PET (Polyethylene Terephthalate) molecular probe targeting LAT1 as well as preparation method and application of PET molecular probe

Abstract

The invention belongs to the field of nuclear medicine imaging agents, and relates to a PET molecular probe targeting LAT1, a preparation method and application thereof. The probe has a structure as shown in a formula I. The LAT1-targeted PET molecular probe provided by the invention has efficient marking performance, has specific uptake on brain glioma, improves the imaging background, particularly improves the uptake in the abdomen, has high tumor background, and has clearer diagnosis background and efficacy on brain glioma and abdominal tumors with high expression of LAT1, such as intestinal cancer.

Description

Technical Field

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

[0002] Gliomas originate from the neuroectoderm and are the most common primary malignant tumors, with high mortality and disability rates. The average survival time for patients with anaplastic gliomas (WHO grade III) is 2–5 years, while for glioblastoma patients it is only 15–17 months.

[0003] Currently, contrast-enhanced MRI, with its excellent soft tissue contrast, high spatial resolution, and multiplanar reconstruction capabilities, has become the preferred method for clinical diagnosis of brain tumors. It can effectively determine and identify the location, differentiation degree, and potential blood-brain barrier (BBB) ​​disruption of the tumor. However, in some cases, such as after radiotherapy, the nonspecificity caused by BBB leakage limits the application of MRI. In these situations, PET imaging supplemented with specific radionuclide tracers can accurately reflect the physiological, biochemical, and metabolic information of tumor cells, assisting MRI in diagnosing and differentiating tumors.

[0004] The most commonly used PET tracer is currently 18 F-FDG, but 18 F-FDG PET has significant limitations in diagnosing and differentiating brain tumors: (1) low-grade gliomas and normal white matter show little difference in 18F-FDG uptake, resulting in a low detection rate for low-grade gliomas; (2) 18 F-FDG uptake in high-grade tumors is less than or similar to that in normal gray matter; (3) due to 18 The nonspecificity of F-FDG means that not only malignant tumors have high uptake values, but some benign lesions such as inflammation can also show high uptake values; (4) normal cerebral cortex to 18 High uptake of F-FDG makes it difficult to delineate brain tumor boundaries and the extent of tumor invasion. These limitations hinder the development of effective treatments. 18 Application of F-FDG in brain tumor imaging.

[0005] With the development of amino acid radiotracers, the study of amino acid metabolic pathways has been paid more and more attention. L-type amino acid transporter 1 (LAT1) is a large neutral amino acid transporter, which is obviously highly expressed in brain glioma but lacks expression in normal brain. LAT1 can specifically transport some amino acids such as tyrosine, so it becomes an ideal clinical nuclear medicine diagnostic target for brain glioma. It has important clinical value to construct a nuclear medicine diagnostic probe based on the tyrosine targeting LAT1. In addition to the specific high expression in brain glioma, LAT1 is also highly expressed in many tumors such as pancreatic cancer, cholangiocarcinoma, gastric cancer and breast cancer, which is related to tumor invasion, metastasis and drug resistance. Therefore, the construction of a nuclear medicine PET probe based on tyrosine targeting LAT1 can have the advantages of easy crossing of the blood-brain barrier and excellent in vivo metabolic performance, which can play an important role in the diagnosis of brain glioma and other non-brain tumors. SUMMARY

[0006] It has important clinical value to construct a nuclear medicine PET molecular probe with the advantages of crossing the BBB and excellent in vivo metabolic performance, which can improve the diagnostic clarity and background in brain glioma and non-brain tumors, and improve the tumor diagnosis rate. The present application introduces a PEG side chain on the functional group of the para-phenolic hydroxyl group of the tyrosine targeting LAT1, which can improve the metabolic performance of the probe while ensuring the targeting performance of the probe, reduce the problem of high abdominal organ uptake caused by too high liposolubility, improve the contrast of the imaging image, facilitate the diagnosis efficiency of tumors, especially abdominal tumors, and improve the contrast between brain glioma tumors and background. The Micro-PET / CT imaging results of the probe of the present application show that the probe has better metabolic performance and lower background than 18 F-FET (a commonly used amino acid probe for brain glioma in clinic) can maintain tumor-specific uptake while improving metabolic performance and imaging background.

[0007] To achieve the above object, a first aspect of the present application provides a PET molecular probe targeting LAT1, which has the structure shown in formula I:

[0008]

[0009] A second aspect of the present application provides a preparation method of the PET molecular probe targeting LAT1, which is prepared by nucleophilic substitution of a compound shown in formula II: 18 F labeling method,

[0010]

[0011] According to a preferred embodiment of the present application, the compound shown in formula II is prepared by the following synthesis route:

[0012]

[0013] In particular, the synthesis method of the probe comprises the following steps:

[0014] (1) mixing compound 1, base and triethylene glycol di-p-toluene sulfonate, and reacting under first reaction conditions to obtain compound 2;

[0015] (2) slowly adding tetrabutylammonium fluoride in a tetrahydrofuran solution containing compound 2, and reacting under second reaction conditions to obtain compound 3;

[0016] (3) stirring the CF3COOH solution of compound 3 at room temperature to obtain compound TT3;

[0017] (4) carrying out 18 F labeling on compound TT3 to obtain the probe.

[0018] More particularly, the first reaction conditions comprise: stirring conditions, temperature of 50-70℃, and time of 2-4h.

[0019] More particularly, the second reaction conditions comprise: stirring conditions, temperature of 50-70℃, and time of 0.5-2h.

[0020] More particularly, the step of 18 F labeling comprises:

[0021] (a) passing a solution containing 18 F - ]F - H2 18 O through a QMA column, and 18 F - ]F - adsorbing on the QMA column;

[0022] (b) passing the QMA column with a K 2,2,2 / K2CO3 eluent, and eluting 18 F - ]F - into a reaction tube, and evaporating the liquid to dryness under nitrogen blowing at 85-95℃;

[0023] (c) adding anhydrous acetonitrile into the reaction tube, and evaporating to dryness repeatedly for multiple times to obtain dry 18 F - ]F - / K 2,2,2 / K2CO3 complex, then sealing the reaction tube with a cover, and cooling the complex;

[0024] (d) The labeled precursor was dissolved in anhydrous acetonitrile and added to the reaction tube, and vortexed to mix; the reaction tube was placed in a water bath at 85-95°C, and reacted for 10-20 minutes; after the reaction was completed, the reaction tube was placed in an ice water bath to cool the reaction solution;

[0025] (e) High-purity water was added to the cooled reaction solution to dilute it, and the reaction solution was purified using a Sep-Pak Light C18 column, and the Sep-Pak Light C18 column was washed with high-purity water, and the radiolabeled probe intermediate before preparation was eluted into a vial using acetonitrile

[0026] (f) The radiolabeled probe intermediate before preparation was purged with nitrogen and concentrated, filtered through a filter membrane, and transferred to a Radio-HPLC sample bottle, and the radiolabeled probe intermediate was collected using Radio-HPLC and concentrated using a Sep-Pak Light C18 column, eluted with ethanol, and blown dry, acid hydrolyzed with trifluoroacetic acid, and the trifluoroacetic acid was blown dry to obtain the product 18 F-TT3.

[0027] The third aspect of the present application provides use of the PET molecular probe targeting LAT1 in the preparation of a tumor imaging reagent. Preferably, the tumor is a brain glioma or an abdominal tumor, and more preferably, the abdominal tumor.

[0028] The present application provides a PEG chain based on LAT1 transport amino acid tyrosine, which improves the membrane permeability on the basis of the para-phenolic hydroxyl group, improves the probe brain and abdominal metabolism background on the basis of ensuring the LAT1 targeting performance of the probe, and improves the imaging clarity. The PET molecular probe targeting LAT1 provided by the present application has high labeling performance, has specific uptake for brain glioma, improves the imaging background, especially the abdominal uptake, has a high tumor background, and has a clearer diagnostic background and performance for brain glioma and abdominal tumors with high expression of LAT1, such as intestinal cancer.

[0029] Other features and advantages of the present application will be described in detail in the following specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0030] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which:

[0031] Figure 1-1 The synthesis routes of the labeled precursor compound 2 and the cold compound TT3 are shown.

[0032] Figures 1-2 to 1-4 The mass spectra of compounds 2, 3, and TT3 are shown, respectively.

[0033] Figure 2 It shows 18 Radio-HPLC quality control results of F-TT3 intermediate.

[0034] Figure 3 It shows 18 Radio-HPLC quality control results of F-TT3 products.

[0035] Figure 4 It shows 18 Micro-PET / CT imaging results of F-TT3 in 87U tumor model mice.

[0036] Figure 5 It shows 18 Micro-PET / CT imaging results of F-FET in 87U tumor model mice.

[0037] Figure 6 The immunohistochemical expression of LAT1 in colorectal cancer is shown. Detailed Implementation

[0038] 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.

[0039] 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.

[0040] Example 1

[0041] Synthesis and characterization of labeled precursor 2 and cold compound TT3, synthetic route as follows: Figure 1-1 As shown:

[0042] 1. Synthesis of Compound 2

[0043] K₂CO₃ (1.23 g, 8.90 mmol, 3.0 eq) and triethylene glycol di-p-toluenesulfonate (1.63 g, 3.56 mmol, 1.2 eq) were added to a DMF (22 mL) solution of compound 1 (1 g, 2.97 mmol, 1.0 eq). The mixture was stirred at 60 °C for 3 h, quenched with water (50 mL), and extracted with EA (50 mL × 3). The combined organic layer was washed with water (50 mL) and brine (50 mL), dried on Na₂SO₄, filtered, and concentrated to dryness. Compound 2 (0.56 g, 30.0% yield) was purified by silica gel column chromatography (PE / EA = 1:1) as a white gel. LRMS calc. for C 31 H45 NO 10 S[M+Na] + 646.2626, found 646.3 (see Figure 1-2 ).

[0044] Synthesis of compound 3

[0045] To a solution of compound 2 (200 mg, 0.32 mmol, 1.0 eq) in THF (10 mL) was added TBAF (0.64 mL, 0.64 mmol, 2.0 eq) slowly at 60 °C with stirring, and the reaction was stirred at 60 °C for 1 h, quenched with water (10 mL), and extracted with EA (20 mL x 3). The combined organic layers were washed with water (20 mL) and brine (20 mL), dried over Na2S04, filtered, and concentrated to dryness. Purification by silica gel column chromatography (PE / EA = 1 : 1) gave compound 3 (65 mg, 43% yield) as a white gum. LRMS calc. for C 24 H 38 FNO7[M+Na] + 494.2530, found 494.1 (see Figure 1-3 ).

[0046] Synthesis of compound TT3

[0047] A solution of compound 3 (50 mg, 0.11 mmol, 1.0 eq) in CF3COOH (3 mL) was stirred at room temperature for 4 h, the solvent was removed under reduced pressure, and lyophilized by Pre-HPLC to give compound TT3 (22 mg, 65%) as a white gum. LRMS calc. for C 15 H 22 FNO5[M+H] + 316.1560, found 316.1 (see Figure 1-4 ).

[0048] Example 2

[0049] 18 Synthesis of F-TT3 18 F radiolabeling:

[0050] (1) Pass a solution of H2 18 F - ]F - in H2 18 O through a QMA column, and adsorb [ 18 F - ]F - on the QMA column.

[0051] (2) Prepare K2,2,2 K2CO3 eluent: 1 g K 2,2,2 Dissolved in 77 mL acetonitrile, 231 mg K2CO3 dissolved in 7.7 mL high purity water, mixed well.

[0052] (3) Using 1 mL K 2,2,2 / K2CO3 eluent through QMA column, and 18 F - ]F - Eluted into the reaction tube. The liquid was evaporated to dryness at 90°C under nitrogen purge.

[0053] (4) 1 mL of anhydrous acetonitrile was added to the reaction tube, and evaporated to dryness again. This process was repeated 3 times. Dry 18 F - ]F - / K 2,2,2 / K2CO3 complex, the reaction tube was sealed and the complex was cooled.

[0054] (5) Using a syringe to take 1 mL of anhydrous acetonitrile and dissolve 1 mg of labeling precursor, add to the reaction tube, vortex well. The reaction tube was at 90°C for 15 minutes. After the reaction was completed, the reaction tube was placed in an ice water bath to cool the reaction solution.

[0055] (6) 9 mL of high purity water was added to the cooled reaction solution for dilution, and the reaction solution was purified using a Sep-Pak Light C18 column, and 9 mL of high purity water was used to rinse the above Sep-Pak Light C18 column, and 0.5 mL of acetonitrile was used to elute the radioactive product intermediate (before preparation) into a Schlenk flask.

[0056] (7) The prepared product was purged with nitrogen and concentrated, filtered with a filter membrane, and transferred to a Radio-HPLC sample bottle, and Radio-HPLC was used to prepare the radioactive probe intermediate.

[0057] (8) The intermediate of the radioactive probe was collected and concentrated using a Sep-Pak Light C18 column, eluted with 0.5 mL of ethanol, and blown dry, 0.5 mL of trifluoroacetic acid was added for acid hydrolysis (50°C, 5 min), and the trifluoroacetic acid was blown dry to obtain the product 18 F-TT3.

[0058] Example 3

[0059] 18 Quality control of F-TT3:

[0060] Radio-HPLC results show that the intermediate of the probe 18The retention time of F-TT3-mid is 16.07 min Figure 2 , 18 The retention time of F-TT3 probe is 9.41 min Figure 3 The radiochemical purity of the probe is higher than 99%, which can be used for biological evaluation.

[0061] Example 4

[0062] 18 F-TT3 and 18 Micro-PET / CT imaging of F-FET:

[0063] 18 F-FET is a commonly used amino acid probe for clinical glioma (J Nucl Med. 2022 Apr; 63(4): 522-527. doi: 10.2967 / jnumed.121.262051).

[0064] U87-MG tumor-bearing mice and KM mice were placed in the anesthesia chamber and anesthetized with isoflurane (1.5%, 1 L / min oxygen). The labeled probes 18 F-TT3 / physiological saline solution and 18 F-FET / physiological saline solution (7.4 MBq, 200 μL) was injected into the mice through the tail vein, and micro-PET / CT imaging was performed at 30 min and 60 min, respectively, to observe the metabolism of the probes in the mice and draw the ROI to measure the SUVmax of each organ and tumor site. In micro-PET imaging, 18 F-TT3 probe has significant uptake in the tumor lesion of U87 tumor-bearing mice, and the SUVmax reaches 0.65±0.02 at 30 min. The probe is metabolized faster, and the SUVmax in the tumor decreases to 0.43±0.01 at 60 min. The trend of tumor-muscle ratio (T / M) of the probe is consistent with that of F-FET, and the T / M reaches 2.07±0.09 at 30 min, while it decreases to 1.49±0.09 at 60 min. The probe uptake in the abdomen is lower than 18 F-FET at 30 min and 60 min, and the probe uptake in the abdomen position decreases significantly at 60 min (as shown in Figure 4 ). 18 F-FET probe has higher uptake in the tumor lesion of U87 tumor-bearing mice, and the SUVmax reaches 2.00±0.08 at 30 min. The uptake value decreases with time, and the SUVmax in the tumor decreases to 1.66±0.18 at 60 min. The trend of T / M of the probe is consistent, and the T / M reaches 6.00±0.21 at 30 min, while it decreases to 5.48±0.47 at 60 min, but18 F-FET had a significant uptake in the abdomen due to its high lipid solubility, which was much higher than 18 F-TT3 at both 30 min and 60 min. Figure 5 ).

[0065] Example 5

[0066] Immunohistochemistry of colon cancer tumor model mice

[0067] The tumor tissues of HT29 and LS174T tumor-bearing mice were fixed with 5% tissue fixative and embedded with a slice thickness of 5 μm. Before the experiment, the slices were deparaffinated and then rehydrated with xylene and alcohol in a gradient. To block the activity of endogenous peroxidase, the tumor slices were treated with 3% H2O2for 10 minutes. Then, an antigen retrieval solution was added and heated in a microwave oven for 10 minutes. After washing with PBS several times, the slices were blocked with goat serum at room temperature for 40 minutes. After sealing, the primary antibody was added at 1:200 and incubated overnight at 4°C. The next day, the slices were removed and washed with PBS several times. The secondary antibody was added and incubated at room temperature for 30 minutes. Then, hydrochloric acid identification was performed using a DAB substrate kit and hematoxylin staining. After gradient alcohol dehydration, the staining results were observed using neutral gum sealing. As shown in Figure 6 , the experimental results confirmed that both tumors highly expressed LAT1 protein. The tumor tissues showed strong positive LAT1 high expression, and the positive staining was distributed in most of the tumor cells, and clear and continuous cell membrane coloring was observed under a high-power lens, indicating that LAT1 was highly expressed in the colon cancer tumor.

[0068] The above has described various embodiments of the present application, and the above description is exemplary and is not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and changes will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.

Claims

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

2. The method for preparing the LAT1-targeting PET molecular probe according to claim 1, characterized in that, The probe is a nucleophilically substituted compound of Formula II. 18 Prepared by the F-labeling method 3. The method for preparing a LAT1-targeting 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 a LAT1-targeting PET molecular probe according to claim 3, characterized in that, The method for synthesizing the probe includes the following steps: (1) Compound 1, base and triethylene glycol di-p-toluenesulfonate were mixed and reacted under the first reaction conditions to obtain compound 2; (2) In a tetrahydrofuran solution containing compound 2, tetrabutylammonium fluoride was slowly added and the reaction was carried out under the second reaction conditions to obtain compound 3; (3) The CF3COOH solution of compound 3 was stirred at room temperature to react and obtain compound TT3; (4) Compound TT3 was subjected to... 18 The probe is obtained by marking it with F.

5. The preparation method according to claim 4, characterized in that, The first reaction conditions include: stirring, temperature of 50-70℃, and time of 2-4h.

6. The preparation method according to claim 4, characterized in that, The second reaction conditions include: stirring, temperature of 50-70℃, and time of 0.5-2h.

7. The preparation method according to claim 4, characterized in that, The 18 The steps for marking F include: (a) Make it contain [ 18 F - ]F - H2 18 O solution is passed through a QMA column, [ 18 F - ]F - Adsorbed on the QMA column; (b) Using K 2,2,2 / K2CO3 eluent is passed through a QMA column to [ 18 F - ]F - Rinse the liquid into the reaction tube and evaporate it to dryness under nitrogen purging at 85-95℃. (c) Add anhydrous acetonitrile to the reaction tube, evaporate again until dry, repeat several times to obtain dry [[ 18 F - ]F - / K 2,2,2 The K2CO3 complex was then formed, the reaction tube was sealed, and the complex was cooled. (d) Dissolve the labeled precursor in anhydrous acetonitrile and add it to the reaction tube, then vortex to mix. Incubate the reaction tube at 85-95℃ for 10-20 minutes. After the reaction is complete, place the reaction tube in an ice-water bath to cool the reaction solution. (e) Dilute the cooled reaction solution with high-purity water, purify the reaction solution using a Sep-Pak Light C18 column, rinse the Sep-Pak Light C18 column with high-purity water, and then rinse the unprepared radioprobe intermediate into a vial with acetonitrile. (f) The radioprobe intermediate before preparation was purged with nitrogen and concentrated, filtered through a filter membrane, and transferred to a Radio-HPLC vial. The radioprobe intermediate was collected using Radio-HPLC and concentrated using a Sep-Pak Light C18 column. After rinsing with ethanol and drying, it was acid-hydrolyzed with trifluoroacetic acid, and then dried again to obtain the product. 18 F-TT3.

8. The application of the LAT1-targeting PET molecular probe of claim 1 in the preparation of tumor imaging reagents.

9. The application according to claim 8, characterized in that, The tumor is either a glioma or an abdominal tumor.

10. The application according to claim 9, characterized in that, The abdominal tumor is intestinal cancer.

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