PD-L1 targeted biphenyl radioactive tracer and application thereof

Abstract

The invention relates to a PD-L1 targeted biphenyl radioactive tracer and application thereof, and belongs to the technical field of PET imaging. The invention provides a series of PD-L1 targeted biphenyl radioactive tracers which comprise [18F] LG-2, [18F] LG-3, [18F] LG-13, [18F] LG-14, [18F] LG-15 and the like, the series of biphenyl radioactive tracers have good in-vitro stability, can be specifically combined with PD-L1, and are relatively good in targeting specificity, and therefore, the series of biphenyl radioactive tracers can be applied to the field of radioactive tracers. The series of biphenyl radioactive tracers can noninvasively, accurately, dynamically and systemically monitor the expression level and change of PD-L1 in tumors from the molecular level in real time through PET imaging, and then the curative effect of PD-1 / PD-L1 immunotherapy on tumor patients is effectively evaluated.

Description

Technical Field

[0001] The present invention relates to a PD-L1-targeted biphenyl-based radioactive tracer and its application, belonging to the technical field of PET imaging. Background Art

[0002] Immunotherapy is a treatment method that eliminates cancer cells by enhancing or remodeling the body's own immune system and utilizing its own immune mechanism, and has become a treatment strategy that has attracted much attention. Among them, immune checkpoint blockade of programmed death receptor 1 (PD-1) and its ligand (PD-L1) is the main research direction of current cancer immunotherapy. The PD-1 / PD-L1 pathway controls the induction and maintenance of immune tolerance in the tumor microenvironment. After the PD-1 / PD-L1 signaling pathway is activated, it promotes the apoptosis of antigen-specific T cells, thereby inhibiting the T cell-mediated immune response, enabling tumor cells to escape immune surveillance and undergo immune escape. Inhibitors that block the interaction between PD-1 and PD-L1 can restore the T cell-mediated immune response.

[0003] At present, antibody-based inhibitors targeting PD-1 / PD-L1 have achieved remarkable results in the field of cancer immunotherapy. However, at the present stage, the biggest problem faced clinically is that not all patients can benefit from immunotherapy. A large amount of clinical data shows that only 20% - 30% of cancer patients can respond to immunotherapy. PD-L1 is a key biomarker for the treatment of immune checkpoint inhibitors, and the detection of its expression level is of great significance for guiding immunotherapy strategies, predicting treatment responses, and evaluating patient prognosis. However, due to the high heterogeneity of PD-L1 expression in tumors, the commonly used immunohistochemistry method in clinical practice cannot accurately and comprehensively evaluate the expression of PD-L1, and thus cannot accurately screen patients who will benefit (see the literature "The impact of a pathologist's personality on the interobserver variability and diagnostic accuracy of predictive PD-L1 immunohistochemistry in lung cancer. Lung Cancer. 2022; 166: 143 - 9.").

[0004] Positron emission tomography (PET) imaging has become a powerful tool for non-invasive visualization and quantification of molecular expression in vivo. Currently, a variety of PET imaging tracers targeting PD-L1 have been developed. For example, 89 Zr-Pembrolizumab, 89Antibody-based imaging tracers such as Zr-Nivolumab have received extensive attention due to their high specificity. However, the inherent disadvantages of antibodies, such as long half-life, poor tumor penetrability, high cost, etc., limit the practical application of antibody-based imaging tracers in PET imaging targeting PD-L1, which has prompted the development of small molecule-based imaging tracers targeting PD-L1. Small molecule-based imaging tracers can well compensate for the defects of antibody-based imaging tracers and have the advantages of fast imaging, good tumor penetrability, short metabolic half-life, low manufacturing cost, etc. For example, 68 Ga]WL-12 and 18 F]BMS986192 have been verified to have good targeting effects in in vivo imaging and have great potential in the rapid detection of PD-L1 expression.

[0005] In recent years, many reports have shown that small molecules containing a biphenyl backbone have high affinity for PD-L1 (see the literature "Design, synthesis, and evaluation of o-(biphenyl-3-ylmethoxy)nitrophenyl derivatives as PD-1 / PD-L1 inhibitors with potent anticancer efficacy in vivo. Journal of Medicinal Chemistry, 2021, 64(11): 7646-7666."). Starting from small molecules containing a biphenyl backbone, more small molecule imaging tracers with good performance may be designed for the detection of PD-L1. However, the currently reported fluorine-18 ( 18 F)-labeled small molecule imaging tracers containing a biphenyl backbone, such as 18 F]LN, 18 F]LG-1 and 18 F]LGSu-1, etc. (see the literature "One-step radiosynthesis and initial evaluation of a small molecule PET tracer for PD-L1 imaging. Bioorg Med Chem Lett. 2020; 30(24): 127572.", "Promising potential of a 18"F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging. Bioorg Chem. 2021;115:105294." and "Preparation and Bioevaluation of 18 F-Labeled Small-Molecular Radiotracers via Sulfur(VI) Fluoride Exchange Chemistry for Imaging of Programmed Cell Death Protein Ligand 1 Expression in Tumors. Mol Pharm. 2023;20(8):4228-4235."), there are defects in poor tumor specificity, which severely limits the practical application of such small-molecule imaging tracers in PET imaging targeting PD-L1. There is an urgent need to further improve the F-18 ( 18 F)-labeled small-molecule imaging tracers containing a biphenyl skeleton to improve their poor tumor specificity. Summary of the Invention

[0006] To solve the above problems, the present invention provides a PD-L1-targeted biphenyl-based radiotracer, and the biphenyl-based radiotracer has the following structure:

[0007]

[0008] In the formula, R is -CN, -OH, -NO2 or -NH2, and the value of n is an integer from 3 to 7.

[0009] In one embodiment of the present invention, the value of n is 3 or 7.

[0010] In one embodiment of the present invention, the labeling precursor of the biphenyl-based radiotracer has the following structure:

[0011]

[0012] Alternatively, the labeling precursor of the biphenyl-based radiotracer has the following structure:

[0013]

[0014] Alternatively, the labeling precursor of the biphenyl-based radiotracer has the following structure:

[0015]

[0016] Alternatively, the labeling precursor of the biphenyl-based radioactive tracer has the structure shown below:

[0017]

[0018] The present invention also provides a method for preparing the above-mentioned biphenyl-based radioactive tracer, which is characterized in that the method includes: mixing the labeling precursor of the biphenyl-based radioactive tracer and a radioactive labeling compound, and then performing a copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction to obtain the biphenyl-based radioactive tracer;

[0019] The radioactive labeling compound has the structure shown below:

[0020]

[0021] In the formula, n is an integer ranging from 3 to 7.

[0022] In an embodiment of the present invention, n is 3 or 7.

[0023] In an embodiment of the present invention, the preparation method of the labeling precursor includes: mixing compound 3, tris(hydroxymethyl)aminomethane, AcOH and sodium cyanoborohydride and reacting them to obtain compound 2; mixing compound 2, potassium carbonate, potassium iodide and propargyl bromide and reacting them to obtain the labeling precursor;

[0024] The labeling precursor has the structure shown below:

[0025]

[0026] Compound 3 has the structure shown below:

[0027]

[0028] Compound 2 has the structure shown below:

[0029]

[0030] In an embodiment of the present invention, the preparation method of the labeling precursor includes: mixing compound 9, diethyl (tosyloxymethyl)phosphonate and cesium carbonate and reacting them to obtain compound 10; mixing compound 10, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride and reacting them to obtain compound 11; mixing compound 11, potassium carbonate, potassium iodide and 3-bromopropyne and reacting them to obtain compound 12; mixing compound 12 and trimethylsilyl bromide and reacting them to obtain the labeling precursor;

[0031] The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below:

[0032]

[0033] Compound 9 has the structure shown below:

[0034]

[0035] Compound 10 has the structure shown below:

[0036]

[0037] Compound 11 has the structure shown below:

[0038]

[0039] Compound 12 has the structure shown below:

[0040]

[0041] In one embodiment of the present invention, the preparation method of the labeling precursor includes: mixing compound 14, methyl 3-bromomethylbenzoate and cesium carbonate and reacting them to obtain compound 15; mixing compound 15, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride and reacting them to obtain compound 16; mixing compound 16, potassium carbonate, potassium iodide and 3-bromopropyne and reacting them to obtain compound 17; mixing compound 17 and LiOH and reacting them to obtain the labeling precursor;

[0042] The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below:

[0043]

[0044] Compound 14 has the structure shown below:

[0045]

[0046] Compound 15 has the structure shown below:

[0047]

[0048] Compound 16 has the structure shown below:

[0049]

[0050] Compound 17 has the structure shown below:

[0051]

[0052] In one embodiment of the present invention, the method for preparing the labeling precursor includes: mixing compound 19, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride, and reacting them to obtain compound 20; mixing compound 20, potassium carbonate, potassium iodide and 3-bromopropyne, and reacting them to obtain the labeling precursor;

[0053] The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below:

[0054]

[0055] Compound 19 has the structure shown below:

[0056]

[0057] Compound 20 has the structure shown below:

[0058]

[0059] The present invention also provides the application of the above-mentioned biphenyl-based radioactive tracer in the preparation of a cell programmed death-ligand 1 imaging agent or in the preparation of a product for evaluating the effectiveness of PD-1 / PD-L1 immunotherapy.

[0060] In one embodiment of the present invention, the evaluation of the effectiveness of PD-1 / PD-L1 immunotherapy includes predicting the PD-1 / PD-L1 immunotherapy response and / or evaluating the PD-1 / PD-L1 immunotherapy prognosis.

[0061] In one embodiment of the present invention, the product is a detection kit.

[0062] The present invention also provides an imaging agent targeting cell programmed death-ligand 1, and the imaging agent contains the above-mentioned biphenyl-based radioactive tracer.

[0063] The present invention also provides a product for evaluating the effectiveness of PD-1 / PD-L1 immunotherapy, the product contains the above-mentioned biphenyl-based radioactive tracer, or the product contains the above-mentioned imaging agent.

[0064] In one embodiment of the present invention, the evaluation of the effectiveness of PD-1 / PD-L1 immunotherapy includes predicting the PD-1 / PD-L1 immunotherapy response and / or evaluating the PD-1 / PD-L1 immunotherapy prognosis.

[0065] In one embodiment of the present invention, the product is a detection kit.

[0066] The technical solution of the present invention has the following advantages:

[0067] The present invention provides a series of biphenyl-based radioactive tracers targeting PD-L1, including 18 F]LG-2, 18 F]LG-3, 18 F]LG-13, 18 F]LG-14, and 18 F]LG-15, etc. This series of biphenyl-based radioactive tracers has good in vitro stability, can specifically bind to PD-L1, and has good targeting specificity. It can be seen that this series of biphenyl-based radioactive tracers can non-invasively and accurately monitor the expression level and changes of PD-L1 in tumors in real time, dynamically, and systemically at the molecular level through PET imaging, and then effectively evaluate the efficacy of PD-1 / PD-L1 immunotherapy for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 : Synthetic routes of non-radioactive compounds LG-2 and LG-3.

[0069] Figure 2 : Synthetic route of non-radioactive compound LG-13.

[0070] Figure 3 : Synthetic route of non-radioactive compound LG-14.

[0071] Figure 4 : Synthetic route of non-radioactive compound LG-15.

[0072] Figure 5 : Mass spectrum of compound 2.

[0073] Figure 6 : Mass spectrum of compound 1.

[0074] [[ID=4३]] Figure 7 : Mass spectrum of compound LG-2.

[0075] Figure 8 : Mass spectrum of compound LG-3.

[0076] Figure 9 : 1 1H NMR spectrum of compound 2.

[0077] Figure 10 : 1 1H NMR spectrum of compound 1.

[0078] Figure 11 : 1 1H NMR spectrum of compound LG-2.

[0079] Figure 12 : The 1 H NMR spectrum of compound LG-3.

[0080] Figure 13 : The mass spectrum of compound 10.

[0081] Figure 14 : The mass spectrum of compound 11.

[0082] Figure 15 : The mass spectrum of compound 12.

[0083] Figure 16 : The mass spectrum of compound 13.

[0084] Figure 17 : The mass spectrum of compound 15.

[0085] Figure 18 : The mass spectrum of compound 17.

[0086] Figure 19 : The mass spectrum of compound 18.

[0087] Figure 20 : The mass spectrum of compound 21.

[0088] Figure 21 : Radiotracer 18 F]LG-2 and 18 The radiolabeling process of F]LG-3.

[0089] Figure 22 : Radiotracer 18 F]LG-13, 18 F]LG-14 and 18 The radiolabeling process of F]LG-15.

[0090] Figure 23 : Tracer 18 F]LG-2 and 18 The HPLC analysis results of F]LG-3. Figure 23 Among them, (A) LG-2 (UV: λ = 254 nm) / 18 The HPLC chromatogram of F]LG-2 (radio); (B) LG-3 / 18 The HPLC chromatogram of F]LG-3.

[0091] Figure 24 : Tracer 18 F]LG-2 and 18 The stability study results of F]LG-3. Figure 24 Among them, (A) 18Radioactive HPLC chromatograms of F]LG-2 in PBS (pH 7.4) and mouse serum at 1 h and 2 h; (B) 18 Radioactive HPLC chromatograms of F]LG-3 in PBS (pH 7.4) and mouse serum at 1 h and 2 h.

[0092] Figure 25 : tracer 18 F]LG-13, 18 F]LG-14 and 18 Results of the stability study of F]LG-15. Figure 25 Among them, (A) 18 Radioactive HPLC chromatograms of F]LG-13 in PBS (pH 7.4) and mouse serum at 1 h and 2 h; (B) 18 Radioactive HPLC chromatograms of F]LG-14 in PBS (pH 7.4) and mouse serum at 1 h and 2 h; (C) 18 Radioactive HPLC chromatograms of F]LG-15 in PBS (pH 7.4) and mouse serum at 1 h and 2 h.

[0093] Figure 26 : Results of the cytotoxicity experiments of non-radioactive compounds LG-2 and LG-3. Figure 26 Among them, (a) Cell viability of LG-2 incubated with A375-hPD-L1 cells for 24 hours; (b) Cell viability of LG-3 incubated with A375-hPD-L1 cells for 24 hours.

[0094] Figure 27 : tracer 18 F]LG-2 and 18 Results of the cellular uptake study of F]LG-3. Figure 27 Among them, (A) 18 Uptake of F]LG-2 in B16-F10 tumor cells with and without non-radioactive compound LG-2; (B) [[ID=3,9]] 18 [[ID=,40]]Uptake of F]LG-3 in B16-F10 tumor cells with and without non-radioactive compound LG-3; (C) 18 Uptake of F]LG-2 in A375-hPD-L1 and A375 tumor cells with and without non-radioactive compound LG-2; (D) 18 Uptake of F]LG-3 in A375-hPD-L1 and A375 tumor cells with and without non-radioactive compound LG-3. N.S indicates no significant difference, *p<0.05, **p<0.01, ***p<0.001.

[0095] Figure 28 : tracer18 F]LG-13, [ 18 F]LG-14 and [ 18 F] Results of cellular uptake studies of LG-15. Figure 28 In, (A)[ 18 F] LG-13 uptake in B16-F10 tumor cells with and without the non-radioactive compound LG-13; (B) [ 18 F] LG-14 uptake in B16-F10 tumor cells with and without the non-radioactive compound LG-14; (C) [ 18 F] Uptake of LG-15 in B16-F10 tumor cells with and without the non-radioactive compound LG-15. ****p<0.001.

[0096] Figure 29 : tracer[ 18 F]LG-2 and [ 18 F] PET imaging and quantitative analysis results of LG-3 in B16-F10 tumor-bearing mice (n=3). Figure 29 Injection 18 F]LG-2 or [ 18 F] Coronal images of B16-F10 tumor-bearing mice after LG-3 (~5 MBq); (B) Quantitative analysis of PET images based on [ 18 F]LG-2 and [ 18 F] LG-3 uptake values in tumor and muscle tissues of B16-F10 tumor-bearing mice; (C) [ 18 F]LG-2 and [ 18 F] LG-3 uptake ratio in tumor and muscle tissue of B16-F10 tumor-bearing mice; (D) quantitative analysis of PET images based on [ 18 F]LG-2 and [ 18 F] LG-3 uptake in the intestinal tissue of B16-F10 tumor-bearing mice; (E) [ 18 F]LG-2 and [ 18 F] LG-3 uptake ratio in tumor and intestinal tissues of B16-F10 tumor-bearing mice; (F) Based on quantitative analysis of PET images [ 18 F]LG-2 and [ 18 F] LG-3 uptake in liver tissue of B16-F10 tumor-bearing mice; (G) [ 18 F]LG-2 and [ 18 F] Uptake ratio of LG-3 in tumor and liver tissues of B16-F10 tumor-bearing mice.

[0097] Figure 30: Effect of blockade of non-radioactive compound LG-2 or LG-3 on the uptake of tracer 18 18 18 18 Figure 30 In (a), static PET images of 18 18 18 18 18 18 18 18

[0098] Figure 31 : PET imaging and quantitative analysis results of tracer 18 18 Figure 31 In (A), coronal images of B16-F10 tumor-bearing mice after injection of 18 18 18 18 18 18

[0099] Figure 32 : PET imaging and quantitative analysis results of tracer 18 18 Figure 32 In (A), coronal images of B16-F10 tumor-bearing mice after injection of 18 18 18 18 18 18

[0100] Figure 33 : PET imaging and quantitative analysis results of tracer 18 18 Figure 33 In (A), coronal images of B16-F10 tumor-bearing mice after injection of 18 1818 Uptake values of [¹⁸F]LG-15 in tumor and muscle tissues of B16-F10 tumor-bearing mice; (C) Effect of LG-15 blockade on 18 Uptake of [¹⁸F]LG-15 in tumor tissues; (D) 18 Uptake ratio of [¹⁸F]LG-15 in tumor and muscle tissues of B16-F10 tumor-bearing mice.

[0101] Figure 34 : Tracer 18 PET imaging and quantitative analysis results of [¹⁸F]LG-3 in different tumor-bearing mice (n = 3). Figure 34 Among them, (A) Static PET imaging of A375-hPD-L1, A375-hPD-L1 / A375, and A375 tumor-bearing mice after injection of 18 [¹⁸F]LG-3; (B) Expression levels of PD-L1 in different tumors detected by Western Blot; (C) Quantitative analysis based on PET images to obtain 18 Uptake values of [¹⁸F]LG-3 in different tumors; (D) Uptake values of [¹⁸F]LG-3 in different tumors measured by a gamma counter 18 ; (E) Pearson correlation analysis between the tumor uptake values obtained by quantitative analysis of PET images and the tumor uptake values measured by a gamma counter; (F) Pearson correlation analysis between the tumor uptake values obtained by quantitative analysis of PET images and the tumor PD-L1 expression levels detected by Western blot. ***p < 0.001.

[0102] Figure 35 : Monitoring of PD-L1 changes induced by chemotherapy using the tracer 18 [¹⁸F]LG-3 by PET imaging. Figure 35 Among them, (A) Treatment protocol for NCI-H1299 tumor-bearing mice with CDDP; (B) PET imaging study of NCI-H1299 tumor-bearing mice before and after CDDP treatment; (C) Quantitative analysis based on PET images to obtain 18 Uptake values of [¹⁸F]LG-3 in tumors of NCI-H1299 tumor-bearing mice. ***p < 0.001.

[0103] Figure 36 : Antitumor activity of compound LG-12 in B16-F10 tumor-bearing mice (n = 4). Figure 36 Among them, (A) Tumor photos of control group and LG-12 treatment group mice on the 1st and 7th days; (B) Tumor volume growth curves of control group and LG-12 treatment group mice.

[0104] Figure 37 : Using the tracer 18LG-3 monitors the changes in PD-L1 caused by the treatment with a PD-L1 inhibitor (i.e., compound LG-12) through PET imaging. Figure 37 Among them, (A) the treatment protocol for B16-F10 tumor-bearing mice with a PD-L1 inhibitor; (B) the 18 PET imaging study of B16-F10 tumor-bearing mice that received or did not receive LG-12 treatment 2 hours after injection of 18 LG-3; (C) the uptake value of LG-3 in the tumors of B16-F10 tumor-bearing mice obtained based on quantitative analysis of PET images; (D) autoradiography analysis of tumor tissues after PET imaging on the 7th day; (E) the expression level of PD-L1 in tumors detected by Western Blot on the 7th day. **P<0.01, ***P<0.001, ****P<0.0001.

[0105] Figure 38 : The results of the affinity experiment of the tracer 18 LG- and 18 LG-3. Figure 38 Among them, (a) 18 the results of the saturation binding assay of LG-2 with A375-hPD-L1 cells; (b) 18 the results of the saturation binding assay of LG-3 with A375-hPD-L1 cells.

[0106] Figure 39 : The results of the pharmacokinetic study of the tracer 18 LG-2 and 18 LG-3. Figure 39 Among them, (a) 18 the pharmacokinetic curve of LG-2 in mice; (b) 18 the pharmacokinetic curve of LG-3 in mice.

[0107] Figure 40 : The biodistribution map of the tracer 18 LG-2 or 18 LG-3 in B16-F10 tumor-bearing mice within 1 hour. Specific implementation mode

[0108] The following embodiments are provided to better further understand the present invention, which are not limited to the described optimal implementation mode, and do not constitute a limitation to the content and protection scope of the present invention. Any product that is the same as or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other existing technologies falls within the protection scope of the present invention.

[0109] For those without specific experimental steps or conditions noted in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For the reagents or instruments without the manufacturer noted, they are all conventional reagent products that can be obtained through commercial purchase.

[0110] In terms of chemical characterization, electrospray ionization mass spectrometry (ESI-MS) analysis was performed using a quadruple tandem mass spectrometer ZMD4000 LC / MS. High-performance liquid chromatography (HPLC) and radio-high-performance liquid chromatography (radio-HPLC) were carried out using a Waters pump, and a C18 chromatographic column (250 mm × 4.6 mm, 10 μm, Phenomenex) was equipped with an ultraviolet-visible absorption spectrum (UV-Vis) and a radioactivity detector. 1 H / 13 1H nuclear magnetic resonance (NMR) spectra were analyzed using a Bruker 500 MHz NMR spectrometer (Bruker, Germany). Dynamic and static images were acquired using an Inveon small animal micro-PET scanner (Siemens AG, Germany).

[0111] Examples 1-1 to 1-2: Non-radioactive compounds LG-2, LG-3

[0112] This example provides non-radioactive compounds LG-2 and LG-3, and the non-radioactive compounds have the structures shown below:

[0113]

[0114] Example 2-1: A method for preparing non-radioactive compound LG-2

[0115] This example provides a method for preparing the non-radioactive compound LG-2 described in Example 1-1 (the synthesis route of the non-radioactive compound LG-2 is shown in Figure 1 ), and the specific steps are as follows:

[0116] Step 1: Refer to the literature "Promising potential of a 18 18F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging. Bioorg Chem. 2021;115:105294." to synthesize compound 3.

[0117] Step 2: Dissolve compound 3 (0.64 g, 1.2 mmol), tris(hydroxymethyl)aminomethane (0.29 g, 2.4 mmol) and glacial acetic acid (AcOH, 0.3 mL, 4.8 mmol) in DMF (N,N-dimethylformamide, 8 mL), then stir and react at room temperature (25 °C) for 1 h to obtain reaction product A; after adding sodium cyanoborohydride (0.3 g, 4.8 mmol) to reaction product A, continue to stir and react at 60 °C for 24 h to obtain reaction product B; add saturated sodium chloride solution (50 mL) to reaction product B, a large amount of white solid precipitates. After the precipitation is complete, centrifuge and collect the precipitate (0.38 mg); concentrate the precipitate by rotary evaporation and take the supernatant; purify the supernatant by column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound 2 in the form of a white solid (0.38 g, yield 49.7%). The mass spectrum and hydrogen spectrum data of compound 2 are as follows (the mass spectrum result of compound 2 is shown in Figure 5 , and the hydrogen spectrum result is shown in Figure 9 ):

[0118] 1 H NMR (500 MHz, DMSO-d6, δ: ppm) δ 8.05 (s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.44 (d, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.81–6.71 (m, 2H), 5.33 (s, 2H), 5.29 (s, 2H), 4.29 (s, 6H), 3.60 (s, 6H), 2.24 (s, 3H). ESI-MS (m / z): 631 [M+H] + , 653 [M+Na] + .

[0119] Step 3: Dissolve the compound (0.15 g, 0.24 mmol), potassium carbonate (0.10 g, 0.18 mmol) and potassium iodide (11 mg, 0.06 mmol) in DMF (3 mL) to obtain a solution. After adding 3-bromopropyne (0.08 mL, 0.48 mmol) to the solution, react at room temperature for 4 h to obtain a reaction product. After adding saturated sodium chloride solution (50 mL) to the reaction product, extract three times with EtOAc (50 mL × 3), and take the organic phase. Dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by column chromatography (DCM / MeOH = 20 / 1, v / v) to obtain compound 1 (50 mg, yield 31.4%). The mass spectrum and hydrogen spectrum data of compound 1 are as follows (the mass spectrum result of compound 1 is shown in Figure 6 , and the hydrogen spectrum result is shown in Figure 10 ):

[0120] 1 H NMR (500 MHz, DMSO-d6, δ: ppm) δ 7.96 (s, 1H), 7.84 (dd, J = 14.2, 7.8 Hz, 2H), 7.62 (t, J = 7.8 Hz, 1H), 7.47 (s, 1H), 7.47–7.40 (m, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.17 (dd, J = 7.7, 1.5 Hz, 1H), 7.03 (s, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.79–6.75 (m, 2H), 5.29 (s, 2H), 5.19 (s, 2H), 4.35 (d, J = 5.2 Hz, 2H), 4.29 (s, 4H), 4.04 (s, 2H), 3.63 (d, J = 5.3 Hz, 6H), 3.03–3.00 (m, 1H), 2.24 (s, 3H). The extra proton at δ 7.96, 2.89 and 2.73 (1:3:3) was assigned to DMF. ESI-MS (m / z): 691 [M+Na] + .

[0121] Step 4: Synthesize compound 8a with reference to the literature "Xu L, Zhang L, Liang B, Zhu S, Lv G, Qiu L, Lin J. Design, synthesis, and biological evaluation of a small-molecule PET agent for imaging PD-L1 expression. Pharmaceuticals, 2023, 16(2): 213."; dissolve compound 1 (0.04 g, 0.06 mmol), compound 8a (0.01 g, 0.06 mmol), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.026 g, 0.06 mmol) and CuSO4·5H2O (0.002 g, 0.06 mmol) in DMF / H2O (4 mL / 1 mL) to obtain a solution; after adding sodium ascorbate (0.03 g, 0.12 mmol) to the solution, react at 45 °C for 3 h under the protection of N2 to obtain a reaction product; add saturated sodium chloride solution (30 mL) to the reaction product, extract three times with EtOAc (20 mL × 3), and take the organic phase; dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant; purify the supernatant by column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound LG-2 (0.045 g, yield 84.9%). The mass spectrum and hydrogen spectrum data of compound LG-2 are as follows (the mass spectrum result of compound LG-2 is shown in Figure 7 , and the hydrogen spectrum result is shown in Figure 11 ):

[0122] 1 H NMR (500 MHz, DMSO-d6, δ: ppm) δ 8.00 (s, 1H), 7.86 (dd, J = 11.8, 7.9 Hz, 2H), 7.69 (s, 1H), 7.62 (t, J = 7.8 Hz, 1H), 7.55 (s, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.24 (t, J = 7.6 Hz, A), 6.93 (dd, J = 8.2, 1.5 Hz, 1H), 6.86 (s, 1H), 6.81–6.71 (m, 2H), 5.19 (s, 4H), 4.53 (dd, J = 5.6, 2.6 Hz, 2H), 4.48–4.41 (m, 2H), 4.38 (s, 2H), 4.29 (s, 4H), 3.80 (s, 6H), 3.70 (t, J = 5.4 Hz, 2H), 3.65–3.61 (m, 2H), 3.49 (d, J = 4.1 Hz, 10H), 2.23 (s, 3H). ESI-MS (m / z): 891 [M + H]+ ,913[M+Na] + .

[0123] Example 2-2: A method for preparing non-radioactive compound LG-3

[0124] This example provides a method for preparing the non-radioactive compound LG-3 described in Example 1-2 (the synthetic route of non-radioactive compound LG-2 is shown in Figure 1 ), and the specific steps are as follows:

[0125] Compound 8b was synthesized with reference to the literature "Xu L, Zhang L, Liang B, Zhu S, Lv G, Qiu L, Lin J. Design, synthesis, and biological evaluation of a small-molecule PET agent for imaging PD-L1 expression. Pharmaceuticals, 2023, 16(2): 213."

[0126] On the basis of Example 2-1, step four was replaced with: Dissolve compound 1 (0.12 g, 0.18 mmol), compound 8b (0.09 g, 0.18 mmol), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.08 g, 0.18 mmol) and CuSO4·5H2O (0.05 g, 0.18 mmol) in DMF / H2O (4 mL / 1 mL) to obtain a solution; after adding sodium ascorbate (0.03 g, 0.12 mmol) to the solution, react at 45 °C for 3 h under the protection of N2 to obtain a reaction product; after adding saturated sodium chloride solution (30 mL) to the reaction product, extract three times with EtOAc (20 mL×3), and take the organic phase; dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant; purify the supernatant by column chromatography (DCM / MeOH = 6 / 1, v / v) to obtain compound LG-3 (0.13 g, yield 72.3%). The mass spectrum and 1H NMR data of compound LG-3 are as follows (the mass spectrum result of compound LG-3 is shown in Figure 8 , and the 1H NMR result is shown in Figure 12 ):

[0127] 11H NMR (500 MHz, DMSO-d6, δ: ppm) δ 7.98 (s, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.68 (s, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.40 (d, J = 7.4 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 6.93 (d, J = 8.2 Hz, 1H), 6.88 (s, 1H), 6.78 (d, J = 2.1 Hz, 1H), 6.75 (dd, J = 8.2, 2.1 Hz, 2H), 5.18 (s, 4H), 4.57–4.52 (m, 2H), 4.49–4.41 (m, 2H), 4.37 (s, 2H), 4.29 (s, 4H), 4.03 (t, J = 7.1 Hz, 2H), 3.80 (s, 2H), 3.71–3.64 (m, 4H), 3.61–3.58 (m, 2H), 3.52 (d, J = 4.2 Hz, 6H), 3.51–3.43 (m, 20H), 2.23 (s, 3H). ESI-MS (m / z): 1066 [M+H] + , 1088 [M+Na] + .

[0128] Example 1-3: A non-radioactive compound LG-13

[0129] This example provides a non-radioactive compound LG-13, and the non-radioactive compound has the following structure:

[0130]

[0131] Example 2-3: A method for preparing the non-radioactive compound LG-13

[0132] This example provides a method for preparing the non-radioactive compound LG-13 described in Example 1-3 (the synthetic route of the non-radioactive compound LG-13 is shown in Figure 2 ), and the specific steps are as follows:

[0133] Step 1: Refer to the literature "Preparation and Bioevaluation of 18F-Labeled Small-Molecular Radiotracers via Sulfur(VI)Fluoride Exchange Chemistry for Imaging of Programmed Cell Death Protein Ligand 1 Expression in Tumors. Mol Pharm. 2023;20(8):4228-4235. "Synthesize compound 9."

[0134] Step 2: Dissolve compound 9 (310 mg, 0.6 mmol), diethyl (tosyloxymethyl)phosphonate (232 mg, 0.7 mmol), and cesium carbonate (488 mg, 1.5 mmol) in a mixed solvent of ACN (acetonitrile, 25 mL) and THF (tetrahydrofuran, 5 mL), and then react at 80 °C for 4 h to obtain a reaction product; Concentrate the reaction product by rotary evaporation and take the supernatant; Purify the supernatant by silica gel column chromatography (Hex / EA = 2 / 3, v / v) to obtain compound 10 (220 mg, yield: 55%) (the mass spectrometry results of compound 10 are shown in Figure 13 ).

[0135] Step 3: Dissolve compound 10 (200 mg, 0.3 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (109 mg, 0.9 mmol) in N,N-dimethylformamide (DMF, 4 mL) to obtain a solution; Add AcOH (55 μL, 0.9 mmol) and sodium cyanoborohydride (57 mg, 0.9 mmol) to the solution and react overnight at room temperature (16 h) to obtain a reaction product; Mix the reaction product with water (50 mL), extract with EtOAc (20 mL × 3), and take the organic phase; Dry the organic phase over anhydrous sodium sulfate, then concentrate by rotary evaporation and take the supernatant; Purify the supernatant by silica gel column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound 11 (90 mg, yield: 39%) (the mass spectrometry results of compound 11 are shown in Figure 14 ).

[0136] Step 4: Dissolve compound 11 (90 mg, 0.1 mmol), potassium carbonate (48 mg, 0.3 mmol), and potassium iodide (6.6 mg) in DMF (5 mL) to obtain a solution. After adding 3-bromopropyne (28 mg, 0.2 mmol) to the solution, react at 50 °C for 4 h to obtain a reaction product. After adding saturated sodium chloride solution (40 mL) to the reaction product, extract three times with EtOAc (20 mL × 3), and take the organic phase. Dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by silica gel column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound 12 (42 mg, yield: 45%) (the mass spectrometry results of compound 12 are shown in Figure 15 ).

[0137] Step 5: Dissolve compound 12 (25.6 mg, 0.03 mmol) in a mixed solvent of anhydrous ACN (1 mL) and anhydrous THF (1 mL) to obtain a solution. After adding trimethylsilyl bromide (25 μL, 0.18 mmol) to the solution, react at 35 °C for 8 h to obtain a reaction product. Purify the reaction product by semi-preparative high performance liquid chromatography to obtain compound 13 (1 mg, yield: 4%) (the mass spectrometry results of compound 13 are shown in Figure 16 ).

[0138] Step 6: Dissolve compound 13 (0.04 g, 0.05 mmol), compound 8a (0.04 g, 0.1 mmol), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.092 g, 0.02 mmol), and CuSO4·5H2O (0.053 g, 0.02 mmol) in DMF / H2O (4 mL / 1 mL) to obtain a solution. After adding sodium ascorbate (0.084 g, 0.04 mmol) to the solution, react at 45 °C for 3 h under the protection of N2 to obtain a reaction product. After adding saturated sodium chloride solution (50 mL) to the reaction product, extract three times with EtOAc (15 mL × 3), and take the organic phase. Dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound LG-13 (0.52 g, yield 85.2%).

[0139] Examples 1-4: A non-radioactive compound LG-14

[0140] This example provides a non-radioactive compound LG-14, and the non-radioactive compound has the following structure:

[0141]

[0142] Example 2-4: A method for preparing non-radioactive compound LG-14

[0143] This example provides a method for preparing the non-radioactive compound LG-14 described in Examples 1-4 (for the synthetic route of the non-radioactive compound LG-14, see Figure 3 ). The specific steps are as follows:

[0144] Step 1: Synthesize compound 14 with reference to the literature "Bioorg Med Chem Lett. 2020; 30(24): 127572."

[0145] Step 2: Dissolve compound 14 (224 mg, 0.5 mmol), methyl 3-(bromomethyl)benzoate (375 mg, 1.6 mmol), and cesium carbonate (712 mg, 2.2 mmol) in a mixed solvent of N,N-dimethylformamide (5 mL) and THF (tetrahydrofuran, 3 mL), and then react overnight at room temperature (16 h) to obtain a reaction product; mix the reaction product with water (40 mL), extract with EtOAc (15 mL × 3), and take the organic phase; dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant; purify the supernatant by silica gel column chromatography (Hex / EA = 2 / 1, v / v) to obtain compound 15 in the form of a white solid (190 mg, yield: 62%) (for the mass spectrometry results of compound 15, see Figure 17 ).

[0146] Step 3: Dissolve compound 15 (190 mg, 0.3 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (165 mg, 1.3 mmol) in N,N-dimethylformamide (4.5 mL) to obtain a solution; add AcOH (82 μL, 1.3 mmol) and sodium cyanoborohydride (85 mg, 1.3 mmol) to the solution, and then react overnight at room temperature (16 h) to obtain a reaction product; mix the reaction product with water (50 mL), extract with EtOAc (20 mL × 3), and take the organic phase; dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant; purify the supernatant by silica gel column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound 16 (158 mg, yield: 70%).

[0147] Step 4: Dissolve compound 16 (158 mg, 0.2 mmol), potassium carbonate (99 mg, 0.7 mmol), and potassium iodide (12.4 mg, 0.07 mmol) in N,N-dimethylformamide (4 mL) to obtain a solution. After adding 3-bromopropyne (88 mg, 0.7 mmol) to the solution, react at 50 °C for 4 h to obtain a reaction product. Mix the reaction product with water (40 mL), extract with EtOAc (20 mL × 3), and take the organic phase. Dry the organic phase over anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by silica column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound 17 (31.5 mg, yield: 19%) (for the mass spectrometry results of compound 17, see Figure 18 ).

[0148] Step 5: Dissolve compound 17 (31.5 mg, 0.04 mmol) in anhydrous THF (1 mL) to obtain a solution. After adding MeOH containing 1 M LiOH to the solution, react at 28 °C for 1 h to obtain a reaction product. Neutralize the reaction product to neutral with 1 M HCl first, then mix with water (20 mL), extract with EtOAc (10 mL × 3), and take the organic phase. Dry the organic phase over anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by silica column chromatography (DCM / MeOH = 8 / 1, v / v, v / v) to obtain compound 18 (10 mg, yield: 32%) (for the mass spectrometry results of compound 18, see Figure 19 )

[0149] Step 6: Dissolve compound 18 (0.04 g, 0.05 mmol), compound 8a (0.04 g, 0.1 mmol), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.101 g, 0.02 mmol), and CuSO4·5H2O (0.058 g, 0.02 mmol) in DMF / H2O (4 mL / 1 mL) to obtain a solution. After adding sodium ascorbate (0.092 g, 0.04 mmol) to the solution, react at 45 °C for 3 h under the protection of N2 to obtain a reaction product. After adding saturated sodium chloride solution (50 mL) to the reaction product, extract three times with EtOAc (15 mL × 3), and take the organic phase. Dry the organic phase over anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound LG-14 (0.54 g, yield 85.6%).

[0150] Examples 1-5: A non-radioactive compound LG-15

[0151] This example provides a non-radioactive compound LG-15, and the non-radioactive compound has the structure shown below:

[0152]

[0153] Examples 2-5: A method for preparing the non-radioactive compound LG-15

[0154] This example provides a method for preparing the non-radioactive compound LG-15 described in Examples 1-5 (the synthetic route of the non-radioactive compound LG-15 is shown in Figure 4 ), and the specific steps are as follows:

[0155] Step 1: Refer to the literature "Preparation and Bioevaluation of 18 F-Labeled Small-Molecular Radiotracers via Sulfur(VI)Fluoride Exchange Chemistry for Imagingof Programmed Cell Death Protein Ligand 1Expression in Tumors.Mol Pharm.2023;20(8):4228-4235." to synthesize Compound 19.

[0156] Step 2: Dissolve Compound 19 (1 g, 1.7 mmol) and 2-amino-2-(hydroxymethyl)propane-1,3-diol (815 mg, 6.7 mmol) in N,N-dimethylformamide (8 mL) to obtain a solution; add AcOH (418 μL, 7 mmol) and sodium cyanoborohydride (418 mg, 6.6 mmol) to the solution, and react overnight at room temperature (16 h) to obtain a reaction product; mix the reaction product with water (100 mL), extract with EtOAc (70 mL×3), and take the organic phase; dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant; purify the supernatant by silica gel column chromatography (DCM / MeOH = 15 / 1, v / v) to obtain Compound 20 (807 mg, yield: 68%).

[0157] Step 3: Dissolve compound 20 (412 mg, 0.6 mmol), potassium carbonate (243 mg, 1.8 mmol), and potassium iodide (30 mg, 0.2 mmol) in N,N-dimethylformamide (6 mL) to obtain a solution. After adding 3-bromopropyne (214 mg, 1.8 mmol) to the solution, react at room temperature for 4 h to obtain a reaction product. Mix the reaction product with water (50 mL), extract with EtOAc (40 mL×3), and take the organic phase. Dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by silica gel column chromatography (DCM / MeOH = 30 / 1, v / v) to obtain compound 21 (102 mg, yield: 24%) (the mass spectrometry results of compound 21 are shown in Figure 20 ).

[0158] Step 4: Dissolve compound 21 (0.04 g, 0.05 mmol), compound 8a (0.04 g, 0.1 mmol), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.094 g, 0.02 mmol), and CuSO4·5H2O (0.054 g, 0.02 mmol) in DMF / H2O (4 mL / 1 mL) to obtain a solution. After adding sodium ascorbate (0.085 g, 0.04 mmol) to the solution, react at 45 °C for 3 h under the protection of N2 to obtain a reaction product. After adding saturated sodium chloride solution (50 mL) to the reaction product, extract three times with EtOAc (15 mL×3), and take the organic phase. Dry the organic phase with anhydrous sodium sulfate first, then concentrate by rotary evaporation, and take the supernatant. Purify the supernatant by column chromatography (DCM / MeOH = 10 / 1, v / v) to obtain compound LG-15 (0.53 g, yield 86.3%).

[0159] Examples 2-1 to 2-2: Biphenyl-based radioactive tracers 18 F]LG-2, 18 F]LG-3

[0160] This example provides biphenyl-based radioactive tracers 18 F]LG-2, 18 F]LG-3, and the biphenyl-based radioactive tracers have the following structures:

[0161]

[0162] Examples 3-1 to 3-2: A method for preparing biphenyl-based radioactive tracers 18 F]LG-2, 18 F]LG-3

[0163] This example provides the preparation methods of the biphenyl-based radioactive tracers described in Examples 2-1 to 2-2 18 F]LG-2, 18 F]LG-3 (see Figure 21 for the radiolabeling process of Compound 1), and the specific steps are as follows:

[0164] On a medical cyclotron (Jiuyiyuan Particle Technology Co., Ltd.), the nucleophile 18 O(p,n) 18 F is generated by a nuclear reaction 18 F-F - ; Use a Sep-Pak light QMA chromatographic column (Waters) to capture 18 F-F - generated by the cyclotron (activate the QMA column with NaHCO3 (0.5 M, 10 mL) and water (10 mL) before capture, and then blow out the residual moisture in the column with a syringe); Use 1.5 mL of Kryptofix 2.2.2 and K2CO3 solution to elute 18 F - from the QMA column into the reaction flask; Evaporate the eluted solution with a nitrogen stream at 100 °C; After drying, add 2 mL of anhydrous acetonitrile and evaporate and dry the solution twice to ensure that all moisture is removed; Dissolve Compound 7a or Compound 7b (2 mg, synthesize Compound 7a and Compound 7b with reference to the literature "Promising potential of a 18 F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging. Bioorg Chem. 2021;115:105294.") in dimethyl sulfoxide (0.7 mL) and add it to the reaction flask, and react with 18 F - at 100 °C for 30 min; After the reaction is completed, purify the reaction product by semi-preparative high-performance liquid chromatography (HPLC), dilute with water after obtaining the product peak, concentrate with a C18 light Sep-Pak chromatographic column, and then elute with DMF (0.8 mL) into the second reaction flask; Pre-activate the C18 chromatographic column with ethanol (10 mL) and water (10 mL); First add Compound 1 (0.1 mg) to the reaction flask, then add CuSO4·5H2O (0.3 mg), tris(hydroxypropyltriazolyl)methylamine (THPTA, 0.5 mg) and sodium ascorbate (0.3 mg), and react at 45 °C for 30 min under nitrogen protection; After the reaction is completed, analyze the 18 F-labelled product by radioactive HPLC, and the analysis results are shown inFigure 23 ; The product was purified by semi-preparative high performance liquid chromatography, diluted with water, concentrated using a C18 light Sep-Pak column, eluted with ethanol (0.8 - 1.0 mL), and the eluate was collected to obtain the biphenyl-based radioactive tracer 18 F]LG-2, 18 F]LG-3.

[0165] From Figure 23 the results, it can be seen that the retention times of the tracer and the corresponding non-radioactive compound in high performance liquid chromatography are the same, indicating that 18 F]LG-2 and 18 F]LG-3 were successfully obtained by the CuAAC labeling method. Among them, 18 the total RCY of 18 F]LG-2 was 15%, 18 and that of 18 F]LG-3 was 18%. After purification by semi-preparative high performance liquid chromatography,

[0166] Example 2-3 to 2-5: Biphenyl-based radioactive tracers 18 F]LG-13, 18 F]LG-14, 18 F]LG-15

[0167] This example provides biphenyl-based radioactive tracers 18 F]LG-13, 18 F]LG-14, 18 F]LG-15, and the biphenyl-based radioactive tracers have the following structures:

[0168]

[0169] Example 3-3 to 3-5: A method for preparing biphenyl-based radioactive tracers 18 F]LG-13, 18 F]LG-14, 18 F]LG-15

[0170] This example provides the preparation method of the biphenyl-based radioactive tracers 18 F]LG-13, 18 F]LG-14, 18 F]LG-15 described in Example 2-3 to 2-5 (the radiolabeling process of compounds LG-13, LG-14, and LG-15 is shown in Figure 22 ), and the specific steps are as follows:

[0171] On the basis of Examples 3-1 to 3-2, compound 7b was used, and compound 1 was replaced with compound 13, compound 18 or compound 21 respectively to obtain biphenyl-based radioactive tracers 18 18 18

[0172] Experimental Example 1: In vitro stability experiment of PD-L1-targeted biphenyl-based radioactive tracers

[0173] This experimental example provides an in vitro stability experiment of PD-L1-targeted biphenyl-based radioactive tracers, and the specific process is as follows:

[0174] Experiment 1: The biphenyl-based radioactive tracers 18 18 18 18 18 Figures 24 - 25 .

[0175] Experiment 2: The biphenyl-based radioactive tracers 18 18 18 18 18 Figures 24 - 25 .

[0176] Experimental results: From Figures 24 - 25 , it can be seen that after incubation for 2 h, the integrity rates of the five tracers in PBS and serum were both greater than 98%, indicating that the five tracers had good stability in vitro.

[0177] ​​​​​​​​​​​​​Experimental Example 2: Experiment on the Lipid-Water Partition Coefficient of PD-L1-Targeted Biphenyl Radioactive Tracers

[0178] This experimental example provides an experiment on the lipid-water partition coefficient of PD-L1-targeted biphenyl radioactive tracers, and the specific process is as follows:

[0179] Absorb radioactive tracer 18 F]LG-2 or 18 F]LG-3 (0.7 MBq) is dissolved in 1 mL of deionized water and 1 mL of n-octanol respectively, and then fully mixed to make the tracer evenly distributed in the aqueous phase and n-octanol to obtain a mixed solution; the mixed solution is centrifuged at 4000 g for 5 min to completely separate the two phases; 500 μL is taken from each of the upper organic phase and the lower aqueous phase into a radioimmunoassay tube, and the radioactivity of the tracer in the two phases is detected with a gamma counter, and according to the formula: Log P = Log(C O / C W ), calculate the lipid-water partition coefficient, where C O represents the radioactivity of the tracer in the organic phase, and C W represents the radioactivity of the tracer in the aqueous phase; after sampling, 500 μL is added to each of the organic phase and the aqueous phase to make the volume 1 mL, and this operation is repeated three times, and the average value of three groups of data is taken to calculate the lipid-water partition coefficient.

[0180] Experimental results: The distribution of radioactive tracers 18 F]LG-2 and 18 F]LG-3 in the aqueous phase and n-octanol phase is shown in Table 1, and the measured LogP values of the two tracers are 1.71 ± 0.09 and 1.40 ± 0.06 respectively.

[0181] Table 1 Distribution of radioactive tracers 18 F]LG-2 and 18 F]LG-3 in the aqueous phase and n-octanol phase

[0182]

[0183]

[0184] Experimental Example 3: Cytotoxicity Experiment of PD-L1-Targeted Biphenyl Radioactive Tracers

[0185] This experimental example provides a cytotoxicity experiment of PD-L1-targeted biphenyl radioactive tracers, and the specific process is as follows:

[0186] B16-F10 cells (mouse melanoma cell line, purchased from the Cell Bank of the Chinese Academy of Sciences) are seeded at a density of 1 × 10 4After inoculating into a 96-well plate with the inoculum volume of the holes, incubate overnight (16 h) in a 37 °C, 5% CO₂ incubator using DMEM medium (purchased from Biological Industries, Israel); after the incubation, add LG-2 or LG-3 with final concentrations of 0.195, 0.39, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50 μM (the solvent is DMEM medium at 100 μL / well) into the 96-well plate, and continue to incubate in a 37 °C, 5% CO₂ incubator for 24 h; after the incubation, add MTT reagent with a concentration of 5 mg / mL into the 96-well plate at an addition amount of 20 μL / well, and continue to incubate in a 37 °C, 5% CO₂ incubator for 4 h; after the incubation, aspirate all the solutions in the wells, add DMSO into the 96-well plate at an addition amount of 150 μL / well, and shake for 10 min; after the shaking, use an enzyme-linked immunosorbent detector (MD / M5e, VEDENG) to detect the absorbance at 490 nm, and calculate the cell survival rate according to the formula: cell survival rate = (absorbance of experimental wells / absorbance of blank wells), and then evaluate the cytotoxicity. The calculation results are shown in Figure 26 .

[0187] Experimental results: Figure 26 The results show that both non-radioactive tracers LG-2 or LG-3 have low toxicity to A375-hPD-L1 cells. Among them, the half-maximal inhibitory concentration IC 50 of LG-2 and LG-3 are 20.24 ± 6.9 μM and 28.32 ± 3.4 μM, respectively. When the cells are incubated with 6.25 μM of LG-2 or LG-3 for 24 hours, the cell survival rate is still greater than 90%. When the concentrations of LG-2 and LG-3 are 12.5 μM and 25 μM, respectively, the cell survival rate is about 60%. Therefore, the usage concentrations of LG-2 and LG-3 in subsequent experiments need to be controlled below 6.25 μM.

[0188] Experimental Example 4: Cellular uptake experiment of PD-L1-targeted biphenyl-based radioactive tracers

[0189] This experimental example provides a cellular uptake experiment of PD-L1-targeted biphenyl-based radioactive tracers, and the specific process is as follows:

[0190] Experiment 1: Seed B16-F10 cells (mouse melanoma cell line, purchased from the Cell Bank of the Chinese Academy of Sciences) at 3×10 6After inoculating at an inoculum density of cells / mL into DMEM medium (purchased from Biological Industries, Israel) containing 1% (v / v) penicillin-streptomycin double antibody (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) and 10% (v / v) fetal bovine serum (purchased from Biological Industries, Israel), incubate in a 37°C, 5% CO₂ incubator for 12 h until the B16-F10 cells are in the logarithmic growth phase; inoculate the B16-F10 cells in the logarithmic growth phase at an inoculum density of 2.6×10 5 / well into a six-well plate containing DMEM medium (1500 μL) supplemented with 1% (v / v) penicillin-streptomycin double antibody and 10% (v / v) fetal bovine serum, and then incubate in a 37°C, 5% CO₂ incubator for 12 h; divide the wells in the six-well plate into two groups, namely the blocking group and the non-blocking group. Among them, for the blocking group: aspirate the medium, first add compound LG-2, LG-3, LG-13, or LG-14 or LG-15 (50 μM, 1 mL, with the solvent being DMEM medium) into the wells of the blocking group respectively, incubate in a 37°C, 5% CO₂ incubator for 30 min, and then add biphenyl-based radioactive tracer 18 F]LG-2, 18 F]LG-3, 18 F]LG-13, 18 F]LG-14 or 18 F]LG-15 (3.7×10 -2 MBq, 200 μL, with the solvent being DMEM medium), incubate in a 37°C, 5% CO₂ incubator for 0.5, 1, 2 h, then rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3 M NaOH to lyse the B16-F10 cells for 10 min to obtain a lysate. For the non-blocking group: directly add biphenyl-based radioactive tracer 18 F]LG-2, 18 F]LG-3, 18 F]LG-13, 18 F]LG-14 or 18 F]LG-15 (3.7×10 -2 MBq, 1200 μL, with the solvent being DMEM medium) into the wells of the non-blocking group respectively, and incubate in a 37°C, 5% CO₂ incubator for 0.5, 1, 2 h; after incubation, first aspirate the medium, then rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3 M NaOH to lyse the B16-F10 cells for 10 min to obtain a lysate; collect the lysate and detect the radioactivity in the cells using a γ counter (1470Wizard, Perkins Elmer). The detection results are shown in Figures 27 - 28 .

[0191] Experiment 2: On the basis of Experiment 1, the B16-F10 cells were replaced with A375-hPD-L1 cells (a human melanoma cell line with high expression of PD-L1 by gene transfection, see the literature "Lv G, Sun X, Qiu L, Sun Y, Li K, Liu Q, Zhao Q, Qin S, Lin J. PET Imaging of Tumor PD-L1 Expression with a Highly Specific Nonblocking Single-Domain Antibody[J]. Joural of Nuclear Medicine, 2020, 61:117-122.") and A375 cells (a human malignant melanoma cell line, purchased from the Cell Bank of the Chinese Academy of Sciences) respectively to detect the uptake of biphenyl-based radioactive tracers in cells with different PD-L1 expression levels. The detection results are shown in Figure 27 .

[0192] Experimental results: To evaluate the binding of biphenyl-based radioactive tracers to PD-L1 in vitro, a cell uptake experiment was conducted in the mouse melanoma cell line B16-F10. As Figure 27 shown in A of Figure 27 and B of 18 , the uptake rates of both tracers in B16-F10 cells were relatively high. 18 The uptake rate of 18 F]LG-2 increased from 7.43 ± 0.01% at 0.5 h to 11.25 ± 0.02% at 1 h. 18 The uptake rate of 18 F]LG-2-3 increased from 3.69 ± 0.09% at 0.5 h to 9.11 ± 0.15% at 1 h. After 2 hours of culture, the cell uptake rates remained at relatively high levels. 18 The uptake rates of Figure 28 F]LG-2 and 18 F]LG-3 were 11.77 ± 0.02% and 9.02 ± 0.48% respectively. After adding the corresponding non-radioactive compounds, 18The cellular uptake of F]LG-13 was significantly reduced, being 0.25 ± 0.01% AD, 0.03 ± 0.07% AD, and 0.24 ± 0.12% AD, respectively. 18 F]LG-14 showed low cellular uptake values in B16-F10. After co-incubation with the non-radioactive compound LG-14, the cellular uptake became even lower, decreasing from 1.71 ± 0.25% AD at 0.5 h to 0.46 ± 0.14% AD, from 1.83 ± 0.12% AD at 1 h to 0.37 ± 0.01% AD, and from 2.13 ± 0.22% AD at 2 h to 0.25 ± 0.08% AD. 18 F]LG-15 had higher cellular uptake in B16-F10, with a cellular uptake of 4.70 ± 0.13% AD at 0.5 h and reaching a maximum uptake of 4.95 ± 0.04% AD at 2 h. In the presence of the non-radioactive compound LG-15, 18 the uptake values of F]LG-15 in B16-F10 were significantly reduced and remained at around 1.00% AD within 2 h. The experimental results showed that, 18 F]LG-13, 18 F]LG-14, and 18 F]LG-15 had good targeting specificity for PD-L1 in vitro.

[0193] To evaluate the uptake of biphenyl-based radioactive tracers in cells with different PD-L1 expression levels, cellular uptake assays were performed in the gene-transfected human malignant melanoma cell line A375-hPD-L1 with high PD-L1 expression and the human malignant melanoma cell line A375. As Figure 27 shown in C of Figure 27 and D of 18 F]LG-2 and 18 F]LG-3 had similar uptake trends in the A375-hPD-L1 cell line. At 0.5 h, the uptake rate of F]LG-2 was 10.21 ± 1.79%, and at 2 h it was 18.05 ± 1.30%; at 0.5 h, the uptake rate of F]LG-3 was 6.37 ± 0.18%, and at 2 h it was 18.74 ± 1.07%; while after being blocked by the non-radioactive compound, at 2 h 18 the uptake rate of F]LG-2 decreased to 3.83 ± 1.37%, 18 the uptake rate of F]LG-3 decreased to 2.17 ± 0.95%. In contrast, the binding rate of the tracer in the A375 cell line was 6-fold lower than that in the A375-hPD-L1 cell line, and after 2 h, the cellular uptake of F]LG-2 was 3.49 ± 1.99%, 18 the uptake rate of F]LG-3 decreased to 2.17 ± 0.95%. In contrast, the binding rate of the tracer in the A375 cell line was 6-fold lower than that in the A375-hPD-L1 cell line, and after 2 h, the cellular uptake of F]LG-2 was 3.49 ± 1.99%, 18 F]LG-3 was 2.17 ± 0.95%. 18 F]LG-2 was 3.49 ± 1.99%, 18The [F]LG-3 was 2.88 ± 0.21%, which corresponded to the difference in the basal PD-L1 expression. All the research results indicated that 18 [F]LG-2 and 18 [F]LG-3, these two tracers were targeted to PD-L1.

[0194] In the previous cellular uptake studies of the radiotracers 18 [F]LGSu-1, 18 [F]LGSu-2 (see the literature "Mol Pharm. 2023; 20(8): 4228 - 4235.") and 18 [F]LGT-1, 18 [F]LGT-2 (see the literature "Bioorg Med Chem Lett. Published online March 12, 2025."), after blocking with non-radioactive blockers, there was no obvious difference in the uptake values. In contrast, 18 [F]LG-2, 18 [F]LG-3, 18 [F]LG-13, 18 [F]LG-14 and 18 [F]LG-15 had obvious cell blocking effects, showing the advantages of these five radiotracers targeting PD-L1 in vitro.

[0195] Experimental Example 5: Imaging Experiment of Biphenyl-based Radiotracers Targeting PD-L1

[0196] This experimental example provided an imaging experiment (PET imaging and autoradiography) of biphenyl-based radiotracers targeting PD-L1. The specific process was as follows:

[0197] Experiment 1: B16-F10 cells were implanted subcutaneously into the upper right axilla of female 4 - 5-week-old BALB / c nude mice (purchased from Changzhou Cavens Experimental Animal Co., Ltd.) at a dose of 4×10 6 cells / mouse, obtaining a B16-F10 tumor-bearing nude mouse model. The tumor diameter was monitored every other day. When the tumor volume reached 100 mm 3 (The tumor volume calculation formula: 1 / 2 × long diameter × short diameter 2) After that, the B16-F10 tumor-bearing nude mouse model was divided into two groups, namely the blocking group and the non-blocking group. Blocking group: After anesthetizing the tumor-bearing mice in the blocking group with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min, fix the limbs and tail of the tumor-bearing mice, and inject the compounds LG-2, LG-3 or LG-15 (100 μM, 200 μL) dissolved in physiological saline through the tail vein respectively. 30 minutes after the injection of the blocker, inject the biphenyl radioactive tracer 18 F]LG-2, 18 F]LG-3 or 18 F]LG-15 (~5 MBq, diluted with 100 μL of physiological saline) through the tail vein respectively. Non-blocking group: After anesthetizing the tumor-bearing mice in the blocking group with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min, fix the limbs and tail of the tumor-bearing mice, and inject the biphenyl radioactive tracer 18 F]LG-2, 18 F]LG-3 or 18 F]LG-15 (~5 MBq, diluted with 100 μL of physiological saline) through the tail vein respectively. For the blocking group, static imaging (scanning for 10 minutes) was performed 60 minutes after the injection of the probes 18 F]LG-2 and 18 F]LG-3, and static imaging was performed 90 minutes after the injection of the probe 18 F]LG-15. For the non-blocking group, 1-hour dynamic and 120-minute static imaging (scanning for 10 minutes) were performed for the probes 18 F]LG-2 and 18 F]LG-3, and 2-hour dynamic and 150-minute and 180-minute static imaging (scanning for 10 minutes) were performed for the probe 18 F]LG-15. Quantitative evaluation was performed on the regions of interest (ROIs) of the tumor site and other important organs through the ASIPro image processing software to verify whether the biphenyl radioactive tracer can identify PD-L1-positive tumors. The imaging and quantitative analysis results are shown in Figures 29 - 30 and Figure 33 . Referring to the same method as the biphenyl radioactive tracer 18 F]LG-2, 18 F]LG-3 or 18 F]LG-15, PET imaging experiments were carried out on the biphenyl radioactive tracers 18 F]LG-13 and 18 F]LG-14. The difference is that the blocking group was not set, and dynamic scanning was performed within 2 hours after injection. The imaging and quantitative analysis results are shown in Figures 31 - 32 .

[0198] Experiment 2: On the basis of Experiment 1, replace B16-F10 cells with A375-hPD-L1 (high PD-L1 expression level), A375-hPD-L1 / A375 (medium PD-L1 expression level, obtained by mixing A375-hPD-L1 and A375 cells at a cell ratio of 1:1 and inoculating mice to obtain tumors with medium PD-L1 expression level), and A375 (low PD-L1 expression level) cells respectively. Inject the probe 18 F]LG-3 (~5 MBq, diluted with 100 μL of normal saline) into the tail veins of different tumor-bearing mice according to the method of Experiment 1 and perform PET imaging scans; after the PET imaging is completed, measure the radioactivity in the tumor tissues of different tumor-bearing mice with a γ counter, detect the PD-L1 expression level in the tumor tissues of different tumor-bearing mice by Western blotting, and perform Pearson correlation analysis on the obtained results and the tumor uptake values obtained by quantitative analysis of the PET images to further verify whether the biphenyl-based radioactive tracer can identify tumors with different PD-L1 expression levels. The relevant experimental results are shown in Figure 34 . Among them, the measurement method of the γ counter is as follows:

[0199] Euthanize the mice and dissect them 2 h after the probe injection, strip the target tissues, weigh the target tissues and place them in a radioimmunoassay tube, and measure the radioactivity of the samples with a γ counter (1470 WIZER, Perkins Elmer). The biodistribution of the probe is expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0200] The Western blotting method for detecting the PD-L1 expression level is as follows:

[0201] After washing the tumor tissue with physiological saline, dry the water first, then lyse it at 4°C using RIPI lysis buffer (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) containing 1% PMSF (phenylmethylsulfonyl fluoride, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), and take the supernatant for later use. The protein concentration of the supernatant was determined by the BCA method. The loading buffer was added to the supernatant, and then the mixture was boiled at 100°C for 5 minutes to obtain a protein sample. The protein sample was subjected to SDS-PAGE gel electrophoresis, and then the protein sample was transferred to a 0.22μm thick polyvinylidene fluoride (PVDF) membrane by electrophoresis (4°C). After the transfer, the protein-loaded PVDF membrane was blocked with 5% (w / v, g / 100 mL) skim milk powder at room temperature for 1 hour, and then incubated with human PD-L1 antibody [EPR19759] (1:1500, Abcam, Cat.#ab213524) or mouse PD-L1 antibody (1:1500, eBioscience, Cat.#14-5982-82), GAPDH (1:1000, Bio-Thera Biotechnology, Cat.#AF1186) at 4°C overnight (16 h). After primary antibody incubation, the membrane was washed with TBST buffer and then incubated with goat anti-rabbit secondary antibody (purchased from Santa Cruz Biotechnology, diluted 1500-fold with 5% skim milk powder for incubation, where % refers to w / w, mass ratio) or goat anti-mouse secondary antibody (purchased from Santa Cruz Biotechnology, diluted 1500-fold with 5% skim milk powder for incubation) at room temperature for 1 hour. Finally, the membrane was developed using an ECL chemiluminescence kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for chemiluminescence imaging.

[0202] Experiment 3: Based on Experiment 1, B16-F10 cells were replaced with NCI-H1299 cells (human non-small cell lung cancer cell line, purchased from the Chinese Academy of Sciences Cell Bank) to obtain NCI-H1299 tumor-bearing mice. NCI-H1299 tumor-bearing mice (n=3) were injected with CDDP (cisplatin, injection dose 5 mg / kg) via the tail vein once every 2 days for a total of 3 injections. The control group mice were injected with an equal amount of normal saline. On day 0 (i.e., before administration) and day 7 after the first administration, the NCI-H1299 tumor-bearing mice were injected with the probe [ 18 F]LG-3 (~5MBq, diluted in 100μL saline) and PET imaging scans were performed to explore the tracer [ 18 F] Whether LG-3 can monitor chemotherapy-induced PD-L1 changes through PET imaging, the relevant experimental results are shown in Figure 35 .

[0203] Experiment 4: B16-F10 tumor-bearing mice were constructed according to the method of Experiment 1. Compound LG-12 (injection dose 5 mg / kg, compound LG-12 refers to the literature "Synthesis and immunotherapy efficacy of a PD-L1 small-molecule inhibitor combined with its131I-iodide labelled isostructural compound. Bioorganic Chemistry. 2024; 153: 107810.") was injected intraperitoneally into B16-F10 tumor-bearing mice once every 2 days for a total of 3 injections. The control group of mice was injected with an equal amount of normal saline. During the injection period, the tumor size was measured every other day, and the tumor volume was calculated according to the formula: tumor volume = (length × width 2 ) / 2, and calculate the tumor volume to verify the antitumor activity of compound LG-12 in B16-F10 tumor-bearing mice. The relevant experimental results are shown in Figure 36 On the 0th day (i.e. before administration) and the 7th day after the first administration, the probe was injected into the tail vein of B16-F10 tumor-bearing mice according to the method of Experiment 1. 18 F]LG-3 (~5MBq, diluted in 100μL saline) was used for PET imaging scans; after PET imaging, the expression levels of PD-L1 in tumor tissues of different tumor-bearing mice were detected by Western blotting, and the results were compared with the tumor uptake values obtained by quantitative analysis of PET images to explore the effect of tracer [ 18 F] Whether LG-3 can monitor PD-L1 changes caused by LG-12 treatment through PET imaging, the relevant experimental results are shown in Figure 37 .

[0204] Experimental Results: To investigate the ability of biphenyl radiotracers to detect PD-L1 expression in vivo, PET imaging was performed on B16-F10 tumor-bearing mice. Figure 29 A and Figure 29 As shown in B, [ 18 F]LG-2 and [ 18 Both tracers, F]LG-3, were rapidly observed in the tumor. 30 min after injection, [ 18 F]LG-2 and [ 18 F]LG-3 reached the maximum uptake value, which was 6.45±0.38%ID / mL and 5.64±0.02%ID / mL respectively. The tumor uptake value remained relatively stable within 60 minutes after injection ([ 18 F]LG-2 was 6.03±0.36%ID / mL, [ 18The uptake value of [F]LG-3 was 5.85 ± 0.13% ID / mL and remained at a relatively high level within 120 min after injection. During the scan, the uptake value in muscle was relatively lower than that in tumor. 18 The uptake value of [F]LG-2 was 1.20 ± 0.15% ID / mL - 3.12 ± 0.56% ID / mL. 18 The uptake value of [F]LG-3 was 1.96 ± 0.26% ID / mL - 2.36 ± 0.52% ID / mL. 18 [F]LG-2 and 18 [F]LG-3 had maximum tumor-to-muscle (T / M) ratios of 2.89 ± 0.24 and 2.93 ± 0.29, respectively, indicating comparable specific uptake of the two tracers by tumors. Figure 29 (C in...). Meanwhile, in the presence of the corresponding non-radioactive compounds LG-2 and LG-3, the uptake values of tumors for 18 [F]LG-2 and 18 [F]LG-3 decreased significantly to 2.98 ± 0.07% ID / mL and 2.36 ± 0.13% ID / mL, respectively, 1 hour later. Figure 30 ) This further demonstrated the specificity of the two tracers for PD-L1.

[0205] In addition to tumors, 18 [F]LG-2 and 18 [F]LG-3 also showed high activity in the kidneys and bladder, indicating their excretion through the urinary system. However, 18 [F]LG-2 and 18 [F]LG-3 showed different behaviors in the intestine and liver. At 60 min after injection, 18 [F]LG-2 had significantly higher accumulation in the intestine, with an uptake value of 38.72 ± 11.21% ID / mL, while 18 [F]LG-3 had an uptake value that was almost halved, at 19.07 ± 4.82% ID / mL. Figure 29 (D in...). Similarly, in the early stage after injection of 18 [F]LG-2, the liver had a high uptake of 18 [F]LG-2, up to 40.59 ± 4.01% ID / mL, but it gradually decreased to 8.65 ± 1.22% ID / mL after 60 min. However, 18 [F]LG-3 had a significantly lower liver uptake rate during the scan, decreasing from 6.99 ± 0.37% ID / mL at 5 min to 4.12 ± 0.23% ID / mL at 60 min. Figure 29 At 60 min, 18The tumor-to-intestine uptake ratio (T / I) and tumor-to-liver uptake ratio (T / L) of FLG-2 were 0.15 ± 0.06 and 0.69 ± 0.08, respectively. 18 The tumor-to-intestine uptake ratio (T / I) and tumor-to-liver uptake ratio (T / L) of FLG-3 were 0.31 ± 0.08 and 1.42 ± 0.18 ( Figure 29 E in Figure 29 and 18 G in 18 ). The above research results showed that 18 the tumor uptake rates of FLG-2 and 18 FLG-3 were comparable, but

[0206] the uptake rate of FLG-3 in non-target tissues, especially in the intestine and liver, was lower, and it could generate clearer tumor images. Therefore, Figure 31 it was observed from 18 that there was no obvious uptake of FLG-13 in tumors. The maximum uptake value reached 1.73 ± 0.07% ID / mL at 120 min. At this time, the T / M value also reached the maximum of 1, which was only comparable to the muscle uptake, indicating poor target specificity. 18 The uptake of FLG-14 mainly accumulated in the intestine, and the tumor uptake was low. The maximum tumor uptake value was only 0.40 ± 0.01% ID / mL at 60 min, and the T / M value remained at about 1.2 within 2 h, and no clear tumor contour could be observed ( Figure 32 ). 18 After 60 min of tail vein injection of FLG-15, clear tumor imaging was observed. The tumor uptake increased slowly with time and reached the maximum uptake value of 4.24 ± 0.04% ID / mL at 120 min, and then gradually decreased. The T / M value remained at about 2 within 180 min and reached the maximum T / M value of 2.6 at 180 min. After blocking with the non-radioactive compound LG-15, the tumor uptake decreased significantly. The uptake value at 90 min decreased from 4.22 ± 0.07% ID / mL to 2.22 ± 0.03% ID / mL, indicating 18 that FLG-15 had good tumor specificity ( Figure 33 ). The above results showed that 18 as a small molecule PET probe for PD-L1, FLG-15 also had great potential.

[0207] In tumor-bearing mice with A375-hPD-L1 (high), A375-hPD-L1 / A375 (medium), and A375 (low), 18PET imaging of F]LG-3 was performed to further verify whether biphenyl-based radiotracers can identify tumors with different PD-L1 expression levels. As Figure 34 shown in A of Figure 34 and C of

[0208] , it was clearly observed that the A375-hPD-L1 tumor with the highest PD-L1 expression level showed the highest uptake value at 2 h, which was 6.56 ± 0.07% ID / mL; while the tumor uptake value of A375-hPD-L1 / A375 tumor-bearing mice was nearly half less than that of A375-hPD-L1 tumor-bearing mice, and the measurement result was 2.95 ± 0.53% ID / mL. The A375 tumor with the lowest PD-L1 expression level had a very weak radioactive signal (1.45 ± 0.23% ID / mL), and almost no tumor uptake of the probe was observed. 18 Since an increase in signal of F]LG-3 was observed in tumors with elevated PD-L1 expression levels, a comprehensive quantitative analysis was subsequently performed to clarify the correlation between the in vivo tumor uptake determined by region of interest (ROI) analysis, the Western blot (WB) expression level, and the tumor uptake quantified by in vitro and in vivo gamma counting. As Figure 34 shown in B of Figure 34 and D of Figure 34 and F of Figure 34 , the relevant PD-L1 WB expression level of A375-hPD-L1 tumor was 2.09 ± 0.25, and the tumor uptake rate was 5.23 ± 0.22% ID / g; the relevant PD-L1 WB expression level of A375-hPD-L1 / A375 tumor was 0.89 ± 0.02, and the tumor uptake rate was 2.63 ± 0.15% ID / g; the relevant PD-L1 WB expression level of A375 tumor was 0.03 ± 0.01, and the tumor uptake rate was 0.13 ± 0.04% ID / g. As expected, a significant correlation was observed between the in vivo tumor uptake and the in vitro tumor uptake (R2 = 0.93, p < 0.0001) and the PD-L1 expression level (R2 = 0.94, p < 0.0001) in the Pearson correlation analysis ( 18 The results showed that F]LG-3 can be used to detect the expression of PD-L1 in vivo.

[0209] Numerous studies have shown that chemotherapy with drugs such as cisplatin (CDDP) can regulate the expression level of PD-L1 in malignant tumors (Reference: Cisplatin promotes the expression level of PD-L1 in the microenvironment of hepatocellular carcinoma through YAP1. Molecular and Cellular Biochemistry. 2020;475:79-91.). Precise monitoring of the dynamics of PD-L1 can provide guidance for the combined application of immunotherapy and chemotherapy. Therefore, using 18 PET imaging of [¹⁸F]LG-3 to monitor the dynamic changes in PD-L1 levels in NCI-H1299 xenograft tumors of human lung cancer after CDDP treatment to further verify whether biphenyl-based radiotracers can sensitively monitor changes in PD-L1 in tumors. As Figure 35 shown in A to Figure 35 C in 18 , due to the low expression level of PD-L1, there was almost no 18 accumulation of [¹⁸F]LG-3 in the tumor before CDDP treatment. At 30 min, 60 min, and 90 min after injection of 18 [¹⁸F]LG-3, the tumor uptake values were 1.02 ± 0.16% ID / g, 1.62 ± 0.23% ID / g, and 1.28 ± 0.08% ID / g, respectively. In contrast, after three treatments with CDDP (5 mg / kg), the 18 [¹⁸F]LG-3 signal in the tumor increased significantly, and the tumor uptake values were 3.28 ± 0.16% ID / g, 4.07 ± 0.12% ID / g, and 3.41 ± 0.05% ID / g, respectively. The significant increase in tumor uptake of 18 [¹⁸F]LG-3 may be due to the increased expression level of PD-L1 induced by CDDP. In summary, 18 [¹⁸F]LG-3 can sensitively monitor changes in PD-L1 in tumors and has important potential in guiding immunotherapy.

[0210] To evaluate 18Evaluation of the efficacy of PD-L1 blockade in tumors by F]LG-3. The PD-L1 small molecule inhibitor LG-12 (see the literature "Synthesis and immunotherapy efficacy of a PD-L1 small-molecule inhibitor combined with its 131I-iodide labelled isostructural compound. Bioorganic Chemistry. 2024;153:107810.") was injected into B16-F10 tumor-bearing mice at a dose of 5 mg / kg every other day for 3 times, and PET imaging of 18 F]LG-3 was performed on the 1st and 7th days. As Figure 36 shown, compared with the control group (83 ± 27 vs. 93 ± 11 mm 3 ), tumor growth in the treatment group was significantly inhibited after 3 injections (82 ± 5 vs. 445 ± 38 mm 3 ), indicating that LG-12 has good immunotherapeutic effects. As shown in B of Figure 37 and C of Figure 37 , the tumor uptake in the control group and the LG-12 treatment group was comparable on the 1st day (3.95 ± 0.22% ID / mL vs. 3.49 ± 0.15% ID / mL). However, on the 7th day, the uptake value in the control group increased to 4.64 ± 0.03% ID / mL, while the uptake value in the LG-12 treatment group decreased to 1.88 ± 0.08% ID / mL. Autoradiographic experimental analysis after PET imaging also confirmed the significant difference in activity between the two groups of tumors on the 7th day ([[]]END]] Figure 37 in D). The results showed that the immunotherapy based on LG-12 reduced the expression of PD-L1 in tumors. The tumor PD-L1 expression levels of the two groups of patients were detected by Western blot on the 7th day, and the results showed that the PD-L1 level in the LG-12 treatment group was lower ([[]]END]] Figure 37 in E). All these results indicate that PET imaging with 18 F]LG-3 can accurately evaluate the PD-L1 blockade effect in immunotherapy.

[0211] Experimental Example 6: Affinity experiment of PD-L1-targeted biphenyl-based radiotracers

[0212] This experimental example provides an affinity experiment of PD-L1-targeted biphenyl-based radiotracers, and the specific process is as follows:

[0213] B16-F10 cells were seeded at 1 × 10 per well 5After the cells were seeded into 24-well plates at a density of 100 μg / mL, they were incubated in a 37°C, 5% CO2 incubator with DMEM containing 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum for 12 hours until the B16-F10 cells were in the logarithmic growth phase. The experiment was started after the incubation was completed. The original culture medium was replaced with DMEM culture medium 1 hour before the start of the experiment. The experimental design includes two groups: non-specific binding group and specific binding group, with three parallel groups in each group. In the non-specific binding group, the cells were pre-incubated with non-radioactive tracers LG-2 and LG-3 (final concentration 20 μM, solvent is 600 μL DMEM culture medium) at 37°C for 1 hour; after the pre-incubation, the tracers [ 18 F]LG-2 and [ 18 F]LG-3 (final concentrations of 1.17, 2.34, 4.68, 9.37, 18.75, 37.5, 75, 150, and 300 nM) was added to the corresponding wells, and the two groups were incubated at 37°C for 1 hour. After the incubation, the cells were washed twice with cold (4°C) PBS buffer (500 μL / well) to stop the uptake process, and then the cells were lysed with 1 M NaOH (500 μL / well, 10 min). The CPM value was measured using a γ counter, and the KD value was calculated based on the difference between the nonspecific binding group and the specific binding group. The experimental results are shown in Figure 38 .

[0214] The tracer was further evaluated by competition binding experiments [ 18 F]LG-2 and [ 18 F] The dissociation constant (KD, Figure 38 ), calculated by Scatchard analysis [ 18 The KD value of LG-2 binding to PD-L1 was 34.00±2.97nM. 18 The KD value of LG-3 binding to PD-L1 is 33.98±6.98nM. 18 F]LG-2 and [ 18 There is no significant difference in the binding affinity between F]LG-3 and PD-L1, indicating that the introduction of PEG chains of different lengths does not change the binding ability of the targeting structure to PD-L1, and it still has good targeting ability to PD-L1.

[0215] Experimental Example 7: Pharmacokinetics of PD-L1-targeted biphenyl radiotracers

[0216] This experimental example provides a pharmacokinetic study of a PD-L1-targeted biphenyl radiotracer. The specific process is as follows:

[0217] Female BALB / c normal mice (n = 3) were injected via the tail vein with 18 F]LG-2 and 18 F]LG-3 (~5.5 MBq, 100 μL normal saline), and blood was collected by cutting the tail; at the time points of 1, 2, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120, 150, and 180 min, blood at the tail end of the mouse vein was collected with a cotton ball, and the weight (wet weight) of the blood collected at each time point was immediately weighed. The radioactivity count of the sample was detected using a gamma counter, and the radioactive tracer 18 F]LG-2 and 18 F]LG-3 with the same volume was used as the calibration reference. With the blood drug concentration as the ordinate and time as the abscissa, a pharmacokinetic curve was plotted. The experimental results are shown in Figure 39 .

[0218] To study the absorption, distribution, metabolism, and excretion processes of the radioactive tracers 18 F]LG-2 and 18 F]LG-3 in vivo, pharmacokinetic studies were conducted on 18 F]LG-2 and 18 F]LG-3. Pharmacokinetics mainly studies the law of the dynamic change of the blood drug concentration of drugs in living organisms. The results are as Figure 39 shown. 18 F]LG-2 and 18 F]LG-3 both conform to the two-compartment model in the in vivo metabolism process, with elimination half-lives of 63.7 minutes and 48.4 minutes respectively, and are cleared relatively quickly in vivo. And according to the pharmacokinetic and PET imaging results, there are differences in the elimination behaviors of the two tracers in mice. Combining their metabolic characteristics, 18 F]LG-2 may be mainly metabolized via the liver and kidneys, while 18 F]LG-3 tends to be cleared by the kidneys.

[0219] Experimental Example 8: Biodistribution Experiment of PD-L1-Targeted Biphenyl Radioactive Tracers

[0220] This experimental example provides a biodistribution experiment of PD-L1-targeted biphenyl radioactive tracers, and the specific process is as follows:

[0221] B16-F10 tumor-bearing mice (n = 6) were injected via the tail vein with the radioactive tracer 18 F]LG-2 or 18 F]LG-3 (~5.5 MBq, with the solvent being 100 μL normal saline), and the radioactive tracer 18 F]LG-2 or 18F]LG-3 was used as the attenuation correction control; the mice were sacrificed and dissected 1 h after injection, and samples of tumors, blood, and major organs (heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, bone, muscle, brain) were taken for weighing. After weighing, the samples were placed in radioimmunoassay tubes, and the radioactivity of the samples was measured using a gamma counter (1470 WIZER, PerkinsElmer). The experimental results are shown in Figure 40 . 18 F]LG-2 or 18 The biodistribution of F]LG-3 was expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0222] To further study the biodistribution and targeting specificity of the radioactive tracers 18 F]LG-2 and 18 F]LG-3 in mice, a biodistribution experiment was conducted in B16-F10 tumor-bearing mice 1 h after injection. As Figure 40 shown, 18 F]LG-2 (6.81 ± 0.97%ID / g) and 18 F]LG-3 (8.16 ± 2.68%ID / g) had tumor uptake values consistent with the results obtained by PET imaging. Similarly, compared with 18 F]LG-3, 18 F]LG-2 showed higher accumulation in the liver and intestine, with uptake values in the liver, large intestine, and small intestine of 18.66 ± 2.05%ID / g, 19.13 ± 0.86%ID / g, and 12.67 ± 0.22%ID / g, respectively, while 18 F]LG-3 had uptake values in the liver, large intestine, and small intestine of 4.27 ± 1.41%ID / g, 7.82 ± 1.48%ID / g, and 7.64 ± 2.53%ID / g. Conversely, compared with 18 F]LG-3 (16.51 ± 1.93%ID / g), 18 F]LG-2 (4.94 ± 0.45%ID / g) had significantly lower kidney activity. These results all verified that the two tracers were metabolized through different pathways, which was consistent with the results shown by pharmacokinetics. The above research results all showed that 18 F]LG-2 and 18 F]LG-3 had similar tumor uptake values, but 18 F]LG-3 showed better in vivo distribution characteristics, with significantly lower uptake values in non-target tissues such as the intestine and liver. Based on 18 the improved targeting selectivity of F]LG-3, its ability to detect PD-L1 in vivo will be further studied in the future.

[0223] Obviously, the above embodiments are merely examples for clear illustration and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all implementation manners here. And the obvious changes or alterations derived therefrom still fall within the protection scope of the present invention.

Claims

1. A PD-L1-targeted biphenyl-based radioactive tracer, characterized in that, The biphenyl-based radioactive tracer has the structure shown below: In the formula, the R is -CN, -OH, -NO2 or -NH2, and the value of n is an integer from 3 to 7.

2. The biphenyl-based radioactive tracer according to claim 1, characterized in that, The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: Alternatively, the labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: Alternatively, the labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: Alternatively, the labeling precursor of the biphenyl-based radioactive tracer has the structure shown below:

3. A method for preparing the biphenyl-based radioactive tracer according to claim 1 or 2, characterized in that, The method includes: mixing the labeling precursor of the biphenyl-based radioactive tracer and a radioactive labeling compound, and then performing a copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction to obtain the biphenyl-based radioactive tracer; The radioactive labeling compound has the structure shown below: In the formula, the value of n is an integer from 3 to 7.

4. The method according to claim 3, characterized in that The preparation method of the labeling precursor includes: mixing compound 3, tris(hydroxymethyl)aminomethane, AcOH and sodium cyanoborohydride and reacting them to obtain compound 2; mixing compound 2, potassium carbonate, potassium iodide and propargyl bromide and reacting them to obtain the labeling precursor; The labeling precursor has the structure shown below: Compound 3 has the structure shown below: Compound 2 has the structure shown below:

5. The method according to claim 3, characterized in that, The preparation method of the labeling precursor includes: mixing compound 9, diethyl (tosyloxymethyl)phosphonate and cesium carbonate and reacting them to obtain compound 10; mixing compound 10, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride and reacting them to obtain compound 11; mixing compound 11, potassium carbonate, potassium iodide and 3-bromopropyne and reacting them to obtain compound 12; mixing compound 12 and trimethylsilyl bromide and reacting them to obtain the labeling precursor; The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: Compound 9 has the structure shown below: Compound 10 has the structure shown below: Compound 11 has the structure shown below: Compound 12 has the structure shown below:

6. The method according to claim 3, wherein The preparation method of the labeling precursor includes: mixing compound 14, methyl 3-(bromomethyl)benzoate and cesium carbonate and reacting them to obtain compound 15; mixing compound 15, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride and reacting them to obtain compound 16; mixing compound 16, potassium carbonate, potassium iodide and 3-bromopropyne and reacting them to obtain compound 17; mixing compound 17 and LiOH and reacting them to obtain the labeling precursor; The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: Compound 14 has the structure shown below: Compound 15 has the structure shown below: Compound 16 has the structure shown below: Compound 17 has the structure shown below:

7. The method according to claim 3, characterized in that, The preparation method of the labeling precursor includes: mixing compound 19, 2-amino-2-(hydroxymethyl)propane-1,3-diol, AcOH and sodium cyanoborohydride and reacting them to obtain compound 20; mixing compound 20, potassium carbonate, potassium iodide and 3-bromopropyne and reacting them to obtain the labeling precursor; The labeling precursor of the biphenyl-based radioactive tracer has the structure shown below: The compound 19 has the structure shown below: The compound 20 has the structure shown below:

8. Use of the biphenyl-based radioactive tracer according to claim 1 or 2 in the preparation of a programmed cell death-ligand 1 imaging agent or in the preparation of a product for evaluating the effectiveness of PD-1 / PD-L1 immunotherapy.

9. An imaging agent targeting programmed cell death ligand 1, characterized in that, The imaging agent contains the biphenyl-based radioactive tracer according to claim 1 or 2.

10. A product for evaluating the effectiveness of PD-1 / PD-L1 immunotherapy, characterized in that, The product contains the biphenyl-based radioactive tracer according to claim 1 or 2, or the product contains the imaging agent according to claim 9.

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