolic patterns and targeting characteristics of Zr-DZ-HX1 in a HeLa tumor-bearing mouse model of cervical cancer. Results are as follows: Figure 6 As shown in Table 1:

[0052] Table 1 89 In vivo distribution of Zr-DZ-HX1 in HeLa cervical cancer-bearing mice within 168 h The results show that 4 hours after administration, 89Zr-DZ-HX1 rapidly distributed to the liver (25.76 ± 3.04% ID / g), blood (16.00 ± 6.17% ID / g), and kidneys (7.83 ± 1.15% ID / g); followed by accumulation in the spleen (5.91 ± 1.56% ID / g) and lungs (4.62 ± 1.15% ID / g). Over time, hepatic radioactive uptake did not significantly decrease, remaining at 14.46 ± 9.44% ID / g at 24 h and maintaining at 14.39 ± 5.37% ID / g at 168 h. The renal uptake value gradually increased from 7.83 ± 1.15%ID / g at 4 h to 15.42 ± 1.87%ID / g at 96 h, and then decreased to 9.53 ± 1.37%ID / g at 168 h, indicating that the liver plays a role in the continuous metabolism process, while the kidneys are mainly responsible for clearance in the middle and late stages.

[0053] Radioactive uptake in tumor tissue gradually increased from 5.70 ± 0.59% ID / g at 4 h to 7.40 ± 2.28% ID / g at 48 h, peaking at 7.74 ± 2.00% ID / g at 96 h, and still showing a high uptake value (4.21 ± 0.10% ID / g) at 168 h. 89 Zr-DZ-HX1 exhibits highly efficient tumor targeting and long-term retention. Radioactive uptake in bone remained consistently high, decreasing from 2.96 ± 1.38%ID / g at 4 h to 2.44 ± 0.96%ID / g at 48 h, reaching 2.56 ± 0.34%ID / g at 96 h, and still remaining at 2.16 ± 0.98%ID / g at 168 h, suggesting the presence of... 89 Zr delabeling was observed. Radioactivity distribution in the stomach, small intestine, muscle, and brain remained consistently low, at 1.26 ± 0.12%ID / g, 1.12 ± 0.12%ID / g, 0.66 ± 0.11%ID / g, and 0.09 ± 0.05%ID / g at 168 h, respectively. The radioactivity retention characteristics in tumor tissue indicate good drug targeting and a long peak uptake time, enabling sustained distribution within the tumor tissue.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A radioactive metal chelate, characterized in that, The radioactive metal chelate is a complex formed by the chelation of the compound shown in Formula I with a radioactive metal ion through a deferoxamine chelating group. Formula I; The radioactive metal ions are selected from zirconium-89; The method for preparing the radioactive metal chelate includes reacting the compound of Formula I with radioactive metal ions in a buffer solution with a pH of 6.0 to 7.

0.

2. The use of the radioactive metal chelate according to claim 1 in the preparation of tumor imaging diagnostic agents.

3. The application according to claim 2, characterized in that, The tumor is either cervical cancer or lung cancer.

4. A pharmaceutical composition, characterized in that, It comprises the radioactive metal chelate of claim 1 and a pharmaceutically acceptable carrier.

5. The method for preparing the radioactive metal chelate according to claim 1, characterized in that, This includes reacting the compound shown in Formula I with radioactive metal ions in a buffer solution with a pH of 6.0 to 7.0; The preparation method of the compound shown in Formula I includes the following steps: Compound DZ-1 is reacted with an activating reagent under alkaline conditions and in a first organic solvent to generate an activated intermediate compound; The activated intermediate compound was coupled with deferoxamine in the presence of a second organic solvent and an organic base to obtain the compound. The compound DZ-1 has the structure shown in formula a: Formula a.

6. The preparation method according to claim 5, characterized in that, The activating agent is N,N,N′,N′-tetramethyl-O-(N-succinimide)urea tetrafluoroborate, the first organic solvent is dichloromethane, and the alkaline conditions are provided by triethylamine.

7. The preparation method according to claim 5, characterized in that, The second organic solvent is dimethyl sulfoxide, and the organic base in the coupling reaction is N,N-diisopropylethylamine.

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