PD-l1 small-molecule inhibitor, preparation method therefor, and use thereof

Abstract

The present invention relates to a PD-L1 small-molecule inhibitor, a preparation method therefor, and use thereof, and pertains to the technical field of biopharmaceuticals. The present invention provides an iodine-131-labeled PD-L1 small-molecule inhibitor designated [131I]LG-12 and a non-radioactive PD-L1 small-molecule inhibitor designated LG-12, wherein both the iodine-131-labeled PD-L1 small-molecule inhibitor [131I]LG-12 and the non-radioactive PD-L1 small-molecule inhibitor LG-12 can effectively prevent and / or treat cancer, and the combined use of [131I]LG-12 and LG-12 has a synergistic effect. Such nuclide / immune combination therapy strategies on the basis of PD-L1 small-molecule inhibitors have great potential in cancer treatment. Therefore, [131I]LG-12 and LG-12 have great application prospects in the preparation of drugs for preventing and / or treating cancer.

Description

A PD-L1 small molecule inhibitor, its preparation method and application Technical Field

[0001] This invention relates to a PD-L1 small molecule inhibitor, its preparation method, and its application, belonging to the field of biomedical technology. Background Technology

[0002] Cancer, also known as malignant tumors, is caused by the malignant proliferation of cells and includes melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer, and prostate cancer. Cancer treatment often employs a comprehensive approach, primarily combining surgery, chemotherapy, and radiotherapy—three traditional treatment methods—and, when necessary, incorporating targeted therapy, biological therapy, and anti-tumor immunotherapy. Among these, anti-tumor immunotherapy works by enhancing the body's own immune function to inhibit or kill cancer cells, significantly reducing the severe side effects of traditional treatments for cancer patients.

[0003] Antitumor immunotherapy is a cancer treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle, restoring the body's normal antitumor immune response. Immune checkpoint programmed cell death receptor 1 (PD-1) is a major research direction in antitumor immunotherapy. Studies have found that overexpressed programmed cell death ligand 1 (PD-L1) on the surface of tumor cells binds to PD-1 on the surface of effector T cells, inhibiting T cell attack on tumor cells and allowing tumor cells to escape immune responses. PD-1 / PD-L1 immune checkpoint inhibitors can restore the T cell antitumor immune response by blocking the binding of PD-L1 on the surface of tumor cells to PD-1 on T cells, thereby inhibiting rapid tumor proliferation and metastasis. Currently, the phenomenon that combining immune checkpoint inhibitors with other antitumor methods can effectively improve the low response rate of immunotherapy has been widely confirmed.

[0004] Targeted radionuclide therapy (TRT) is a cancer-targeted treatment method that utilizes targeted carriers to deliver therapeutic radionuclides to tumors, where they decay and release ionizing radiation, thereby destroying tumor tissue. Studies have shown that TRT can enhance the efficacy of immunotherapy in vivo by inducing immunogenic cell death (ICD), releasing tumor neoantigens, and stimulating anti-tumor immune responses. A recent study reported that low-dose radiation can remodel the tumor microenvironment, which is beneficial for anti-tumor immunity. Simultaneously, immunotherapy may also make tumors more sensitive to TRT. Therefore, combining immune checkpoint inhibitors with TRT may produce synergistic effects, leading to better cancer treatment outcomes. However, at present, there are no reports of PD-L1 small molecule inhibitors that simultaneously function as both immune checkpoint inhibitors and targeted radionuclide therapy drugs. Summary of the Invention

[0005] To address the above problems, this invention provides an iodine-131-labeled PD-L1 small molecule inhibitor, which has the following structure:

[0006] Where R is In one embodiment of the present invention, when R is At that time, the iodine-131-labeled PD-L1 small molecule inhibitor has the following structure:

[0007] The present invention also provides a non-radioactive PD-L1 small molecule inhibitor, wherein the non-radioactive PD-L1 small molecule inhibitor has the following structure:

[0008] Where R is

[0009] In one embodiment of the present invention, when R is At that time, the non-radioactive PD-L1 small molecule inhibitor has the following structure:

[0010] This invention also provides a method for preparing the above-mentioned iodine-131-labeled PD-L1 small molecule inhibitor, the method comprising: dissolving compound 2 and 4-methoxy(diacetoxyiodine)benzene in a solvent, and then reacting them under nitrogen protection to obtain compound 3; and further processing compound 3. 131 Radiolabeling of I yields an intermediate compound. 131 I]1; intermediate compound [ 131 I]1, Compound 4 and sodium cyanoborohydride were dissolved in a solvent to obtain a solution; glacial acetic acid was added to the solution and the reaction was carried out to obtain the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor.

[0011] The compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, sulfoalanine, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane (Tris), or glucosamine.

[0012] Compound 2 has the following structure:

[0013] The 4-methoxy(diacetoxyiodide)benzene has the following structure:

[0014] Compound 3 has the following structure:

[0015] The intermediate compound [ 131 I]1 has the following structure:

[0016] In one embodiment of the present invention, the preparation method of compound 2 includes: dissolving compound 1, tetrakis(triphenylphosphine)palladium and hexa-n-butylditin in a solvent, and then heating the mixture under nitrogen protection to obtain compound 2;

[0017] Compound 1 has the following structure:

[0018] In one embodiment of the present invention, the preparation method of compound 1 includes: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent and reacting them to obtain compound 1;

[0019] Compound 5 has the following structure:

[0020] The 3-bromomethylbenzonitrile has the following structure:

[0021] In one embodiment of the present invention, the preparation method of compound 5 includes: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarbonate dropwise to the solution under stirring in an ice bath and nitrogen to obtain a reaction solution; and reacting the reaction solution to obtain compound 5.

[0022] Compound 7 has the following structure:

[0023] Compound 6 has the following structure:

[0024] The present invention also provides a method for preparing the above-mentioned non-radioactive PD-L1 small molecule inhibitor, the method comprising: dissolving compound 1, compound 4 and sodium cyanoborohydride in a solvent to obtain a solution; adding glacial acetic acid to the solution and reacting to obtain the above-mentioned non-radioactive PD-L1 small molecule inhibitor;

[0025] The compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, sulfoalanine, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane (Tris), or glucosamine.

[0026] Compound 1 has the following structure:

[0027] In one embodiment of the present invention, the preparation method of compound 1 includes: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent and reacting them to obtain compound 1;

[0028] Compound 5 has the following structure:

[0029] The 3-bromomethylbenzonitrile has the following structure:

[0030] In one embodiment of the present invention, the preparation method of compound 5 includes: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarbonate dropwise to the solution under stirring in an ice bath and nitrogen to obtain a reaction solution; and reacting the reaction solution to obtain compound 5.

[0031] Compound 7 has the following structure:

[0032] Compound 6 has the following structure:

[0033] The present invention also provides the use of the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor or the above-mentioned non-radioactive PD-L1 small molecule inhibitor in the preparation of medicaments for the prevention and / or treatment of cancer.

[0034] In one embodiment of the present invention, the drug contains the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and the above-mentioned non-radioactive PD-L1 small molecule inhibitor.

[0035] In one embodiment of the present invention, the prevention and / or treatment of cancer includes promoting tumor death by inhibiting the PD-1 / PD-L1 pathway and / or inducing immunogenic death (ICD) in tumor cells.

[0036] In one embodiment of the present invention, promoting tumor death by inhibiting the PD-1 / PD-L1 pathway includes inhibiting PD-1 / PD-L1 interaction by specifically binding to PD-L1 in the tumor and / or reducing the expression level of PD-L1 in the tumor, activating the anti-tumor immune function of T cells, enhancing the killing ability of T cells against tumors, and thereby promoting tumor death.

[0037] The induction of immunogenic death in tumor cells includes promoting the release of high-mobility group box 1 (HMGB1) and calreticulin (CRT) from tumor cells, thereby inducing immunogenic death in tumor cells.

[0038] In one embodiment of the present invention, the activation of the anti-tumor immune function of T cells includes increasing CD4+ in tumor tissue. 4+ The proportion of T cells and the increase in CD4+ in tumor tissue 8+ T cell ratio and / or promotion of T cell secretion of interferon-γ.

[0039] In one embodiment of the present invention, the tumor includes a solid tumor; the solid tumor includes melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and / or prostate cancer.

[0040] The present invention also provides a medicament for the prevention and / or treatment of cancer, the medicament containing the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and / or the above-mentioned non-radioactive PD-L1 small molecule inhibitor.

[0041] In one embodiment of the present invention, the drug contains the above-mentioned iodine-131 labeled PD-L1 small molecule inhibitor and the above-mentioned non-radioactive PD-L1 small molecule inhibitor.

[0042] In one embodiment of the present invention, the prevention and / or treatment of cancer includes promoting tumor death by inhibiting the PD-1 / PD-L1 pathway, and / or inducing immunogenic death of tumor cells.

[0043] In one embodiment of the present invention, promoting tumor death by inhibiting the PD-1 / PD-L1 pathway includes inhibiting PD-1 / PD-L1 interaction by specifically binding to PD-L1 in the tumor and / or reducing the expression level of PD-L1 in the tumor, activating the anti-tumor immune function of T cells, enhancing the killing ability of T cells against tumors, and thereby promoting tumor death.

[0044] The induction of immunogenic death in tumor cells includes promoting the release of high-mobility group box 1 (HMGB1) and calreticulin (CRT) from tumor cells, thereby inducing immunogenic death in tumor cells.

[0045] In one embodiment of the present invention, the activation of the anti-tumor immune function of T cells includes increasing CD4+ in tumor tissue. 4+ The proportion of T cells and the increase in CD4+ in tumor tissue 8+ T cell ratio and / or promotion of T cell secretion of interferon-γ.

[0046] In one embodiment of the present invention, the tumor includes a solid tumor; the solid tumor includes melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and / or prostate cancer.

[0047] The technical solution of this invention has the following advantages:

[0048] This invention provides an iodine-131 labeled small molecule inhibitor of PD-L1. 131 [I] LG-12 and non-radioactive PD-L1 small molecule inhibitor LG-12, iodine-131 labeled PD-L1 small molecule inhibitor [ 131 LG-12 and the non-radioactive PD-L1 small molecule inhibitor LG-12 have the following advantages:

[0049] First, the PD-1 / PD-L1 TR-FRET experimental results show that the EC50 of the non-radioactive PD-L1 small molecule inhibitor LG-12... 50 The value was 34.55±3.21 nM, indicating that LG-12 has high inhibitory activity against PD-1 / PD-L1 interaction, can significantly inhibit tumor growth, and shows good anti-tumor immunotherapy effect;

[0050] Second, in vitro stability experiments showed that the iodine-131-labeled PD-L1 small molecule inhibitor [ 131 [I]LG-12 remained stable in vitro for 12 hours, indicating that, 131 I]LG-12 has the advantage of strong in vitro stability;

[0051] Third, cellular uptake and biodistribution experiments showed that iodine-131-labeled PD-L1 small molecule inhibitors [ 131 The maximum uptake of LG-12 in mouse melanoma cells B16-F10 was 5.33 ± 0.33% AD, and it could be significantly blocked by the non-radioactive PD-L1 small molecule inhibitor LG-12 (2.56 ± 0.06% AD). Simultaneously, 1 hour after tail vein injection, the uptake of iodine-131-labeled PD-L1 small molecule inhibitor […]. 131 The uptake value of LG-12 at the tumor site in B16-F10 tumor-bearing mice was 6.50 ± 1.05% ID / g, indicating that... 131 I]LG-12 can specifically bind to PD-L1 in tumors;

[0052] Fourth, in vivo imaging results showed that 30 minutes after tail vein injection, the iodine-131-labeled PD-L1 small molecule inhibitor […]. 131 [I] LG-12 rapidly accumulates in tumors of B16-F10 tumor-bearing mice, exhibiting 2.3 times higher activity in tumors than in muscle. Furthermore, upon blockade by the non-radioactive PD-L1 small molecule inhibitor LG-12, tumor uptake is significantly reduced, indicating that... 131 I]LG-12 can specifically bind to PD-L1;

[0053] Fifth, the results of the T cell / tumor cell co-culture experiment showed that when the concentrations of the non-radioactive PD-L1 small molecule inhibitor LG-12 were 0.78 μM and 3.125 μM, the survival rates of B16-F10 cells co-cultured with T cells decreased to 0.66±0.06 and 0.55±0.03, respectively. This indicates that LG-12 can activate the anti-tumor immune function of T cells and enhance the tumor-killing ability of T cells in a dose-dependent manner.

[0054] Sixth, the results of the T cell / tumor cell co-culture experiment showed that when the B16-F10 / T cell ratio was 1 / 20, after treatment with non-radioactive PD-L1 small molecule inhibitor LG-12 at concentrations of 0.78 μM and 3.125 μM, the expression level of interferon-γ increased from 34.44±1.39 pg / 100 μL to 40.28±1.97 pg / 100 μL and 76.33±5.24 pg / 100 μL, respectively. When the concentration was 1 / 40, after treatment with non-radioactive PD-L1 small molecule inhibitor LG-12 at concentrations of 0.78 μM and 3.125 μM, the expression level of interferon-γ increased from 53.19 ± 2.85 pg / 100 μL to 83.13 ± 3.64 pg / 100 μL and 110.26 ± 1.72 pg / 100 μL, respectively. This indicates that LG-12 can increase interferon-γ secretion and promote CD4+ expression by blocking the PD-1 / PD-L1 signaling pathway. 8+ T cell activation, which in turn activates the anti-tumor immune function of T cells and enhances the ability of T cells to kill tumors in a dose-dependent manner;

[0055] Seventh, in vivo anti-tumor experiments showed that after intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12, no mice experienced significant weight loss or death during the treatment process, indicating that LG-12 was well tolerated at all doses.

[0056] Eighth, in vivo anti-tumor experiments showed that intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12 significantly reduced tumor volume and weight in mice, and increased CD4 levels in mouse tumor tissue. + T cell percentage and CD8 + The increased proportion of T cells, the decreased expression of PD-L1 in mouse tumor tissue, and the significantly increased expression of interferon-γ in mouse serum indicate that LG-12 has a good in vivo anti-tumor effect.

[0057] Ninth, in vitro cloning experiments showed that, compared with iodine-131-labeled PD-L1 small molecule inhibitors [ 131The number of colonies formed by B16-F10 cells co-cultured with LG-12 was significantly reduced, and the effect was far superior to that with Na+. 131 In B16-F10 cells co-cultured with I, it can be seen that, [ 131 I]LG-12 can significantly inhibit the proliferation of tumor cells and has a good in vitro anti-tumor effect;

[0058] Tenth, experimental results on in vitro radiopharmaceutical stimulation of tumor cells showed that iodine-131-labeled PD-L1 small molecule inhibitors [ 131 Following LG-12 stimulation, the expression level of high-mobility group box 1 (HMGB1) outside B16-F10 cells significantly increased (the significant decrease in HMGB1 expression inside B16-F10 cells indicates a significant increase in HMGB1 expression outside B16-F10 cells). Simultaneously, the expression level of calreticulin (CRT) on the surface of B16-F10 cells significantly increased. Therefore, [ 131 I]LG-12 can induce immunogenic cell death in tumor cells by promoting the release of HMGB1 and CRT proteins, thus exhibiting a good in vitro anti-tumor effect;

[0059] Eleventh, in vivo antitumor experimental results showed that intraperitoneal injection of iodine-131-labeled PD-L1 small molecule inhibitors [ 131 Following LG-12, no significant weight loss or death was observed in any of the mice during the treatment process, indicating that... 131 I]LG-12 has low toxicity to normal tissues;

[0060] Twelfth, in vivo antitumor experimental results showed that intraperitoneal injection of iodine-131-labeled PD-L1 small molecule inhibitors [ 131 Following LG-12 administration, the expression levels of HMGB1 and CRT proteins in mouse tumor tissues significantly increased, and the effect was far superior to that of intraperitoneal injection of Na. 131 In mice of type I, it can be seen that, [ 131 I]LG-12 can induce immunogenic cell death in tumor cells by promoting the release of HMGB1 and CRT proteins, thus exhibiting a good in vivo anti-tumor effect.

[0061] Thirteenth, in vivo antitumor experiments showed that first, intravenous injection of iodine-131-labeled PD-L1 small molecule inhibitors […]. 131 Following intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12, the tumor volume in mice was significantly reduced, and CD4+ levels in the mouse tumor tissue were significantly lower. + T cell percentage and CD8 +The increased proportion of T cells, decreased PD-L1 expression in mouse tumor tissue, and significantly increased interferon-γ expression in mouse serum, with effects far exceeding those observed in mice receiving intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12 alone, demonstrate that... 131 The combination of LG-12 and LG-12 has a synergistic effect, which can significantly inhibit tumor growth, enhance anti-tumor immune response, and has a good in vivo anti-tumor effect.

[0062] In summary, iodine-131-labeled PD-L1 small molecule inhibitors [ 131 LG-12 and the non-radioactive PD-L1 small molecule inhibitor LG-12 can both effectively prevent and / or treat cancer, and, 131 [I] The combination of LG-12 and LG-12 has a synergistic effect. This radionuclide / immunotherapy strategy based on PD-L1 small molecule inhibitors has great potential in cancer treatment. Therefore, [ 131 LG-12 and LG-12 have great potential applications in the preparation of drugs for the prevention and / or treatment of cancer. Attached Figure Description

[0063] Figure 1: Synthesis process of LG-12, a non-radioactive small molecule inhibitor of PD-L1.

[0064] Figure 2: Synthesis process of labeled precursor compound 3.

[0065] Figure 3: ESI-MS analysis of the non-radioactive PD-L1 small molecule inhibitor LG-12.

[0066] Figure 4: ESI-MS analysis of compound 2.

[0067] Figure 5: ESI-MS analysis of labeled precursor compound 3.

[0068] Figure 6: The proton NMR spectrum of compound 5.

[0069] Figure 7: Carbon NMR spectrum of compound 5.

[0070] Figure 8: The proton NMR spectrum of compound 1.

[0071] Figure 9: Carbon NMR spectrum of compound 1.

[0072] Figure 10: Proton NMR spectrum of LG-12, a non-radioactive small molecule inhibitor of PD-L1.

[0073] Figure 11: Carbon NMR spectrum of LG-12, a non-radioactive small molecule inhibitor of PD-L1.

[0074] Figure 12: Proton NMR spectrum of compound 2.

[0075] Figure 13: Carbon NMR spectrum of compound 2.

[0076] Figure 14: Proton NMR spectrum of compound 3.

[0077] Figure 15: Carbon NMR spectrum of compound 3.

[0078] Figure 16: Inhibitory activity of the non-radioactive PD-L1 small molecule inhibitor LG-12 on PD-1 / PD-L1 interaction.

[0079] Figure 17: Intermediate compounds [ 131 I]1(a) and iodine-131 labeled PD-L1 small molecule inhibitors [ 131 I]LG-12(b) Radio-HPLC chromatograms before and after purification.

[0080] Figure 18: Intermediate compounds [ 131 I]1 and iodine-131 labeled PD-L1 small molecule inhibitors 131 I]LG-12 in vitro stability.

[0081] Figure 19: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 I] LG-12 uptake in B16-F10 cells at 1, 2 and 4 h of incubation (***P<0.001).

[0082] Figure 20: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 I] LG-12 in B16-F10 tumor-bearing mice SPECT / CT imaging (A), in vivo biodistribution analysis (B), and autoradiography analysis of tumor and muscle tissue (C) (***P<0.001).

[0083] Figure 21: Cytotoxicity of B16-F10 cells and T cells after incubation with the non-radioactive PD-L1 small molecule inhibitor LG-12 for 48 h.

[0084] Figure 22: Cell viability (A) and interferon-γ content in culture medium of B16-F10 cells after co-incubation with different concentrations of non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 0.78 and 3.125 μM) and B16-F10 cells / T cells (1 / 20 or 1 / 40) (NS: no significant difference, *p<0.05, **p<0.01 and ***p<0.001).

[0085] Figure 23: Tumor inhibition of the non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 5 mg / kg and 20 mg / kg) in a B16-F10 mouse melanoma model (control group n=4, treatment group n=5). In Figure 23, (A) is the tumor growth curve; (B) is the final tumor weight; (C) is the mouse body weight; (D) is a photograph of the dissected tumor (**p<0.01 and ***p<0.001).

[0086] Figure 24: H&E staining analysis of major tissues (heart, liver, spleen, lung, kidney, muscle) after treatment with the non-radioactive PD-L1 small molecule inhibitor LG-12 (scale bar is 50 μM, n=4).

[0087] Figure 25: IHC and H&E staining analysis of tumor tissues (A) and serum IFN-γ levels (B) in B16-F10 tumor-bearing mice after treatment with non-radioactive PD-L1 small molecule inhibitor LG-12 (5 mg / kg and 20 mg / kg) (scale bar: 50 μM, n = 4, *p < 0.05 and **p < 0.01).

[0088] Figure 26: Different doses of iodine-131-labeled PD-L1 small molecule inhibitors [ 131 Inhibitory effect of LG-12 (0, 0.49, 0.98, 1.97, 3.94 and 7.89 KBq / μL) on colony formation in B16-F10 cells.

[0089] Figure 27: Study on tumor ICD induced by iodine-131-labeled PD-L1 small molecule inhibitor LG-12. In Figure 27, (A) shows the results obtained by Western blotting. 131 I) Expression levels of HMGB1 and CRT proteins in B16-F10 cells treated with LG-12 (0 and 1.85 MBq / mL); (B) is [ 131 I]LG-12 or Na 131 (I) (11.1 MBq) antitumor effect in B16-F10 tumor-bearing mouse model; (C) is a tumor-peeling photograph; 131 I]LG-12 or Na 131 IHC analysis of HMGB 1 and CRT in B16-F10 tumor-bearing mice after I (11.1 MBq) treatment (scale bar 50 μM, n = 4).

[0090] Figure 28: [ 131[I] In vivo antitumor activity of LG-12 / LG-12 combination therapy in a B16-F10 tumor-bearing mouse model (n=5). In Figure 28, (a) is a photograph of the tumor after treatment; (b) is the tumor growth curve (insert: comparison between group B and group C); (c) is the serum IFN-γ level of B16-F10 tumor-bearing mice after treatment; (d) is the IHC analysis map of tumor tissue (CD4). + T cells, CD8 + (e) T cells and PD-L1); (e) CD4 in tumor tissue. + T cells, CD8 + Quantitative analysis of T cell and PD-L1 expression (scale bar: 50 μM, n = 4, *p < 0.05, ***p < 0.001). Detailed Implementation

[0091] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0092] For any experimental steps or conditions not specified in the following examples, the procedures or conditions described in the literature in this field can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0093] The PD-1 / PD-L1 binding assay kit (catalog #72038) used in the following examples was purchased from BPS Bioscience. For chemical characterization, electrospray ionization mass spectrometry (ESI-MS) was performed using a quadrupole tandem mass spectrometer ZMD4000 LC / MS (Waters, USA), chromatographic analysis was performed using high-performance liquid chromatography (HPLC) with UV and radioactivity detectors and a C18 column (250 × 4.6 mm, 10 μm, Phenomenex) pump (Waters, USA), and analysis was performed using a Bruker 400 MHz nuclear magnetic resonance spectrometer (Bruker, Germany). 1 H / 13 C nuclear magnetic resonance spectroscopy.

[0094] The cell culture and animal modeling processes involved in the following examples are as follows:

[0095] Tumor cell culture: Mouse melanoma cell line B16-F10 (purchased from the Cell Bank of the Chinese Academy of Sciences) was cultured at 1×10⁻⁶ cells / year. 6The inoculum was evenly distributed in a culture dish and incubated in 10 mL of DMEM medium (purchased from BI) containing 1% (v / v) penicillin-streptomycin (purchased from Shanghai Beyotime) and 10% (v / v) fetal bovine serum (purchased from BI) at 37°C and 5% (v / v) CO2. Cells were ready for in vitro experiments when they reached the logarithmic growth phase and showed good growth.

[0096] T cell culture: Mouse T lymphocytes were extracted from the spleens of BALB / c mice (purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) after sacrifice; CD3+ cells were added to 24-well plates at a concentration of 2 μL / well. + (Use after diluting with antibody diluent to a concentration of 5 μg / mL, CD3) + Both CD28 and antibody dilution buffer were purchased from BioLegend. CD28 (diluted to a concentration of 5 μg / mL with antibody dilution buffer, both purchased from BioLegend) was added at a dosage of 2 μL / well and incubated at 4°C for 24 h to obtain CD3. + 24-well plates coated with CD28 (5 μg / mL) antibody; extracted T lymphocytes were cultured at 3 × 10⁻⁶ cells / mL. 6 Inoculation rate of 1 inoculum per well was applied to CD3. + T lymphocytes were cultured in 24-well plates coated with CD28 (5 μg / mL) antibody at 37°C and 5% (v / v) CO2 in 1640 medium (purchased from BI) containing 10% (v / v) fetal bovine serum (purchased from BI). Once the T lymphocytes aggregated, they were ready for in vitro cell experiments.

[0097] Animal modeling: Female 4-5 week old BALB / c mice (purchased from Cavens Laboratory Animal Company); B16-F10 cells were seeded into the mice (inoculation dose 3×10⁻⁶). 6 The tumor was located in the upper right axilla of the affected area; the tumor diameter was monitored every other day, and when the tumor diameter reached 50–100 mm. 3 Further in vivo experiments were conducted using tumor-bearing mice. All animal research experiments were conducted in accordance with the principles established by the Ethics Committee of the Jiangsu Provincial Institute of Nuclear Medicine.

[0098] Example 1: A non-radioactive small molecule inhibitor of PD-L1, LG-12

[0099] This embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-12, which has the following structure:

[0100] Example 2: A method for preparing a non-radioactive PD-L1 small molecule inhibitor LG-12

[0101] This embodiment provides a method for preparing the non-radioactive PD-L1 small molecule inhibitor LG-12 described in Example 1 (synthetic route is shown in Figure 1). The specific steps are as follows:

[0102] Step 1: Refer to the literature "Dieter Enders; Jeanne Fronert; Tom Bisschops; Florian Boeck. Xiuting; Xie Minhao; Qiu Ling; Lin Jianguo. Promising potential of a 18 F-labeled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging. Bioorganic Chemistry 2021, 115, 105294. (Synthetic compounds 6 and 7)

[0103] Compound 7 has the following structure:

[0104] Compound 6 has the following structure:

[0105] Step 2: Under ice bath conditions, compound 7 (2.56 g, 10 mmol), compound 6 (2.64 g, 10 mmol), and triphenylphosphine (3.9 g, 15 mmol) were dissolved in tetrahydrofuran (THF, 20 mL) to obtain a solution. Under ice bath and nitrogen stirring, diisopropyl azodicarbonate (2 mL, 10 mmol) was added dropwise to the solution. After the addition was complete, a reaction solution was obtained. The reaction solution was stirred at room temperature (25 °C) (150 rpm) for 16 h. After the reaction was complete, a reaction product was obtained. The reaction product was removed by rotary evaporation to obtain a solid product. The solid product was washed with ethyl acetate and then purified by silica gel column chromatography using a mixture of n-hexane / ethyl acetate (n-hexane:ethyl acetate = 2:1, v / v) as the eluent to obtain a white solid compound 5 (3.1 g, yield: 62%).

[0106] Compound 5 has the following structure:

[0107] The 3-bromomethylbenzonitrile has the following structure:

[0108] The proton and carbon NMR spectra of compound 5 are as follows (see Figures 6 and 7 for the proton and carbon NMR spectra):

[0109] 1 H NMR (500MHz, DMSO-d6, δ: ppm) δ = 11.17 (s, 1H), 9.99 (s, 1H), 8.05 (s, 1H), 7.53 (dd, J = 7.6, 1.3, 1H), 7.28 (t, J = 7. 6,1H),7.20(dd,J=7.7,1.4,1H),6.93(d,J=8.2,1H),6.81-6.72(m,3H),5.28(s,2H),4.29(s,4H),2.24(s,3H). 13 C NMR (126MHz, DMSO-d6, δ: ppm) δ = 190.1, 163.5, 162.9, 143.4, 143.0, 142.1, 140.3, 135.0, 134.8, 134. 2,130.2,127.6,125.9,122.6,118.8,118.2,117.3,101.6,75.7,70.3,68.3,64.6,64.6,22.4,16.5.

[0110] Step 3: Compound 5 (2.5 g, 5 mmol), 3-bromomethylbenzonitrile (1.69 g, 7 mmol), and cesium carbonate (4.77 g, 14.6 mmol) were mixed in N,N-dimethylformamide (30 mL) and reacted at room temperature (25 °C) with stirring (150 rpm) for 16 h. After the reaction was completed, the reaction product was obtained. The reaction product was quenched with water, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and then the organic phase was concentrated by rotary evaporation. Finally, the product was purified by silica gel column chromatography using a mixture of n-hexane and ethyl acetate (n-hexane:ethyl acetate = 3:2, v / v) as the eluent to obtain a white solid compound 1 (2.6 g, yield: 85%).

[0111] Compound 1 has the following structure:

[0112] The proton and carbon NMR spectra of compound 1 are as follows (see Figures 8 and 9 for the proton and carbon NMR spectra):

[0113] 1 H NMR (500MHz, DMSO-d6, δ: ppm) δ = 10.17 (s, 1H), 8.05 (s, 2H), 7.87 (dd, J = 20.6, 7.8, 2H), 7.65 (t, J = 7.8, 1H), 7.54 (d, J = 7.2, 1H), 7.27 ( t,J=7.6,1H),7.21(d,J=7.5,1H),7.12(s,1H),6.94(d,J=8.2,1H),6.81-6.75(m,2H),5.41(d,J=44.2,4H),4.29(s,4H),2.28(s,3H). 13 C NMR (126MHz, DMSO-d6, δ: ppm) δ = 187.1, 163.1, 162.8, 162.8, 143.4, 143.0, 142.2, 138.6, 138.3, 134.9, 134.8, 134.5, 132.8, 132 .4,131.5,130.4,130.3,128.0,126.0,122.6,120.7,119.1,118.2,117.3,112.1,99.8,77.7,70.7,69.7,64.6,36.3,31.2,16.7.

[0114] Step 4: Compound 1 (617 mg, 1 mmol), tris(hydroxymethyl)aminomethane (Tris, 242 mg, 2 mmol), and sodium cyanoborohydride (360 mg, 6 mmol) were dissolved in N,N-dimethylformamide (DMF, 9 mL) to obtain a solution. Glacial acetic acid (360 μL) was added to the solution, and the mixture was stirred at room temperature (25 °C) (150 rpm) for 16 h. After the reaction was completed, the reaction product was obtained. The reaction product was quenched with water, extracted with ethyl acetate, dried with anhydrous sodium sulfate, and then the organic phase was concentrated by rotary evaporation. Finally, the product was purified by silica gel column chromatography using a mixture of dichloromethane and methanol (dichloromethane:methanol = 10:1, v / v) as the eluent to obtain a white solid compound LG-12 (223 mg, yield: 31%).

[0115] The proton and carbon NMR spectra of compound LG-12 are as follows (see Figures 10 and 11 for the proton and carbon NMR spectra):

[0116] 1 H NMR (500MHz, DMSO-d6, δ: ppm) δ = 8.03 (s, 1H), 7.92-7.90 (m, 1H), 7.83 (d, J =1.6,2H),7.62(s,1H),7.50(dd,J=7.6,1.4,1H),7.24(d,J=7.5,1H),7.2 1-7.12(m,1H),7.00(s,1H),6.94(d,J=8.2,1H),6.82-6.72(m,2H),5.33( s,2H),5.25(s,2H),4.29(s,4H,),4.15(s,2H),3.59(s,6H),2.27(s,3H). 13 C NMR (126MHz, DMSO-d6, δ: ppm) δ = 143.4, 143.0, 142.1, 138.7, 135.4, 134.8, 134.4, 132.8, 132.2, 131.4, 1 30.2,130.1,128.0,125.9,122.6,119.2,118.2,117.3,112.0,99.6,75.6,70.2,69.3,64.6,64.6,16.6.

[0117] The mass spectrometry results for compound LG-12 are as follows (ESI-MS, see Figure 3): ESI-MS (m / z): 723 [M+H] + .

[0118] Example 3: An iodine-131 labeled PD-L1 small molecule inhibitor [ 131 I]LG-12

[0119] This embodiment provides an iodine-131-labeled PD-L1 small molecule inhibitor. 131 I]LG-12, the iodine-131-labeled PD-L1 small molecule inhibitor [ 131 The LG-12 has the following structure:

[0120] Example 4: A method for preparing an iodine-131 labeled PD-L1 small molecule inhibitor [ 131 Method of I]LG-12

[0121] This embodiment provides the iodine-131-labeled PD-L1 small molecule inhibitor described in Example 3. 131 The preparation method of LG-12 (synthetic route is shown in Figure 2) is as follows:

[0122] Step 1: Synthesize compound 1 according to the method in Example 2.

[0123] Step 2: Compound 1 (234 mg, 0.38 mmol), tetra(triphenylphosphine)palladium (22 mg, 5 mol), and hexa-n-butylditin (217 μL, 0.418 mmol) were dissolved in dry dioxane (5 mL). The mixture was heated to 80 °C and stirred (150 rpm) for 6 h under nitrogen protection. After the reaction was completed, the reaction product was obtained. The reaction product was filtered, and the filtrate was collected. The filtrate was first removed by rotary evaporation to remove dioxane, and then purified by silica gel column chromatography using a mixture of n-hexane and ethyl acetate (n-hexane:ethyl acetate = 4:1, v / v) as the eluent to obtain a colorless oily compound 2 (271 mg, yield: 84%).

[0124] Compound 2 has the following structure:

[0125] The proton and carbon NMR spectra of compound 2 are as follows (see Figures 12 and 13 for the proton and carbon NMR spectra):

[0126] 1H NMR (500MHz, DMSO-d6, δ: ppm) δ = 10.29 (s, 1H), 8.04 (d, J = 1.8, 1H), 7.91-7.82 (m, 2H) ,7.77-7.61(m,2H),7.37(dd,J=7.4,1.7,1H),7.28-7.16(m,2H),7.02(s,1H),6.93(d ,J=8.2,1H),6.78-6.70(m,2H),5.44(s,2H),5.23(d,J=4.2,2H),4.28(s,4H),2.23( s,3H),1.44-1.24(m,6H),1.16(h,J=7.3,6H),0.96-0.81(m,6H),0.76(t,J=7.3,9H). 13 C NMR (126MHz, DMSO-d6, δppm) δ=187.9,169.8,164.1,143.4,143.0,142.3,138 .7,137.5,136.9,135.0,134.9,134.8,133.4,132.9,132.6,132.3,132.1,13 1.5,130.6,130.3,130.3,129.2,126.0,122.4,121.5,119.2,119.1,118.1,117.2,112.1,97.1,70.0,69.3,68.4,64.6,64.6,29.0,27.0,16.4,13.9,9.7.

[0127] The mass spectrometry results for compound 2 are shown in Figure 4 (ESI-MS): ESI-MS (m / z): 804 [M+Na] + .

[0128] Step 3: Compound 2 (390 mg, 0.5 mmol) and 4-methoxy(diacetoxyiodine)benzene (211 mg, 0.5 mmol, CAS: 16308-14-8) were dissolved in acetonitrile (ACN, 10 mL). The mixture was stirred at room temperature (25 °C) for 16 h under nitrogen protection. After the reaction was completed, the reaction product was obtained. The acetonitrile was removed by rotary evaporation of the reaction product. Then, excess diethyl ether (20 mL) was added to precipitate the solid. The solid was then centrifuged and the white solid was collected and dried under vacuum to obtain the labeled precursor compound 3 (214 mg, yield: 48%).

[0129] The 4-methoxy(diacetoxyiodide)benzene has the following structure:

[0130] The labeled precursor compound 3 has the following structure:

[0131] The proton and carbon NMR spectra of the labeled precursor compound 3 are shown below (see Figures 14 and 15 for the proton and carbon NMR spectra):

[0132] 1 H NMR (500MHz, DMSO-d6, δ: ppm) δ = 10.25 (s, 1H), 8.65 (s, 1H), 8.21 (s, 1H), 7.93 (t, J = 1.7, 1H), 7 .90-7.71(m,4H),7.64(t,J=7.8,1H),7.47(d,J=8.1,2H),7.39(td,J=7.3,6.4,2.1,1H),7.35- 7.24(m,3H),7.20(s,1H),7.10(d,J=7.8,2H),7.01-6.87(m,3H),6.87-6.77(m,2H),5.57(s,1H ),5.50(s,1H),5.39(d,J=9.5,2H),4.30(d,J=1.4,4H),3.74(s,3H),3.28(s,3H),2.25(s,3H). 13 C NMR (126MHz, DMSO-d6, δ: ppm) δ = 162.0, 160.7, 157.9, 146.2, 143.5, 143.1, 142.4, 138.4, 138. 0,137.2,137.0,135.9,134.9,134.8,134.7,134.7,132.7,132.3,131.6,131.3,130.8,130.4, 128.9,128.7,128.5,126.2,125.9,122.6,122.4,119.1,118.2,117.6,117.5,117.4,117.4,112.0,105.5,99.8,98.7,97.1,70.8,70.1,69.4,65.4,64.6,56.1,54.0,21.2,16.7,16.7,15.6.

[0133] The mass spectrometry results of labeled precursor compound 3 are shown in Figure 5 (ESI-MS): ESI-MS (m / z): 724 [M-TsO-] + .

[0134] Step 4: Dry the Na with nitrogen gas 131 Solution I (740 MBq) yields a white solid Na. 131 I; in white solid Na 131After adding precursor compound 3 (1.2 mg) dissolved in anhydrous acetonitrile (500 μL) to phase I, the reaction was carried out at 90 °C for 60 min to obtain the reaction product. The reaction product was purified by small silica gel column chromatography using a mixture of n-hexane / ethyl acetate (n-hexane:ethyl acetate = 3:2, v / v) as the eluent to obtain the intermediate compound [ 131 I]1 (500 MBq) was analyzed using radio-HPLC.

[0135] The intermediate compound [ 131 I]1 has the following structure:

[0136] Step 5: Dry the intermediate compound with nitrogen gas. 131 I]1; intermediate compounds dried by nitrogen [ 131 I]1. Tris(hydroxymethyl)aminomethane (Tris, 1.2 mg, 0.01 mmol) and sodium cyanoborohydride (1.8 mg, 0.03 mmol) were dissolved in N,N-dimethylformamide (DMF, 100 μL) to obtain a solution; glacial acetic acid (1.8 μL) was added to the solution, and the mixture was stirred (150 rpm) at room temperature (25 °C) for 10 h. After the reaction was completed, the reaction product was obtained; the reaction product was purified by small silica gel column chromatography using a mixture of dichloromethane / methanol (dichloromethane:methanol = 9:1, v / v) as the eluent to obtain an iodine-131 labeled PD-L1 small molecule inhibitor. 131 I]LG-12, analyzed using radio-HPLC.

[0137] intermediate compounds [ 131 I]1 and iodine-131 labeled PD-L1 small molecule inhibitors 131 The radio-HPLC analysis results of LG-12 before and after purification are shown in Figure 17. As shown in Figure 17, the labeled precursor compound 3 and Na... 131 I reacted in acetonitrile at 90°C to give the intermediate compound [ 131 I]1, radioactive conversion rate 70%, radiochemical purity after purification greater than 98%. Intermediate compound [ 131 I]1 and Tris undergo a reductive amination reaction to yield an iodine-131-labeled PD-L1 small molecule inhibitor. 131 I]LG-12, with a radioactive conversion rate of 50%, and a radiochemical purity of 97% after purification. Iodine-131-labeled PD-L1 small molecule inhibitor [ 131 The total radiochemical yield of I]LG-12 was 7.0 ± 2.4%, and the molar activity was 27 GBq / μmol.

[0138] Example 5: A non-radioactive small molecule inhibitor of PD-L1, LG-2 to LG-11 and LG-13

[0139] This embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-2 to LG-11 and LG-13, which have the following structures:

[0140] Where R is

[0141] Example 6: A method for preparing non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-11 and LG-13

[0142] This embodiment provides a method for preparing the non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-11 and LG-13 described in Example 5. The specific steps are as follows:

[0143] Based on Example 2, the tris(hydroxymethyl)aminomethane (Tris, 242 mg, 2 mmol) in step four was replaced with glycine (250 mg, 3.3 mmol), serine (250 mg, 2.4 mmol), glutamic acid (250 mg, 1.7 mmol), aminomethylphosphonic acid (250 mg, 2.3 mmol), sulfoalanine (250 mg, 1.5 mmol), arginine (250 mg, 1.4 mmol), tyrosine (250 mg, 1.4 mmol), aspartic acid (250 mg, 1.9 mmol), proline (250 mg, 1.9 mmol), and asparagine (250 mg, 1.9 mmol). ), glucosamine (250 mg, 1.4 mmol), to give white solid compounds LG-2 (212 mg, yield: 31%), LG-3 (233 mg, yield: 33%), LG-4 (245 mg, yield: 33%), LG-5 (190 mg, yield: 16%), LG-6 (186 mg, yield: 24%), LG-7 (260 mg, yield: 34%), LG-8 (234 mg, yield: 30%), LG-9 (256 mg, yield: 36%), LG-10 (289 mg, yield: 40%), LG-11 (310 mg, yield: 43%), and LG-13 (178 mg, yield: 23%).

[0144] Example 7: An iodine-131 labeled PD-L1 small molecule inhibitor [ 131 I]LG-2~[ 131 I]LG-11 and [ 131[I]LG-13 This embodiment provides an iodine-131-labeled PD-L1 small molecule inhibitor. 131 I]LG-2~[ 131 I]LG-11 and [ 131 I]LG-13, the iodine-131-labeled PD-L1 small molecule inhibitor [ 131 I]LG-2~[ 131 I]LG-11 and [ 131 The LG-13 has the following structure:

[0145] Where R is

[0146] Experimental Example 1: TR-FRET Experiment of Non-Radioactive PD-L1 Small Molecule Inhibitors LG-2~LG-13

[0147] This experimental example provides a TR-FRET experiment of the non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-13 from Example 1. The specific procedure is as follows:

[0148] The inhibitory effect of non-radioactive PD-L1 small molecule inhibitors LG-2 to LG-13 (analyte compounds) on PD-1 / PD-L1 binding was detected by PD-1 / PD-L1 TR-FRET assay using a PD-1 / PD-L1 binding assay kit in Examples 1 and 5. The experiments were conducted according to the kit instructions. Experimental group: 5 μL of different concentrations of the analyte compounds (100, 25, 6.25, 1.5625, 0.390625, 0.097656, 0.024414, 0.006104, 0.001526, and 0.000381 μM) and 5 μL of PD-L1-biotin (11 μg / mL) were mixed and incubated at room temperature (25°C) for 10 min to obtain the incubation solution. Then, 5 μL of PD-1-Eu (0.2 μg / mL) and 5 μL of dye-labeled material were added to the incubation solution to obtain the mixture. Positive group: 5 μL of PD-L1-Biotin, 5 μL of purified water, 5 μL of PD-1-Eu, and 5 μL of dye-labeled reagent were mixed to obtain a mixture. Negative group: 5 μL of buffer (1×), 5 μL of purified water, 5 μL of PD-1-Eu, and 5 μL of dye-labeled reagent were mixed to obtain a mixture. The three mixtures were incubated at room temperature (25℃) in the dark for 90 min (384-well plate). Fluorescence intensity was read on a molecular device instrument (PerkinElmer EnVision), and absorbance was measured at 620 nm and 665 nm emission wavelengths with an excitation wavelength of 320 nm. Data analysis was performed using the ratio (665 nm absorbance / 620 nm absorbance) and the inhibition rate % = (positive ratio - sample ratio) / (positive ratio - negative ratio) × 100. The analysis results are shown in Figure 16 and Table 1.

[0149] The PD-1 / PD-L1 TR-FRET experimental results (Figure 16 and Table 1) show that the EC50 of the non-radioactive PD-L1 small molecule inhibitor LG-2 50 EC with a value of 837±12.3 nM, LG-3 50 EC with a value of 237±5.56 nM and LG-4 50 EC with a value of 188.34±4.43 nM and LG-5 50 EC with a value of 387±7.21 nM and LG-6 50 EC with a value of 429±4.98 nM, LG-7 50 EC with a value of 252±3.78 nM and LG-8 50 EC50 of LG-9 with a value of 67.83±0.75μM. 50 EC with a value of 168±11.87 nM and LG-10 50 EC with a value of 2.253±0.05μM and LG-11 50EC with a value of 1.756±0.04μM and LG-12 50 EC with a value of 34.55±3.21 nM and LG-13 50 The value was 50.23±2.89 nM, indicating that LG-12 has high inhibitory activity against PD-1 / PD-L1 interaction, can significantly inhibit tumor growth, and shows good anti-tumor immunotherapy effect.

[0150] Table 1 EC50 of different small molecule inhibitors 50 value

[0151] Experimental Example 2: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 In vitro stability test of LG-12

[0152] This experimental example provides the iodine-131-labeled PD-L1 small molecule inhibitor from Example 3. 131 The in vitro stability test of LG-12 was conducted as follows:

[0153] Experiment 1: The intermediate compound obtained in Example 4 [ 131 I]1 (370 KBq) was added to N,N-dimethylformamide (450 μL) solution to obtain a mixture; the mixture was incubated at 37 °C for 36 h; after incubation, the incubation solution was taken and radio-HPLC was used to analyze the in vitro stability of the sample. The analysis results are shown in Figure 18.

[0154] Experiment 2: The iodine-131-labeled PD-L1 small molecule inhibitor from Example 3 [ 131 I]LG-12 (370KBq) was added to PBS buffer (pH=7.4, 0.01M, 450μL) to obtain a mixture; the mixture was incubated at 37℃ for 4h and 12h; after incubation, the incubation solution was taken and radio-HPLC was used to analyze the in vitro stability of the sample. The analysis results are shown in Figure 18.

[0155] As shown in Figure 18, the intermediate compound [ 131 I]1 remained stable in DMF for 36 hours, meeting the requirements for the next reaction; iodine-131 labeled PD-L1 small molecule inhibitor [ 131 I]LG-12 remained stable after incubation in PBS buffer for 12 hours, indicating that both the intermediate and the product have good in vitro stability.

[0156] Experimental Example 3: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 Cellular uptake and biodistribution experiments of LG-12

[0157] This experimental example provides the iodine-131-labeled PD-L1 small molecule inhibitor from Example 3. 131 The cellular uptake and biodistribution experiments of LG-12 were conducted as follows:

[0158] B16-F10 cells were fed at a rate of 2.6 × 10⁻⁶. 5 The inoculum was inoculated into 1500 μL of DMEM medium containing 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum in each well. The plates were then incubated at 37°C and 5% (v / v) CO2 for 16 h. After 16 h of incubation, the wells were divided into two groups: a blocking group and a non-blocking group, with three replicates in each group. For the blocking group: the medium was aspirated, and LG-12 (50 μM, 1 mL, DMEM medium) was added to the wells. The plates were incubated at 37°C and 5% CO2 for 30 min to block the inoculum. Then, […]. 131 I]LG-12(3.7×10 -2 MBq (200 μL, DMEM medium) was incubated at 37°C with 5% (v / v) CO2 for 1, 2, and 4 h, respectively. The B16-F10 cells were then rinsed twice with PBS buffer, and finally lysed with 0.3 M NaOH for 10 min to obtain lysate. Non-blocking group: The medium was aspirated, and 1 mL of DMEM medium and […] were added directly to the wells of the non-blocking group. 131 I]LG-12(3.7×10 -2 B16-F10 cells were incubated in 200 μL of MBq (DMEM medium) at 37°C with 5% (v / v) CO2 for 1, 2, and 4 h, respectively. The cells were then rinsed twice with PBS buffer, and finally lysed with 0.3 M NaOH for 10 min to obtain lysate. The lysate was collected, and intracellular radioactivity was detected using a gamma counter (1470 Wizard, Perkins Elmer). The results are shown in Figure 19.

[0159] As shown in Figure 19, after 1 hour of incubation, [ 131 The cellular uptake value of LG-12 was 3.93 ± 0.15% AD, which decreased to 1.46 ± 0.03% AD after LG-12 blockade; after 4 hours of incubation, [ 131 The cellular uptake of LG-12 increased to 5.33 ± 0.33% AD, and decreased significantly to 2.56 ± 0.06% AD after LG-12 blockade. This result indicates that LG-12 can specifically bind to PD-L1 in tumor cells.

[0160] Experimental Example 4: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 I]LG-12 in vivo imaging experiment

[0161] This experimental example provides the iodine-131-labeled PD-L1 small molecule inhibitor from Example 3. 131 The in vivo imaging experiment of LG-12 was conducted as follows:

[0162] SPECT / CT imaging experiment: Tumor-bearing mice (n=1) were anesthetized with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min. The limbs and tails of the tumor-bearing mice were then fixed, and an iodine-131-labeled PD-L1 small molecule inhibitor was injected into the tumor-bearing mice via the tail vein. 131 I]LG-12 (11.1 MBq, dissolved in 100 μL of physiological saline); after injection, whole-body SPECT / CT imaging of tumor-bearing mice was obtained by static scanning for 30 min. The imaging results are shown in Figure 20A.

[0163] In vivo biodistribution analysis: Tumor-bearing mice (n=4) were anesthetized with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min. The limbs and tails of the tumor-bearing mice were then fixed, and an iodine-131-labeled PD-L1 small molecule inhibitor was injected into the tumor-bearing mice via the tail vein. 131 I]LG-12 (5 MBq, dissolved in 100 μL physiological saline), and take the same volume of [ 131 [I]LG-12 was used as an attenuation correction control; 1 hour after injection, mice were euthanized and dissected, and tumors and major organs (heart, liver, spleen, lungs, kidneys, stomach, intestines, bones, muscles, and brain) were weighed; after weighing, the radioactivity of the samples was measured using a gamma counter (1470 Wizard, Perkins Elmer), and the results are shown in B of Figure 20. 131 The biodistribution of I]LG-12 is expressed as a percentage of the dose injected per gram of tissue (%ID / g).

[0164] Autoradiography analysis of tumor and muscle tissue: Tumor-bearing mice (n=3) were anesthetized with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min. The limbs and tails of the tumor-bearing mice were then fixed, and an iodine-131-labeled PD-L1 small molecule inhibitor was injected into the tumor-bearing mice via the tail vein. 131I]LG-12 (5 MBq, dissolved in 100 μL physiological saline); 1 h after injection, mice were euthanized and dissected, subcutaneous tumor tissue and leg muscles were dissected, washed once with PBS buffer, then embedded at -25°C using cryoemulation medium, and then prepared into tissue sections (30 μm) on glass slides using a cryostat (CM1950, SLEE / MNT). The glass slides were then laid flat on a fluorescent screen for 4 h, and the fluorescent screen was scanned using a Cyclone Plus phosphorus storage screen imaging system (C431200, PerkinElmer) to obtain images. Finally, the images were processed and analyzed using OptiQuant software, and the analysis results are shown in C in Figure 20.

[0165] As shown in Figure 20A, B16-F10 tumor-bearing mice were injected via the tail vein [ 131 After 30 minutes of LG-12 (11.1 MBq), SPECT / CT imaging revealed […]. 131 [I]LG-12 rapidly accumulates at the tumor site. Further research [ 131 Distribution of LG-12 in B16-F10 tumor-bearing mice. As shown in B of Figure 20, [ 131 The uptake of LG-12 in tumor tissue was 6.50 ± 1.05% ID / g, significantly higher than that in most normal tissues, indicating that [ 131 [I]LG-12 has the ability to target PD-L1 in vivo, but its uptake is also high in non-target tissues such as blood, liver, heart, and intestines. This may be due to [ 131 This is due to the high lipophilicity of LG-12. As shown in C of Figure 20, [ 131 [I]LG-12 showed 2.3 times higher activity in tumors than in muscle, further demonstrating [ 131 I]LG-12's targeting of PD-L1.

[0166] Experiment Example 5: Biocompatibility Experiment of Non-Radioactive PD-L1 Small Molecule Inhibitor LG-12

[0167] This experimental example provides a biocompatibility experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 from Example 1, the specific procedure of which is as follows:

[0168] The biocompatibility of the non-radioactive PD-L1 small molecule inhibitor LG-12 from Example 1 was evaluated using the MTT assay. B16-F10 cells were cultured at 1×10⁻⁶ cells per cell line. 4The inoculum was inoculated into 96-well plates containing 100 μL of DMEM medium supplemented with 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum, and incubated at 37°C and 5% (v / v) CO2 for 16 h. After 16 h of incubation, the medium was aspirated, and different concentrations (0, 1.5625, 3.125, 6.25, 12.5, 25, 50 μM) of LG-12 (100 μL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) were added to the wells, and the plates were incubated at 37°C and 5% (v / v) CO2. Incubate in a CO2 incubator for 24 h; after 24 h incubation, aspirate the culture medium and add 20 μL of MTT (diluted with PBS buffer to a concentration of 5 mg / mL before use; MTT and PBS buffer were purchased from Shanghai Beyotime) to the wells, and incubate at 37℃ in a 5% CO2 incubator for 4 h; after 4 h incubation, aspirate the MTT and add 150 μL of DMSO to the wells, and shake for 10 min; after shaking, use a microplate reader to detect the absorbance of the sample at 490 nm (MD / M5e, VEDENG), and calculate the survival rate of B16-F10 tumor cells and T cells according to the formula: survival rate = OD value of sample well / OD value of reference well (the reference well is the well with 0 μM LG-12 added). The experimental results are shown in Figure 21.

[0169] As shown in Figure 21, LG-12 exhibits certain cytotoxic effects on both B16-F10 tumor cells and T cells, with an IC50 value of [missing information]. 50 The values ​​were 28.81 μM and 15.36 μM, respectively; when the LG-12 concentration was 3.13 μM, the survival rates of B16-F10 tumor cells and T cells reached over 96% and 80%, respectively. Therefore, subsequent in vivo experiments controlled the LG-12 concentration to be equal to or lower than 3.13 μM.

[0170] Experiment Example 6: Co-culture of T cells / tumor cells with the non-radioactive PD-L1 small molecule inhibitor LG-12

[0171] This experimental example provides a non-radioactive co-culture experiment of T cells / tumor cells with the PD-L1 small molecule inhibitor LG-12 from Example 1. The specific procedure is as follows:

[0172] CD3 was added to a 96-well plate at a rate of 0.5 μL / well. + (Use after diluting with antibody diluent to a concentration of 5 μg / mL, CD3) +Both CD28 and antibody dilution buffer were purchased from BioLegend. CD28 (diluted to a concentration of 5 μg / mL with antibody dilution buffer, both purchased from BioLegend) was added at a rate of 0.5 μL / well and incubated at 4°C for 24 h to obtain CD3. + 96-well plates coated with CD28 (5 μg / mL) antibody were prepared. The wells of the 96-well plates were divided into two groups: an interaction group and a non-interaction group, with three replicates in each group. For the interaction group: B16-F10 tumor cells (5 × 10⁻⁶) were first added to the wells of the interaction group. 3 Cells / well, 100 μL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum, were incubated at 37°C and 5% (v / v) CO2 for 24 h. Then, different concentrations (0, 0.78, and 3.125 μM) of LG-12 (50 μL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) and different concentrations (1 × 10⁻⁶) of LG-12 were added to the wells of the interaction group. 5 1 cell / well and 2×10 5 T lymphocytes (50 μL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) were added per well and incubated at 37°C in a 5% CO2 incubator for 24 h. Non-interacting group: 100 μL of DMEM medium containing 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum and different concentrations (0, 0.78, and 3.125 μM) of LG-12 (50 μL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) and different concentrations (1×10⁻⁶ cells / well) were added directly to the wells of the non-interacting group. 5 1 cell / well and 2×10 5 T lymphocytes (50 μL per well, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) were incubated at 37°C and 5% CO2 for 24 h. After 24 h of incubation, the supernatant (100 μL) was collected, and the IFN-γ content was detected using a mouse interferon-γ ELISA kit (KE10001, Proteintech). The results are shown in Figure 22, B. CCK-8 reagent (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) was added at a rate of 10 μL / well to the remaining 100 μL of the 96-well plate and incubated at 37℃ in a 5% CO2 incubator for 4 h. After 4 h of incubation, the OD values ​​were detected at 450 nm using a microplate reader (MD / M5e, VEDENG). The survival rate of B16-F10 tumor cells was calculated according to the formula: survival rate = OD value of sample well / OD value of reference well (the reference well is the well with 0 μM LG-12 added). The experimental results are shown in Figure 22A.

[0173] As shown in Figure 22A, LG-12 enhances the PD-1 / PD-L1-mediated T cell activation inhibition mechanism in a dose-dependent manner. Specifically, when B16-F10 cells were co-cultured with T cells at a ratio of 1:20, the cell viability of B16-F10 cells after treatment with LG-12 (0.78 μM and 3.125 μM) was 0.79 ± 0.13 and 0.69 ± 0.02, respectively. When the ratio of B16-F10 to T cells was 1:40 (…),… Without LG-12, the cell survival rate of B16-F10 cells decreased from 0.90±0.06 at a concentration of 1:20 to 0.70±0.12, indicating that increasing the number of T cells can enhance the killing effect on tumor cells. When the LG-12 concentration was 0.78μM and 3.125μM, the survival rate of B16-F10 cells decreased to 0.66±0.06 and 0.55±0.03, respectively, indicating that LG-12 can activate the anti-tumor immune function of T cells.

[0174] To further explore the mechanism by which LG-12 activates anti-tumor immunotherapy by blocking PD-1 / PD-L1 interaction, the expression level of interferon-γ, a functional marker of CD8+ T cell cytotoxicity, was investigated. As shown in Figure 22 (B), treatment with LG-12 resulted in a dose-dependent increase in interferon-γ secretion. Specifically, after treatment with 0.78 μM LG-12 (B16-F10 / T cells = 1 / 20), the expression level of interferon-γ slightly increased from 34.44 ± 1.39 pg / 100 μL to 40.28 ± 1.97 pg / 100 μL. After incubation with 3.125 μM LG-12 (B16-F10 / T cells = 1 / 20), the expression level of interferon-γ... Interferon-γ expression significantly increased from 34.44±1.39 pg / 100 μL to 76.33±5.24 pg / 100 μL. When the B16-F10 / T cell ratio reached 1 / 40, LG-12 (0.78 μM and 3.125 μM) induced an increase in interferon-γ expression from 83.13±3.64 pg / 100 μL to 110.26±1.72 pg / 100 μL, and the interferon-γ level consistently increased with the increase in the proportion of T cells. These results indicate that LG-12 can increase interferon-γ secretion and promote CD8+ T cell activation by blocking the PD-1 / PD-L1 signaling pathway.

[0175] Experimental Example 7: In vivo antitumor experiment of LG-12, a non-radioactive small molecule inhibitor of PD-L1

[0176] This experimental example provides an in vivo antitumor experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 from Example 1. The specific procedure is as follows:

[0177] To evaluate the antitumor activity of LG-12, BALB / c mice were inoculated with B16-F10 cells for 5 days, and the resulting tumor-bearing mice were randomly divided into three groups: a control group, a low-dose treatment group, and a high-dose treatment group (n=5). Mice in the low-dose treatment group (Group B) and the high-dose treatment group (Group C) were intraperitoneally injected with the non-radioactive PD-L1 small molecule inhibitor LG-12 (dissolved in 100 μL of physiological saline) from Example 1 at doses of 5 mg / kg and 20 mg / kg, respectively, every other day for two consecutive weeks (days 1, 3, 5, 7, 9, and 11). Mice in the control group (Group A) were injected with the same volume of physiological saline. During the experiment, tumor size and mouse weight were measured every other day, and the tumor volume was calculated using the formula: Tumor volume = 1 / 2 (length × width). 2 The tumor volume was calculated, and the results are shown in Figure 23. After the experiment (day 14), the tumor-bearing mice were sacrificed. Before sacrifice, the eyeballs of the tumor-bearing mice were enucleated to collect whole blood. The blood samples were allowed to stand at room temperature (25℃) for 30 minutes, then centrifuged at 1000 r / min for 5 minutes. Serum was collected, and the IFN-γ content was detected using a mouse IFN-γ ELISA kit (KE10001, Proteintech). The results are shown in Figure 25, B. After tumor-bearing mice were sacrificed, the heart, liver, spleen, lung, kidney, muscle, and tumor were dissected and fixed in 4% (w / v, g / 100mL) paraformaldehyde solution for 24h. After fixation, the tissues were dehydrated in 15% (w / v, g / 100mL) sucrose solution and 30% (w / v, g / 100mL) sucrose solution for 24h respectively. The dehydrated tissues were first embedded in cryoemulation medium at -25℃, and then prepared into tissue sections (6μm) on glass slides using a cryostat (CM1950, LEICA). The slides were first fixed with fixative (purchased from Wuxi Jiangyuan Industrial Technology and Trade Co., Ltd.) for 10min, then stained with hematoxylin staining solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 10min and eosin staining solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 1min. Finally, the slides were dehydrated, cleared, and mounted. The H&E staining analysis results are shown in Figure 24 and Figure 25, A. Tumor tissue from tumor-bearing mice was fixed in 4% (w / v, g / 100mL) paraformaldehyde for 24h. After fixation, it was dehydrated with ethanol of different concentrations prepared with n-butanol, sequentially in 50% (v / v) ethanol for 2h, 80% ethanol for 3h, 65% ethanol for 1.5h, 50% ethanol for 1h, 30% ethanol for 4h, 10% ethanol for 4h, and pure n-butanol for 24h. After dehydration, the tumor tissue was first embedded in paraffin at 65℃ for 4h, then embedded in paraffin and sectioned (6μm). After dewaxing, the slides were first antigen-retrievald using sodium citrate antigen retrieval solution (purchased from BBI), and then blocked with 5% (v / v) goat serum. The blocked slides were then first coated with the corresponding primary antibody (CD4+). + CD8+ PD-L1, CRT, and HMGB1 antibodies were all diluted 500-fold with antibody dilution buffer before use as working solutions. CD4 + and CD8 + Antibodies were purchased from ProteinTech, PD-L1, CRT, and HMGB1 antibodies from Abcam, and antibody dilution buffers from BioLegend. The working solution was incubated at 25°C for 90 min, and then incubated with the secondary antibody (CD4+). + PD-L1 uses goat anti-mouse secondary antibody, CD8 + CRT and HMGB1 were prepared using goat anti-rabbit secondary antibodies. The antibodies were diluted to a concentration of 4000 times with antibody diluent before being used as working solutions. All antibodies were purchased from R&D Systems, and the antibody diluents were purchased from BioLegend. The working solutions were incubated at 25°C for 30 min, then developed with DAB chromogenic solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Finally, the slides were dehydrated, cleared, and mounted. The immunohistochemical IHC analysis results are shown in Figure 25, A.

[0178] As shown in Figures 23A to 23C, no mice experienced significant weight loss or death during treatment, indicating that all doses of LG-12 were well tolerated. H&E staining analysis further confirmed this (Figure 24). Simultaneously, after 7 days of treatment, the tumor volume in both treatment groups was significantly reduced compared to the control group. After 14 days of treatment, the tumor volume (37±7 mm3) and weight (0.046±0.01 g) in the low-dose (5 mg / kg) treatment group were significantly smaller than those in the control group (volume 1303±203 mm3 and weight 1.88±0.63 g). The anti-tumor effect of the high-dose (20 mg / kg) group was worse than that of the low-dose group, with a tumor volume of 201±44 mm3 and a weight of 0.44±0.22 g. This suggests that anti-tumor immunotherapy based on the small molecule inhibitor LG-12 is not necessarily better the higher the dose; the specific mechanism requires further in-depth research.

[0179] After treatment, mouse tumor tissues were collected for immunohistochemical analysis to observe the differences in the expression of tumor-infiltrating lymphocytes (TILs) and PD-L1. The results showed that LG-12 significantly increased CD4+ expression at low doses. + T cells and CD8 +The infiltration level of T cells and the expression level of PD-L1 were significantly reduced compared with the control group. At the same time, H&E staining of tumor tissue after treatment showed obvious cell apoptosis, indicating that LG-12 has good in vivo anti-tumor effects at low doses. Similarly, the high-dose group of mice had higher levels of CD4+ and CD8+ T cells and lower PD-L1 expression in tumor tissue, but the overall levels were lower than those in the low-dose group, which is consistent with the anti-tumor results (Figure 25, A).

[0180] The expression of interferon-γ in mouse serum was detected using an ELISA kit. The results showed that the expression of interferon-γ in the low-dose treatment group was 153±54 (pg / 100mL), while the expression of interferon-γ in the serum of the high-dose treatment group was 72±23 (pg / 100mL), which was consistent with the results of antitumor and immunohistochemistry (Figure 25, B).

[0181] Experimental Example 8: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 In vitro cloning experiments of I]LG-12

[0182] This experimental example provides the iodine-131-labeled PD-L1 small molecule inhibitor from Example 3. 131 The in vitro cloning experiment of LG-12 was conducted as follows:

[0183] Clonogenic assay: The clonogenic assay is used to evaluate the ability of a single tumor cell to proliferate and form a clone after being exposed to radiation. B16-F10 cells were injected with 1.2 × 10⁻⁶ cells... 3 The inoculum was inoculated into 24-well plates containing 1 mL of DMEM medium supplemented with 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum, and then incubated at 37°C and 5% (v / v) CO2 for 16 h. After 16 h of incubation, the medium was aspirated, and different concentrations (0, 0.148, 0.296, 0.592, 1.184, and 2.368 MBq) of [[] were added to the wells. 131 I]LG-12 (1 mL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) was incubated at 37°C and 5% CO2 for 10 h. After 10 h of incubation, the medium was replaced with fresh medium and the cells were incubated at 37°C and 5% (v / v) CO2 for 9 days. After 9 days of incubation, the B16-F10 cells in the wells were rinsed twice with PBS buffer, fixed with methanol for 20 min, stained with crystal violet (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 15 min, washed off the crystal violet with running water, and finally dried at 37°C and photographed. The photographic results are shown in Figure 26.

[0184] Western Blot Experiment: In order to test [ 131[I] Whether LG-12 can induce ICD in tumors, through [ 131 [I]LG-12 treatment of tumor cells was used to identify features of ICD. One of the key features of ICD is damage-associated molecular patterns (DAMPs), such as high-mobility group box 1 (HMGB1) and calreticulin (CRT). A mixture of RIPA lysis buffer (purchased from Beyotime Biotechnology Co., Ltd.) and benzyl sulfonyl fluoride PMSF (purchased from Beyotime Biotechnology Co., Ltd.) was prepared (RIPA lysis buffer: benzyl sulfonyl fluoride PMSF = 100:1, v / v); B16-F10 cells were incubated at 1.5 × 10⁻⁶ cells / year. 5 The cells were seeded evenly in small culture dishes containing 1 mL of DMEM medium supplemented with 1% (v / v) penicillin-streptomycin and 10% (v / v) fetal bovine serum, and incubated at 37°C and 5% (v / v) CO2 for 16 h. After 16 h of incubation, the medium was aspirated and […]. 131 I]LG-12 (1.85 MBq) (1 mL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) was incubated at 37°C and 5% CO2 for 24 h. After 24 h of incubation, the medium was aspirated, and 200 μL of the mixture was added to lyse B16-F10 cells for 10 min to obtain lysate. The protein concentration in the lysate was quantified using a BCA protein assay kit. The lysate was centrifuged at low temperature (4°C, 12000 r / min for 15 min), and the supernatant was collected. The protein in the supernatant was denatured, cooled, and centrifuged at low temperature. The supernatant sample was loaded and electrophoresed (80 V, 30 min; 120 V, 90 min). The protein sample was transferred to a 0.22 μm polyvinylidene fluoride (PVDF) membrane by transfer (300 mA, 90 min), and then blocked with 5% (w / v, g / 100 mL) skim milk powder for 1 h. Then, the antibodies were incubated with the corresponding primary antibodies (calreticulin CRT, HMGB-1, mouse PD-L1, and β-Actin antibodies; CRT antibody was diluted 1000-fold with antibody diluent before use as working solution; HMGB1 antibody was diluted 10000-fold with antibody diluent before use as working solution; and PD-L1 antibody was diluted 500-fold with antibody diluent before use as working solution. All antibodies were purchased from Abcam, and all antibody diluents were purchased from BioLegend) at 4°C for 24 h. Then, the antibodies were incubated with the secondary antibody (CRT and HMGB1 were treated with goat anti-rabbit secondary antibody, and PD-L1 was treated with goat anti-rat secondary antibody; the antibodies were diluted to a concentration of 4000-fold with antibody diluent before use as working solution. Anti-rabbit or anti-mouse antibodies were purchased from R&D System, and all antibody diluents were purchased from BioLegend) at room temperature (25°C) for 1 h. Finally, the membrane was developed using an ECL kit with a chemiluminescence imaging system. The development results are shown in Figure 27A.

[0185] As shown in Figure 26, [ 131 The inhibitory effect of LG-12 on the proliferation of B16-F10 cells was dose-dependent, and its inhibitory effect was significantly stronger than that of Na. 131 Group I. This result may be due to […]. 131 I]LG-12's targeting of PD-L1.

[0186] As shown in Figure 27A, B16-F10 cells were [… 131 After treatment with LG-12 (1.85 MBq / mL) for 48 h, the intracellular expression of HMGB1 decreased by 2.7-fold, indicating an increase in extracellular HMGB1 expression. Simultaneously, the expression of CRT on the tumor cell surface increased by 1.2-fold, indicating […]. 131 I]LG-12 can induce ICD in tumor cells.

[0187] Experimental Example 9: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 In vivo antitumor experiments of LG-12

[0188] This experimental example provides the iodine-131-labeled PD-L1 small molecule inhibitor from Example 3. 131 The in vivo antitumor experiment of LG-12 was conducted as follows:

[0189] In order to evaluate [ 131 [I]LG-12-targeted radionuclide therapy (TRT) induced immunogenic cell death. Five days after inoculating BALB / c mice with B16-F10 cells, the tumor-bearing mice were randomly divided into three groups: a blank control group, […]. 131 I]LG-12 group, Na 131 Group I (n=4); among which, [ 131 I]LG-12 group and Na 131 Group I mice were injected via tail vein injection [ 131 I]LG-12 (11.1 MBq, dissolved in 100 μL physiological saline) and Na 131I (11.1 MBq, dissolved in 100 μL of physiological saline) was injected into mice in the control group, along with an equal volume of physiological saline. Some tumor-bearing mice were sacrificed 48 hours after treatment. After sacrifice, tumor tissue from tumor-bearing mice was fixed in 4% (w / v, g / 100mL) paraformaldehyde for 24 hours. After fixation, it was dehydrated using ethanol of different concentrations prepared with n-butanol, sequentially in 50% (v / v) ethanol for 2 hours, 80% ethanol for 3 hours, 65% ethanol for 1.5 hours, 50% ethanol for 1 hour, 30% ethanol for 4 hours, 10% ethanol for 4 hours, and pure n-butanol for 24 hours. The dehydrated tumor tissue was first embedded in paraffin at 65°C for 4 hours, then embedded in paraffin and sectioned (6μm). After dewaxing, the slides were first used for antigen retrieval with sodium citrate antigen retrieval solution (purchased from BBI), then blocked with 5% (v / v) goat serum. The blocked slides were then first coated with the corresponding primary antibody (CD4+). + CD8 + PD-L1, CRT, and HMGB1 antibodies were all diluted 500-fold with antibody dilution buffer before use as working solutions. CD4 + and CD8 + All antibodies were purchased from ProteinTech, PD-L1, CRT, and HMGB1 antibodies were purchased from Abcam, and antibody dilution buffer was purchased from BioLegend. The working solution was incubated at 25°C for 90 min, and then incubated with the secondary antibody (CD4+). + PD-L1 uses goat anti-mouse secondary antibody, CD8 + CRT and HMGB1 were prepared using goat anti-rabbit secondary antibodies. The antibodies were diluted to a concentration of 4000 times with antibody diluent before being used as working solutions. All antibodies were purchased from R&D System, and the antibody diluents were purchased from BioLegend. The working solutions were incubated at 25°C for 30 min, then developed with DAB chromogenic solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Finally, the slides were dehydrated, cleared, and mounted. The immunohistochemical IHC analysis results are shown in Figure 27, C.

[0190] As shown in B of Figure 27, with Na 131 Compared with the control group, the treatment group received one injection. 131 I]LG-12 can slowly inhibit tumor growth.

[0191] The secretory expression of HMGB1 and CRT proteins in tumor tissues of three groups of tumor-bearing mice was measured 48 hours after the first injection of the drug. The results showed that Na... 131 Group I showed a slight increase in HMGB1 and CRT secretion levels, but after […] 131 Following LG-12 treatment, the secretion levels of two proteins in the tumor were approximately 2.5 times higher than in the control group, indicating that...131 I]LG-12 can induce ICD both in vivo and in vitro (C in Figure 27).

[0192] Experimental Example 10: [ 131 In vivo antitumor experiments of LG-12 and LG-12 in combination.

[0193] This experimental example provides [ 131 The in vivo antitumor experiment of LG-12 and LG-12 in combination was conducted as follows:

[0194] [ 131 [I]LG-12 can induce tumor cells to express DMAPs, activating tumor-specific cytotoxic T lymphocytes (CTLs) to kill tumor cells. To investigate [ 131 Whether the combined use of LG-12 and LG-12 improves the efficacy of immunotherapy for tumors was investigated. 131 [I] The in vivo antitumor effect of LG-12 combined with LG-12 on B16-F10 tumor-bearing mice. Five days after BALB / c mice were inoculated with B16-F10 cells, the resulting tumor-bearing mice were randomly divided into three groups: saline group (group A), LG-12 group (group B), and […]. 131 I] LG-12 combined with LG-12 (Group C) (n=5); among which, [ 131 Mice in the LG-12 and LG-12 combination therapy group were injected via tail vein on the first day of the experiment. 131 [I]LG-12 (11.1 MBq, dissolved in 100 μL of physiological saline), the other two groups of mice were injected with an equal volume of physiological saline, the LG-12 group and [ 131 Mice in the LG-12 plus / minimum LG-12 group were intraperitoneally injected with LG-12 (dissolved in 100 μL of physiological saline) at a dose of 5 mg / kg on days 1, 3, 6, 9, and 11 of the experiment. Mice in the physiological saline group were injected with the same volume of physiological saline. During the experiment, tumor size and mouse weight were measured every other day, and the tumor volume was calculated using the formula: Tumor volume = 1 / 2 (length × width). 2The tumor volume was calculated, and the results are shown in Figures 28a to 28b. After the experiment (day 16), the tumor-bearing mice were sacrificed. Before sacrifice, whole blood was collected by enucleation. The blood samples were allowed to stand at room temperature (25℃) for 30 minutes, then centrifuged at 1000 rpm for 5 minutes. Serum was collected, and the IFN-γ content was detected using a mouse IFN-γ ELISA kit (KE10001, Proteintech). The results are shown in Figure 28c. After sacrifice, tumor tissue from tumor-bearing mice was fixed in 4% (w / v, g / 100mL) paraformaldehyde for 24 hours. After fixation, it was dehydrated using ethanol of different concentrations prepared with n-butanol, sequentially in 50% (v / v) ethanol for 2 hours, 80% ethanol for 3 hours, 65% ethanol for 1.5 hours, 50% ethanol for 1 hour, 30% ethanol for 4 hours, 10% ethanol for 4 hours, and pure n-butanol for 24 hours. The dehydrated tumor tissue was first embedded in paraffin at 65°C for 4 hours, then embedded in paraffin and sectioned (6μm). After dewaxing, the slides were first used for antigen retrieval with sodium citrate antigen retrieval solution (purchased from BBI), then blocked with 5% (v / v) goat serum. The blocked slides were then first coated with the corresponding primary antibody (CD4+). + CD8 + PD-L1, CRT, and HMGB1 antibodies were all diluted 500-fold with antibody dilution buffer before use as working solutions. CD4 + and CD8 + All antibodies were purchased from ProteinTech, PD-L1, CRT, and HMGB1 antibodies were purchased from Abcam, and antibody dilution buffer was purchased from BioLegend. The working solution was incubated at 25°C for 90 min, and then incubated with the secondary antibody (CD4+). + PD-L1 uses goat anti-mouse secondary antibody, CD8 + CRT and HMGB1 were prepared using goat anti-rabbit secondary antibodies. The antibodies were diluted to a concentration of 4000 times with antibody diluent before being used as working solutions. All antibodies were purchased from R&D System, and the antibody diluents were purchased from BioLegend. The working solutions were incubated at 25°C for 30 min, then developed with DAB chromogenic solution (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), followed by counterstaining with hematoxylin (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Finally, the slides were dehydrated, cleared, and mounted. The immunohistochemical IHC analysis results are shown in Figures 28d to 28e.

[0195] As shown in Figures 28a to 28b, in the saline control group, the tumor volume grew rapidly, reaching 1498.3 mm² at the end of treatment. 3 LG-12 (5 mg / kg) treatment significantly delayed tumor growth (66.7 mm). 3This is consistent with the results of Experiment 6; in the combined treatment group, the tumor volume of tumor-bearing mice was effectively suppressed (27 mm). 3 ), which showed a significant difference compared to LG-12 alone, indicating that [ 131 I] LG-12 combined with LG-12 achieved good radioimmunotherapy results.

[0196] Subsequently, the proportions of CD4+ T cells and CD8+ T cells, as well as PD-L1 levels, in tumor tissue 16 days after treatment were measured to further explore the anti-tumor effect of radioimmunotherapy. CD4+ T cells in the LG-12 treatment group... + T cells and CD8 + The percentages of T cells were 1.9 times and 2 times that of the control group, respectively, while the combined treatment group showed a 2.5-fold upregulation of CD4+ T and CD8+ T cells compared to the control group (Figure 28 d–Figure 28 e). The results indicate that... 131 [I] LG-12 can increase the content of TILs, thereby enhancing the anti-tumor immune response. Furthermore, the expression level of PD-L1 in tumor tissue of the combination therapy group was downregulated by 60% compared to the control group and by 51% compared to the LG-12 treatment group (Figure 28, d–e). In addition, the secretion of interferon-γ in the combination therapy group (285±30 pg / 100 mL) was significantly higher than that in the LG-12 group (177±31 pg / 100 mL), which is one of the reasons why the combination therapy has a stronger inhibitory effect on tumor growth (Figure 28, c).

[0197] In summary, this invention introduces iodine-131 into LG-12 to synthesize radiopharmaceuticals. 131 [I]LG-12 achieves synergistic enhancement of anti-tumor effects through combined radiotherapy and immunotherapy. Among these, due to [ 131 LG-12 and LG-12 are chemically identical and exhibit the same biological properties; therefore, [ 131 LG-12 / LG-12 will be an ideal drug for targeted radiotherapy combined with immunotherapy to fight tumors.

[0198] The results of cellular uptake and biodistribution studies indicate that, 131 [I] LG-12 exhibits high targeting specificity for PD-L1. TR-FRET binding assays confirmed that LG-12 has a high inhibitory effect on the PD-1 / PD-L1 interaction. In vitro and in vivo studies indicate that LG-12 has the potential to enhance T cell responses and activate anti-tumor immune responses by inhibiting the PD-1 / PD-L1 pathway; furthermore, LG-12 increases CD4+. + and CD8 + The level of T cell infiltration is reduced, the expression of PD-L1 in tumor tissue is decreased, and the secretion of IFN-γ is stimulated.

[0199] Compared with LG-12 monotherapy, a single injection [ 131 While LG-12 did not significantly inhibit tumor growth, it could enhance its anti-tumor efficacy by reshaping the immune microenvironment. In vitro and in vivo studies have shown that... 131 [I]LG-12 can induce tumor cells to release HMGB 1 and CRT proteins via ICD, thereby enhancing tumor immunogenicity. It is evident that... 131 The combined use of LG-12 and LG-12 has a synergistic effect with anti-tumor immunotherapy. This strategy upregulates the proportion of tumor-infiltrating CTLs, leading to a decrease in PD-L1 levels in the tumor, thereby producing a good anti-tumor effect.

[0200] therefore,[ 131 [I] LG-12 and LG-12 in combination enhance the anti-tumor immune response. On the one hand, [ 131 [I] LG-12 induces ICD in tumors, triggering the release of DAMPs. DAMPs stimulate the maturation of immature dendritic cells, enhancing their ability to recognize tumors and present antigens. These processes activate tumor-specific CTLs and increase IFN-γ secretion, thereby enhancing tumor immunogenicity. On the other hand, LG-12 blocks the PD-1 / PD-L1 signaling pathway, preventing tumor cell immune escape. Overall, this strategy enhances the anti-tumor immune response by reshaping the tumor immune microenvironment, including enhancing tumor antigen presentation, increasing sensitivity to the anti-tumor immune response, and increasing the proportion of tumor-infiltrating immune cells.

[0201] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. An iodine-131-labeled PD-L1 small molecule inhibitor, characterized in that, The iodine-131-labeled PD-L1 small molecule inhibitor has the following structure: Where R is 2. A non-radioactive PD-L1 small molecule inhibitor, characterized in that, The non-radioactive PD-L1 small molecule inhibitor has the following structure: Where R is 3. A method for preparing the iodine-131-labeled PD-L1 small molecule inhibitor of claim 1, characterized in that, The method includes: dissolving compound 2 and 4-methoxy(diacetoxyiodine)benzene in a solvent, and then reacting them under nitrogen protection to obtain compound 3; and further processing compound 3. 131 Radiolabeling of I yields an intermediate compound. 131 I]1; intermediate compound [ 131 I]1, Compound 4 and sodium cyanoborohydride are dissolved in a solvent to obtain a solution; glacial acetic acid is added to the solution and the reaction is carried out to obtain the iodine-131 labeled PD-L1 small molecule inhibitor as described in claim 1; Compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, sulfoalanine, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane, or glucosamine. Compound 2 has the following structure: The 4-methoxy(diacetoxyiodide)benzene has the following structure: Compound 3 has the following structure: The intermediate compound [ 131 I]1 has the following structure:

4. The method as described in claim 3, characterized in that, The preparation method of compound 2 includes: dissolving compound 1, tetrakis(triphenylphosphine)palladium and hexa-n-butylditin in a solvent, and then heating the mixture under nitrogen protection to obtain compound 2; Compound 1 has the following structure:

5. The method as described in claim 4, characterized in that, The preparation method of compound 1 includes: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent and reacting them to obtain compound 1; Compound 5 has the following structure: The 3-bromomethylbenzonitrile has the following structure:

6. The method as described in claim 5, characterized in that, The preparation method of compound 5 includes: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarbonate dropwise to the solution under stirring in an ice bath and nitrogen to obtain a reaction solution; and reacting the reaction solution to obtain compound 5. Compound 7 has the following structure: Compound 6 has the following structure:

7. A method for preparing the non-radioactive PD-L1 small molecule inhibitor of claim 2, characterized in that, The method includes: dissolving compound 1, compound 4 and sodium cyanoborohydride in a solvent to obtain a solution; adding glacial acetic acid to the solution and reacting to obtain the non-radioactive PD-L1 small molecule inhibitor of claim 2. Compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, sulfoalanine, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane, or glucosamine. Compound 1 has the following structure:

8. The method as described in claim 5, characterized in that, The preparation method of compound 1 includes: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent and reacting them to obtain compound 1; Compound 5 has the following structure: The 3-bromomethylbenzonitrile has the following structure: The preparation method of compound 5 includes: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a solution; adding diisopropyl azodicarbonate dropwise to the solution under stirring in an ice bath and nitrogen to obtain a reaction solution; and reacting the reaction solution to obtain compound 5. Compound 7 has the following structure: Compound 6 has the following structure:

9. The use of the iodine-131 labeled PD-L1 small molecule inhibitor of claim 1 or the non-radioactive PD-L1 small molecule inhibitor of claim 2 in the preparation of a medicament for the prevention and / or treatment of cancer.

10. A drug for the prevention and / or treatment of cancer, characterized in that, The drug contains the iodine-131 labeled PD-L1 small molecule inhibitor of claim 1 and / or the non-radioactive PD-L1 small molecule inhibitor of claim 2.

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