LAT1-targeted phosphoramidated tyrosine PET molecular probe as well as preparation method and application thereof

Abstract

The invention belongs to the field of nuclear medicine imaging agents, and relates to an LAT1 targeted phosphoramidated tyrosine PET molecular probe as well as a preparation method and application thereof. The probe has a structure as shown in a formula I. The LAT1-targeted phosphoramidated tyrosine PET molecular probe provided by the invention can be used for high-specificity uptake of bones, and has excellent in-vivo metabolism performance and bone uptake retention capacity. The targeted PET molecular probe provided by the invention has a clear tumor ratio, can be rapidly metabolized out of a body through a kidney, and has important application value for early and accurate diagnosis of bone tumors and bone metastatic tumors.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine imaging agents, specifically relating to a molecular probe targeting the L-type amino acid transporter (LAT1), its preparation method, and its application. Background Technology

[0002] Bone tumors and bone metastases are tumors that occur in the human skeleton. Primary bone tumors account for less than 1% of all cancerous diseases, while bone metastases account for 15-20% of all metastatic tumors. Bone tumors and bone metastases seriously endanger the life, health, and quality of life of patients, often manifesting as symptoms such as pain and swelling, and in severe cases, pathological fractures, paraplegia, and even death.

[0003] Malignant bone tumors are usually treated with a comprehensive approach, including surgical removal of the tumor as much as possible, chemotherapy before or after surgery to shrink the tumor size and reduce the risk of metastasis, and radiotherapy to control local tumor growth. The main principles of treating bone metastases are to relieve symptoms and improve quality of life, helping patients reduce pain and restore limb function.

[0004] The diagnosis and differential diagnosis of bone tumors often perplex clinicians, leaving them hesitant and uncertain. Bone tumors are diverse, with highly variable presentations. Although they can be classified as benign or malignant, there are no clear boundaries between them; intermediate or borderline lesions can exist, and even the same tumor can exhibit histological features of both benign and malignant tumors simultaneously. To further clarify the diagnosis, pathological and histological observations must be combined with clinical findings and imaging manifestations for a comprehensive analysis, avoiding a narrow focus. It is now a consensus that a correct diagnosis can only be obtained through a comprehensive and organic integration of clinical data, imaging findings, and histopathological results.

[0005] L-type amino acid transporter 1 (LAT1) is a large neutral amino acid transporter highly expressed in various tumors, including glioma, pancreatic cancer, cholangiocarcinoma, gastric cancer, and breast cancer, and is associated with tumor invasion, metastasis, and drug resistance. A research team led by Professor Eiichi Hikai of Gifu Pharmaceutical University in Japan, in collaboration with Professor Katsuyuki Kaneda and Associate Professor Kazuma Ogawa of Kanazawa University, Assistant Professor Hiroki Ochi and Lecturer Shingo Sato of Tokyo Medical and Dental University, Professor Yasuhiro Kobayashi of Matsumoto Dental University, Senior Researcher Yun-Bo Shi of the National Institutes of Health, and Professor Peter M. Taylor of the University of Dundee in the UK, discovered a physiological signal and a new molecular mechanism crucial for maintaining bone health. As an amino acid transporter, LAT1 plays a vital role in maintaining bone health. Amino acid signaling originating from LAT1 in osteoclasts regulates the expression and function of NFATc1 via mTORC1, thus playing a crucial role in bone metabolism.

[0006] Nuclear medicine radionuclide probe PET-CT imaging has the advantages of real-time, dynamic, and high sensitivity, and can accurately diagnose lesions at an early stage. Radionuclide probes can play multiple roles in tumor diagnosis, efficacy evaluation, and prognosis assessment, and have important clinical value for accurate diagnosis. 18 F has a half-life of 109.8 minutes and mainly decays through positron (β+) decay. The positrons released during the decay process annihilate with electrons in the surrounding matter, producing two gamma photons with opposite directions and energies of 0.511 MeV each. It has excellent performance and is the most commonly used radionuclide for positron emission tomography (PET) imaging in clinical practice. 18 Traditional methods for introducing F nuclides into targeted molecular probes mainly focus on the formation of carbon-fluorine bonds. However, C- 18 The F-method typically requires time-consuming, rigorous drying, and complex processes, posing a challenge to its widespread application. Recently, through... 18 F- 19 F element exchange labeling has become a feasible method, offering advantages such as relatively mild radiolabeling conditions and high labeling rates. The key requirement is a suitable elemental carrier, commonly silicon-... 18 F, Boron 18 F, Aluminum 18 F, etc. Phosphorus (P) has a stronger affinity for fluorine, and phosphorus fluoride has a stronger Lewis acid (equivalent to Si-F, BF). Therefore, phosphorus may be a synthetic... 18 A useful radiolabeling group carrier for F radioactive tracers. Professor Zhang Xianzhong's team at Xiamen University utilized P- in 2020... 18The F-labeling method yielded a radiochemical yield greater than 94% within 5 minutes at room temperature. 18 The product is labeled with F and has good in vivo properties.

[0007] Phosphate has a strong coordination effect on calcium ions in the hydroxyapatite lattice of human bone, exhibiting a high level of bone selectivity, especially in bone lesion sites with high bone turnover. Based on the above factors, this invention introduces phosphoryl fluoride on the basis of tyrosine targeting LAT1, and constructs a novel nuclear medicine PET bone imaging probe through element exchange labeling method to solve the problem of early and accurate diagnosis of clinical bone tumors and bone metastases. Summary of the Invention

[0008] The core of improving precision treatment for bone tumors and metastases lies in accurate diagnosis. Nuclear medicine PET / CT imaging can simultaneously assess the metabolic status and anatomical structure of tumors, which is of great value in diagnosing bone metastases and assessing tumor burden. Based on the value of LAT1 in tumors and "maintaining bone health," as well as the high selectivity of phosphate to human bone and phosphorylation... 18 To leverage the high labeling performance of F, this invention introduces phosphoramide fluoride into the structure targeting the tyrosine phenolic hydroxyl group of LAT1, and constructs a novel nuclear medicine PET bone imaging probe through an element exchange labeling method. This probe exhibits convenient radiochemical labeling, high radiochemical labeling rate, and high radiochemical purity. Furthermore, Micro-PET / CT imaging reveals high uptake of the probe in bone with a clear metabolic imaging background.

[0009] To achieve the above objectives, a first aspect of the present invention provides a LAT1-targeting phosphoramidated tyrosine PET molecular probe, the probe having the structure shown in Formula I:

[0010]

[0011] A second aspect of the present invention provides a method for preparing the LAT1-targeted phosphoramidated tyrosine PET molecular probe, wherein the probe is prepared from the compound shown in Formula II by an element-exchange labeling method.

[0012]

[0013] According to a preferred embodiment of the present invention, the compound represented by Formula II is prepared by the following synthetic route:

[0014]

[0015] Specifically, the method for synthesizing the probe includes the following steps:

[0016] (1) Add TEA and INTA to the DCM solution of compound 1 and stir the reaction at room temperature to obtain compound 2;

[0017] (2) KF and TBAC were added to the acetone solution of compound 2 and the mixture was stirred at 40-60°C to obtain compound 3;

[0018] (3) Pd / C was added to the MeOH solution of compound 3 and the reaction was stirred at room temperature under H2 to obtain the compound shown in formula II;

[0019] (4) Make it contain [ 18 F - ]F - H2 18 O solution is passed through a QMA column, [ 18 F - ]F - Adsorbed onto a QMA column, the column was eluent with K2CO3 and passed through the QMA column. 18 F - ]F - The liquid was rinsed into the reaction tube, heated, and purged with nitrogen to evaporate it to dryness, and then subjected to azeotropic dehydration.

[0020] (5) Dissolve the compound of formula II obtained in step (3) in ultra-dry DMSO, and add the resulting solution to the reaction tube obtained in step (4) for reaction. After the reaction is completed, purify the solution using a Sep-Pak Light C18 column to obtain the probe.

[0021] A third aspect of the present invention provides the use of the LAT1-targeted phosphoramidated tyrosine PET molecular probe in the preparation of imaging reagents for bone tumors or bone metastases.

[0022] This invention provides LAT1-targeting molecule tyrosine, phosphate groups (strong coordination groups for calcium ions in the hydroxyapatite lattice of human bone), and P-labeling under mild and efficient conditions. 18 This invention provides a LAT1-targeted bone imaging probe using the F element exchange labeling method. The LAT1-targeted PET molecular probe provided by this invention has mild and efficient labeling performance, high bone uptake, clear imaging background, and high tumor background, and has important clinical value for the accurate diagnosis of bone tumors and bone metastases.

[0023] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0024] The above and other objects, features and advantages of the present invention will become more apparent from the more detailed description of exemplary embodiments of the invention in conjunction with the accompanying drawings.

[0025] Figure 1-1 The synthetic route for TT12 is shown.

[0026] Figures 1-2 to 1-4 The 1H NMR spectra of compounds 2, 3, and TT12 are shown respectively.

[0027] Figure 1-5 The mass spectrum of TT12 is shown.

[0028] Figure 2 It shows 18 Radio-HPLC quality control results for F-TT12 and TT12.

[0029] Figure 3 It shows 18 Micro-PET / CT imaging results of F-TT12 in 87U tumor model mice.

[0030] Figure 4 It shows 18 Changes in F-TT12 uptake over time in spinal and joint tumors in 87U tumor model mice, and 18 Changes in spinal uptake of F-TT12 in tumors and muscles over time in 87U tumor model mice.

[0031] Figure 5 It shows 18 Micro-PET / CT imaging results of F-TT12 in normal Kunming mice.

[0032] Figure 6 It shows 18 Changes in F-TT12 uptake over time in the spine and joints of normal Kunming rats. Detailed Implementation

[0033] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0034] Unless otherwise specified in the examples, all procedures were performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] Example 1

[0036] Synthesis and characterization of the labeled precursor TT12, synthetic route as follows: Figure 1-1 As shown:

[0037] 1. Synthesis of Compound 2

[0038] TEA (1.6 g, 16.3 mmol, 3.0 eq) and INTA (879 mg, 5.4 mmol, 1.0 eq) were added to a DCM (22 mL) solution of compound 1 (2.2 g, 5.4 mmol, 1.0 eq). The mixture was stirred at room temperature for 16 h, and the reaction was quenched with water (50 mL). The mixture was extracted with DCM (50 mL × 3). The combined organic layers were washed with water (50 mL) and brine (50 mL), dried over Na₂SO₄, filtered, and concentrated to dryness. Compound 2 (1.7 g, 59.0% yield, white solid) was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1). The 1H NMR spectrum of compound 2 is shown below. Figure 1-2 As shown. 1 H NMR (300MHz, CDCl3): δ7.43–7.20(m,10H),7.09(d,J=7.5Hz,2H),6.98(d,J=8.4Hz,2H ),5.37–5.00(m,5H),4.74–4.60(m,1H),3.18–2.95(m,1H),2.85(s,3H),2.80(s,3H).

[0039] 2. Synthesis of Compound 3

[0040] KF (109 mg, 1.9 mmol, 5.0 eq) and TBAC (11 mg, 0.06 mmol, 0.1 eq) were added to a 2 mL solution of compound 2 (200 mg, 0.37 mmol, 1.0 eq) in acetone. The mixture was stirred at 50 °C for 16 h. After the reaction was complete, the mixture was cooled, and the reaction was quenched with 10 mL of water. The mixture was extracted with DCM (20 mL × 3). The combined organic layer was washed with 20 mL of water and 20 mL of brine, dried on Na₂SO₄, filtered, and concentrated to dryness. Compound 3 (110 mg, 56.7% yield, white solid) was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 3:1). The 1H NMR spectrum of compound 3 is shown below. Figure 1-3 As shown. 1 H NMR (300MHz, CDCl3): δ7.44–7.27(m,10H),7.05(d,J=8.1Hz,2H),6.96(d,J=8.7Hz,2H),5.29–5 .03(m,5H),4.75–4.62(m,1H),3.18–2.98(m,1H),2.82(d,J=1.8Hz,3H),2.79(d,J=1.8Hz,3H).

[0041] 3. Synthesis of compound TT12

[0042] Pd / C (20 mg) was added to a 3 mL solution of compound 3 (100 mg, 0.2 mmol, 1.0 eq) in MeOH. The mixture was stirred at room temperature under H2 for 4 h, and HPLC showed that the reaction was complete. The mixture was filtered, and the filtrate was concentrated to give TT12 (50 mg, 88.6%, white solid). The 1H NMR spectrum of compound TT12 is shown below. Figure 1-4 As shown, the mass spectrometry is as follows Figure 1-5 As shown. 1 H NMR (300MHz, D2O): δ7.39(d,J=8.7Hz,2H),7.27(d,J=8.4Hz,2H),4.00(dd,J=7.8,5.4Hz,1H),3.4 0–3.23(m,1H),3.22–3.08(m,1H),2.85(d,J=2.1Hz,3H),2.81(d,J=2.1Hz,3H).LCMS:291.0([M+H] + ).

[0043] Example 2

[0044] 18 F-TT12 18 F-radiochemical marker:

[0045] 1. To make containing [ 18 F - ]F - H2 18 O solution is passed through a QMA column, [ 18 F - ]F - Adsorbed onto a QMA column. Elute with 0.2 mL of K2CO3 eluent (3.45 mg / mL) through the QMA column.

[0046] 2. [ 18 F - ]F - Rinse the solution into the reaction tube. Evaporate the liquid to dryness under nitrogen purging at 95°C. Add 1 mL of anhydrous acetonitrile to the reaction tube and perform azeotropic dehydration. Repeat this process three times.

[0047] 3. Dissolve the cold compound TT12 in ultradry DMSO (1 mg / mL), and add 0.1 mL (1 mg / mL) of TT12 in DSMO solution to the reaction tube. React at room temperature for 15 minutes. After the reaction, purify using a Sep-Pak Light C18 column to obtain... 18 The F-TT12 radioactive probe has a radiochemical yield of 92%.

[0048] Example 3

[0049] 18 Quality control of F-TT12:

[0050] 18 The radiochemical purity of F-TT12 was verified using Radio-HPLC analysis, such as... Figure 2 As shown, Radio-HPLC results indicate that the retention time of the labeled precursor (cold compound) TT12 is 5.91 minutes. 18 The F-TT12 probe has a retention time of 6.21 minutes. Its radiochemical purity is higher than 99%, making it suitable for biological evaluation.

[0051] Example 4

[0052] 18 F-TT12 Micro-PET / CT Imaging:

[0053] U87-MG tumor-bearing mice and KM mice were placed in anesthesia chambers and anesthetized using isoflurane (1.5%, 1 L / min oxygen). Labeled probes were then placed... 18 F-TT12 / physiological saline solution (7.4 MBq, 200 μL) was injected into mice via the tail vein. Micro-PET / CT imaging was performed at 30, 60, and 120 minutes to observe the metabolism of the probe in mice, delineate the ROI, and measure the SUVmax of each organ and tumor site. Figure 3 It shows 18 Micro-PET / CT imaging results of F-TT12 in 87U tumor model mice. Figure 4 It shows 18 The changes in F-TT12 uptake over time in spinal and joint tumors in 87U tumor model mice. These changes can be observed in micro-PET imaging. 18 The F-TT12 probe showed high uptake in the bones of U87 tumor-bearing mice. Uptake in the spine increased from 1.65±0.06 (30 min) to 2.49±0.11 (120 min), and uptake in the joints increased from 1.97±0.07 (30 min) to 2.50±0.13 (120 min). Figure 4 It also shows 18 The changes in spinal uptake of F-TT12 in tumors and muscles over time in 87U tumor model mice. It can be seen that... 18 F-TT12 uptake was low in both tumors and muscles, and decreased over time. At 30 minutes post-injection, the probe showed the highest uptake in both tumors and muscles, with an SUVmax of 0.68±0.03 for tumors and 0.50±0.02 for muscles. The tumor-to-muscle ratio was above 1 in both cases, reaching a maximum of 1.41±0.14. Figure 5 It shows18 Micro-PET / CT imaging results of F-TT12 in normal Kunming mice. It can be seen that in KM mice, the probe still has high uptake in the bones. The uptake of the probe in the spine increased from 4.05±0.06 (30 min) to 4.95±0.64 (120 min), and the uptake in the joints increased from 7.60±2.38 (30 min) to 8.96±2.04 (120 min), which is consistent with the uptake of the probe in tumor-bearing mice. Figure 6 It shows 18 The changes in F-TT12 uptake in the spine and joints of normal Kunming rats over time can be observed. 18 F-TT12 exhibits high uptake and long retention in the spine and joints, with minimal reduction in uptake at 120°C and clear background.

[0054] The above experimental results demonstrate that the LAT1 phosphoramidized tyrosine PET molecular probe provided by this invention can specifically maintain high uptake by bone.

[0055] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A LAT1-targeting phosphoramidated tyrosine PET molecular probe, characterized in that, The probe has the structure shown in Formula I:

2. The method for preparing the LAT1-targeted phosphoramidated tyrosine PET molecular probe according to claim 1, characterized in that, The probe was prepared from the compound shown in Formula II using an element-exchange labeling method.

3. The method for preparing the LAT1-targeted phosphoramidated tyrosine PET molecular probe according to claim 2, characterized in that, The compound shown in Formula II was prepared by the following synthetic route:

4. The method for preparing the LAT1-targeted phosphoramidated tyrosine PET molecular probe according to claim 3, characterized in that, The method for synthesizing the probe includes the following steps: (1) Add TEA and INTA to the DCM solution of compound 1 and stir the reaction at room temperature to obtain compound 2; (2) KF and TBAC were added to the acetone solution of compound 2 and the mixture was stirred at 40-60°C to obtain compound 3; (3) Pd / C was added to the MeOH solution of compound 3 and the reaction was stirred at room temperature under H2 to obtain the compound shown in formula II; (4) Make it contain [ 18 F - ]F - H2 18 O solution is passed through a QMA column, [ 18 F - ]F - Adsorbed onto a QMA column, the column was eluent with K2CO3 and passed through the QMA column. 18 F - ]F - The liquid was rinsed into the reaction tube, heated, and purged with nitrogen to evaporate it to dryness, and then subjected to azeotropic dehydration. (5) Dissolve the compound of formula II obtained in step (3) in ultra-dry DMSO, and add the resulting solution to the reaction tube obtained in step (4) for reaction. After the reaction is completed, purify the solution using a Sep-Pak Light C18 column to obtain the probe.

5. The application of the LAT1-targeted phosphoramidated tyrosine PET molecular probe of claim 1 in the preparation of imaging reagents for bone tumors or bone metastases.

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