A PD-L1 small molecule inhibitor and a preparation method and application thereof

Abstract

The present application relates to a kind of PD-L1 Small molecule inhibitor and its preparation method and application, belong to biological medicine technical field.The present application provides the PD-L1 Small molecule inhibitor of iodine-131 marking[ 131 I]LG-12And non-radioactive PD-L1 Small molecule inhibitor LG-12, wherein the PD-L1 Small molecule inhibitor of iodine-131 marking[ 131 I]LG-12And non-radioactive PD-L1 Small molecule inhibitor LG-12Both can effectively prevent and / or treat cancer, and[ 131 I]LG-12And LG-12Combination has synergistic effect, this is based on the nuclide / immune combined treatment strategy of PD-L1 Small molecule inhibitor in cancer treatment has great potential, therefore, 131 I]LG-12And LG-12In the preparation of drug for preventing and / or treating cancer has great application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a PD-L1 small molecule inhibitor and its preparation method and application, belonging to the technical field of biological medicine. BACKGROUND

[0002] Cancer, also known as malignant tumor, is caused by cell malignant proliferation, including melanoma, non-small cell lung cancer, kidney cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer and prostate cancer, etc. Cancer is mainly treated by combining surgery, chemotherapy and radiotherapy, three traditional treatment methods, and if necessary, combined with targeted therapy, biological therapy and anti-tumor immunotherapy, etc. Among them, anti-tumor immunotherapy is to inhibit or kill cancer cells by enhancing the body's immune function, which greatly reduces the serious side effects of traditional treatment methods on tumor patients.

[0003] Anti-tumor immunotherapy is a cancer treatment method that reactivates and maintains tumor-immune circulation, restores the body's normal anti-tumor immune response, and thus controls and eliminates tumors. Immune checkpoint programmed cell death receptor 1 (PD-1) is the main research direction of anti-tumor immunotherapy. Studies have found that the overexpression of programmed cell death ligand 1 (PD-L1) on the surface of tumor cells can combine with PD-1 on the surface of effector T cells to inhibit T cell attack on tumor cells, thereby causing tumor cells to escape from immunity. The PD-1 / PD-L1 immune checkpoint inhibitor can restore the T cell anti-tumor immune response by blocking the combination of PD-L1 on the surface of tumor cells and PD-1 on the surface of T cells, and thus inhibit the rapid proliferation and metastasis of tumors. At present, it has been widely confirmed that the combination of immune checkpoint inhibitors with other anti-tumor methods can effectively improve the low response rate of immunotherapy.

[0004] Targeted radionuclide therapy (TRT) is a cancer targeted therapy method that uses a carrier with targeting properties to transport therapeutic radionuclides to tumor accumulation to release ionizing radiation, thereby destroying tumor tissue. Studies have shown that TRT can improve the effectiveness of immunotherapy by inducing immunogenic cell death (ICD), releasing tumor neoantigens and stimulating anti-tumor immune effects in vivo. A recent study has confirmed that low-dose radiation can reshape the tumor microenvironment, which is conducive to anti-tumor immunity. At the same time, immunotherapy can also make tumors more sensitive to TRT. Therefore, the combination of immune checkpoint inhibitors and TRT may have a synergistic effect, and thus achieve better cancer treatment effect. However, at present, there is no report on PD-L1 small molecule inhibitors that are both immune checkpoint inhibitors and targeted radionuclide therapy drugs. SUMMARY

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

[0006]

[0007] 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:

[0008]

[0009] 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:

[0010]

[0011]

[0012] 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:

[0013]

[0014] 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.

[0015] The compound 4 is glycine, serine, glutamic acid, aminomethylphosphonic acid, sulfoalanine, arginine, tyrosine, aspartic acid, proline, asparagine, tris(hydroxymethyl)aminomethane (Tris), or glucosamine.

[0016] Compound 2 has the following structure:

[0017]

[0018] The 4-methoxy (diacetoxyiodo) benzene has the following structure:

[0019]

[0020] The compound 3 has the following structure:

[0021]

[0022] The intermediate compound[ 131 I]1 has the following structure:

[0023]

[0024] In an embodiment of the present application, the preparation method of the compound 2 comprises: dissolving compound 1, tetrakis (triphenylphosphine) palladium and hexa-n-butyl distannane in a solvent, and then performing a heating reaction under the protection of nitrogen to obtain compound 2.

[0025] The compound 1 has the following structure:

[0026]

[0027] In an embodiment of the present application, the preparation method of the compound 1 comprises: dissolving compound 5, 3-bromomethyl benzonitrile and cesium carbonate in a solvent to perform a reaction to obtain compound 1.

[0028] The compound 5 has the following structure:

[0029]

[0030] The 3-bromomethyl benzonitrile has the following structure:

[0031]

[0032] In an embodiment of the present application, the preparation method of the compound 5 comprises: dissolving compound 7, compound 6 and triphenylphosphine in a solvent under ice bath conditions to obtain a dissolution solution; adding dropwise diisopropyl azodicarboxylate into the dissolution solution under ice bath and nitrogen stirring to obtain a reaction solution; and performing a reaction on the reaction solution to obtain compound 5.

[0033] The compound 7 has the following structure:

[0034]

[0035] The compound 6 has the following structure:

[0036]

[0037] The application further provides a method for preparing the non-radioactive PD-L1 small molecule inhibitor, which comprises the following steps: dissolving compound 1, compound 4 and sodium cyanoborohydride in a solvent to obtain a dissolved solution; and adding glacial acetic acid into the dissolved solution to perform a reaction, so as to obtain the non-radioactive PD-L1 small molecule inhibitor.

[0038] The compound 4 is glycine, serine, glutamic acid, aminomethyl phosphonic acid, sulfopropyl alanine, arginine, asparagine, proline, aspartic acid, asparagine, tris(hydroxymethyl) aminomethane (Tris) or glucosamine.

[0039] The compound 1 has the following structure:

[0040]

[0041] In an embodiment of the application, the method for preparing the compound 1 comprises the following steps: dissolving compound 5, 3-bromomethylbenzonitrile and cesium carbonate in a solvent to perform a reaction, so as to obtain the compound 1.

[0042] The compound 5 has the following structure:

[0043]

[0044] The 3-bromomethylbenzonitrile has the following structure:

[0045]

[0046] In an embodiment of the application, the method for preparing the compound 5 comprises the following steps: dissolving compound 7, compound 6 and triphenyl phosphine in a solvent under ice bath conditions to obtain a dissolved solution; adding dropwise diisopropyl azodicarboxylate into the dissolved solution under ice bath and nitrogen stirring to obtain a reaction solution; and performing a reaction on the reaction solution to obtain the compound 5.

[0047] The compound 7 has the following structure:

[0048]

[0049] The compound 6 has the following structure:

[0050]

[0051] The application further provides application of the iodine-131 labeled PD-L1 small molecule inhibitor or the non-radioactive PD-L1 small molecule inhibitor in preparation of a drug for preventing and / or treating cancer.

[0052] In an embodiment of the present application, the medicament comprises the Iodine-131 labeled PD-L1 small-molecule inhibitor and the non-radioactive PD-L1 small-molecule inhibitor.

[0053] In an embodiment of the present application, the preventing and / or treating cancer comprises promoting tumor death by inhibiting the PD-1 / PD-L1 pathway, and / or, inducing immunogenic cell death (ICD) of tumor cells.

[0054] In an embodiment of the present application, the promoting tumor death by inhibiting the PD-1 / PD-L1 pathway comprises inhibiting the PD-1 / PD-L1 interaction by specifically binding to PD-L1 in the tumor and / or reducing the expression amount of PD-L1 in the tumor, activating the anti-tumor immune function of T cells, enhancing the killing ability of T cells to the tumor, and thereby promoting tumor death.

[0055] The inducing immunogenic cell death of tumor cells comprises promoting the release of high mobility group box 1 (HMGB1) and calreticulin (CRT) from tumor cells, and thereby inducing immunogenic cell death of tumor cells.

[0056] In an embodiment of the present application, the activating the anti-tumor immune function of T cells comprises increasing the proportion of CD 4+ T cells in tumor tissues, increasing the proportion of CD 8+ T cells in tumor tissues, and / or promoting T cells to secrete interferon-γ.

[0057] In an embodiment of the present application, the tumor comprises a solid tumor; the solid tumor comprises melanoma, non-small cell lung cancer, renal cancer, liver cancer, breast cancer, colon cancer, pancreatic cancer, and / or prostate cancer.

[0058] The present application also provides a medicament for preventing and / or treating cancer, the medicament comprising the Iodine-131 labeled PD-L1 small-molecule inhibitor and / or the non-radioactive PD-L1 small-molecule inhibitor.

[0059] In an embodiment of the present application, the medicament comprises the Iodine-131 labeled PD-L1 small-molecule inhibitor and the non-radioactive PD-L1 small-molecule inhibitor.

[0060] In an embodiment of the present application, the preventing and / or treating cancer comprises promoting tumor death by inhibiting the PD-1 / PD-L1 pathway, and / or, inducing immunogenic cell death of tumor cells.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The technical solution of this invention has the following advantages:

[0066] This invention provides an iodine-131 labeled PD-L1 small molecule inhibitor. 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:

[0067] 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 is significantly reduced. 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;

[0068] 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;

[0069] Third, the results of cell uptake and biodistribution experiments show that the iodine-131 labeled PD-L1 small molecule inhibitor[ 131 The maximum uptake value of I]LG-12 in B16-F10 melanoma cells was 5.33 ± 0.33% AD, and the uptake could be significantly blocked by non-radioactive PD-L1 small molecule inhibitor LG-12 (2.56 ± 0.06% AD). Meanwhile, after 1 h of tail vein injection, the uptake value of the iodine-131 labeled PD-L1 small molecule inhibitor[ 131 The uptake value of I]LG-12 in the tumor of B16-F10 tumor-bearing mice was 6.50 ± 1.05% ID / g. It can be seen that, 131 I]LG-12 can specifically bind to PD-L1 in the tumor;

[0070] Fourth, the results of in vivo imaging experiments show that, after 30 min of tail vein injection, the iodine-131 labeled PD-L1 small molecule inhibitor[ 131 I]LG-12 rapidly accumulated in the tumor of B16-F10 tumor-bearing mice, and the activity in the tumor was 2.3 times higher than that in the muscle. After being blocked by non-radioactive PD-L1 small molecule inhibitor LG-12, the uptake in the tumor site was significantly reduced. It can be seen that, 131 I]LG-12 can specifically bind to PD-L1 targets;

[0071] Fifth, the results of T cell / tumor cell co-culture experiments show that, when the concentration of non-radioactive PD-L1 small molecule inhibitor LG-12 was 0.78 μM and 3.125 μM, the survival rate of B16-F10 cells co-cultured with T cells decreased to 0.66 ± 0.06 and 0.55 ± 0.03, respectively. It can be seen that LG-12 can activate the anti-tumor immune function of T cells and enhance the killing ability of T cells to tumors in a dose-dependent manner.

[0072] 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;

[0073] 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.

[0074] 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.

[0075] Ninth, in vitro cloning experiments showed that, compared with iodine-131-labeled PD-L1 small molecule inhibitors [ 131 The 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;

[0076] Tenth, experimental results on in vitro radiopharmaceutical stimulation of tumor cells showed that iodine-131-labeled PD-L1 small molecule inhibitors [ 131The expression of high mobility group box 1 (HMGB1) outside B16-F10 cells is significantly increased after stimulation of ILG-12 (the expression of HMGB1 protein in B16-F10 cells is significantly reduced, which indicates that the expression of HMGB1 protein outside B16-F10 cells is significantly increased), and the expression of calreticulin (CRT) on the surface of B16-F10 cells is significantly increased, so, 131 ILG-12 can induce immunogenic death of tumor cells by promoting the release of HMGB1 protein and CRT protein from tumor cells, and has good in vitro anti-tumor effect;

[0077] Eleventh, the in vivo anti-tumor experiment results show that after intraperitoneal injection of the iodine-131 labeled PD-L1 small molecule inhibitor ILG-12, 131 all mice did not cause significant weight loss or death during the treatment process, so, 131 ILG-12 has low toxicity to normal tissues;

[0078] Twelfth, the in vivo anti-tumor experiment results show that after intraperitoneal injection of the iodine-131 labeled PD-L1 small molecule inhibitor ILG-12, 131 the expression of HMGB1 protein and CRT protein in the tumor tissue of mice is significantly increased, and the effect is much better than that of mice intraperitoneally injected with Na 131 I, so, 131 ILG-12 can induce immunogenic death of tumor cells by promoting the release of HMGB1 protein and CRT protein from tumor cells, and has good in vivo anti-tumor effect;

[0079] Thirteenth, the in vivo anti-tumor experiment results show that after intravenous injection of the iodine-131 labeled PD-L1 small molecule inhibitor ILG-12 and intraperitoneal injection of the non-radioactive PD-L1 small molecule inhibitor LG-12, 131 the tumor volume of mice is significantly reduced, the proportion of CD4 + T cells and the proportion of CD8 + T cells in the tumor tissue of mice are increased, the expression of PD-L1 in the tumor tissue of mice is reduced, and the expression of interferon-γ in the serum of mice is significantly increased, and the effect is much better than that of mice intraperitoneally injected with the non-radioactive PD-L1 small molecule inhibitor LG-12 alone, so, 131 ILG-12 and LG-12 have a synergistic effect, can significantly inhibit tumor growth, enhance anti-tumor immune response, and have good in vivo anti-tumor effect.

[0080] In summary, the iodine-131 labeled PD-L1 small molecule inhibitor ILG-12, 131I]LG-12 and non-radioactive PD-L1 small molecule inhibitor LG-12 can effectively prevent and / or treat cancer, and, 131 I]LG-12 and LG-12 have a synergistic effect, and this nuclide / immune combined treatment strategy based on PD-L1 small molecule inhibitors has great potential in cancer treatment, therefore, 131 I]LG-12 and LG-12 have great application prospects in the preparation of drugs for preventing and / or treating cancer. BRIEF DESCRIPTION OF DRAWINGS

[0081] Figure 1 : Synthesis process of non-radioactive PD-L1 small molecule inhibitor LG-12.

[0082] Figure 2 : Synthesis process of labeled precursor compound 3.

[0083] Figure 3 : ESI-MS analysis chart of non-radioactive PD-L1 small molecule inhibitor LG-12.

[0084] Figure 4 : ESI-MS analysis chart of compound 2.

[0085] Figure 5 : ESI-MS analysis chart of labeled precursor compound 3.

[0086] Figure 6 : Nuclear magnetic resonance hydrogen spectrum chart of compound 5.

[0087] Figure 7 : Nuclear magnetic resonance carbon spectrum chart of compound 5.

[0088] Figure 8 : Nuclear magnetic resonance hydrogen spectrum chart of compound 1.

[0089] Figure 9 : Nuclear magnetic resonance carbon spectrum chart of compound 1.

[0090] Figure 10 : Nuclear magnetic resonance hydrogen spectrum chart of non-radioactive PD-L1 small molecule inhibitor LG-12.

[0091] Figure 11 : Nuclear magnetic resonance carbon spectrum chart of non-radioactive PD-L1 small molecule inhibitor LG-12.

[0092] Figure 12 : Nuclear magnetic resonance hydrogen spectrum chart of compound 2.

[0093] Figure 13 : Nuclear magnetic resonance carbon spectrum chart of compound 2.

[0094] Figure 14NMR spectrum of hydrogen of compound 3.

[0095] Figure 15 NMR spectrum of carbon of compound 3.

[0096] Figure 16 Inhibitory activity of non-radioactive PD-L1 small molecule inhibitor LG-12 on PD-1 / PD-L1 interaction.

[0097] Figure 17 Intermediate compound 131 I] 1(a) and iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12(b) before and after purification.

[0098] Figure 18 Intermediate compound 131 I] 1 and iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 in vitro stability.

[0099] Figure 19 Iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 in B16-F10 cells for 1, 2 and 4 h (***P<0.001).

[0100] Figure 20 Iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 in B16-F10 tumor-bearing mice (A) in vivo biodistribution analysis (B) and autoradiography analysis of tumor and muscle tissues (C) (***P<0.001).

[0101] Figure 21 Cytotoxicity of B16-F10 cells and T cells incubated with non-radioactive PD-L1 small molecule inhibitor LG-12 for 48 h, respectively.

[0102] Figure 22 Cell viability of B16-F10 cells (A) and interferon-γ content in culture medium (B) after co-incubation of different concentrations of non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 0.78 and 3.125 μM) with B16-F10 cells / T cells (1 / 20 or 1 / 40) (N.S.: no significant difference, *p<0.05, **p<0.01 and ***p<0.001).

[0103] Figure 23Tumor inhibitory effect of non-radioactive PD-L1 small molecule inhibitor LG-12 (0, 5 mg / kg and 20 mg / kg) in B16-F10 mouse melanoma model (control group n=4, treatment group n=5). Figure 23 In the figure, (A) is the tumor growth curve; (B) is the final tumor weight; (C) is the mouse body weight; and (D) is a photograph of the dissected tumor (**p<0.01 and ***p<0.001).

[0104] 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: 50 μM, n=4).

[0105] Figure 25 IHC and H&E staining analysis of tumor tissues from B16-F10 tumor-bearing mice after treatment with non-radioactive PD-L1 small molecule inhibitor LG-12 (5 mg / kg and 20 mg / kg) (A) and serum IFN-γ levels in mice (B) (scale bar: 50 μM, n = 4, *p < 0.05 and **p < 0.01).

[0106] 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.

[0107] Figure 27 Study on tumor ICD induced by iodine-131 labeled PD-L1 small molecule inhibitor LG-12. Figure 27 In the image, (A) represents the result determined 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).

[0108] Figure 28 :[ 131In vivo anti-tumor activity of LG-12 / LG-12 combination therapy in B16-F10 tumor-bearing mice model (n=5). Figure 28 In the middle, (a) is the photograph of tumor peeling at the end of treatment; (b) is the tumor growth curve (insert: comparison of 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 chart of tumor tissue (CD4 + T cells, CD8 + T cells and PD-L1); (e) is the quantitative analysis chart of CD4 + T cells, CD8 + T cells and PD-L1 expression (scale: 50 μM, n=4, *p<0.05, ***p<0.001). DETAILED DESCRIPTION

[0109] The following examples are provided to better further understand the present application, and are not limited to the best mode contemplated, do not constitute limitations on the scope of the present application, and any person skilled in the art under the inspiration of the present application or the combination of the present application with other prior art features can derive any product identical or similar to the present application, which falls within the protection scope of the present application.

[0110] The specific experimental steps or conditions in the following examples are not specified, and can be operated according to the conventional experimental steps described in the literature in the art or the conditions. The reagents or instruments used are not specified by the manufacturer, and are conventional reagent products that can be obtained by purchase.

[0111] The PD-1 / PD-L1 binding detection kit (catalog #72038) involved in the following examples is purchased from BPS Bioscience Company. In chemical characterization, electrospray ionization mass spectrometry (ESI-MS) analysis is performed using a quadrupole tandem mass spectrometer ZMD4000 LC / MS (Waters, USA), chromatography analysis is performed using a pump (Waters, USA) equipped with a C18 chromatographic column (250x4.6mm, 10μm, Phenomenex) on a high performance liquid chromatograph (HPLC) and a radioactivity detector, and 1 H / 13 C nuclear magnetic resonance spectrum.

[0112] The cell culture and animal modeling process involved in the following examples are as follows:

[0113] Tumor cell culture: the mouse melanoma cell line B16-F10 (purchased from the Chinese Academy of Sciences Cell Library) is cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS) at 1x10 6The individual inoculation amount was plated on a culture dish, 10 mL of DMEM medium (purchased from BI) containing 1% (v / v) penicillin-streptomycin double antibody (purchased from Shanghai Biyun Tian) and 10% (v / v) fetal bovine serum (purchased from BI) was used, and the culture was carried out in a 37°C, 5% (v / v) CO2 incubator. When the cells were cultured to be in the logarithmic growth phase and the growth state was good, they could be used for in vitro cell experiments.

[0114] T cell culture: After the BALB / c mice (purchased from Changzhou Cavens Experimental Animal Company) were sacrificed, mouse T lymphocytes were extracted from the spleen; CD3 + (used after being diluted to a concentration of 5 μg / mL using an antibody diluent, CD3 + and the antibody diluent were both purchased from BioLegend) was added at an addition amount of 2 μL / well, and CD28 (used after being diluted to a concentration of 5 μg / mL using an antibody diluent, CD28 and the antibody diluent were both purchased from BioLegend) was added at an addition amount of 2 μL / well, and incubated at 4°C for 24 h to obtain a CD3 + / CD28 (5 μg / mL) antibody-coated 24-well plate; the extracted T lymphocytes were plated in the CD3 6 / CD28 (5 μg / mL) antibody-coated 24-well plate at an inoculation amount of 3×10 + / well, 500 μL / well of 1640 medium (purchased from BI) containing 10% (v / v) fetal bovine serum (purchased from BI) was used, and the culture was carried out in a 37°C, 5% (v / v) CO2 incubator. When the T lymphocytes were aggregated into a mass, they could be used for in vitro cell experiments.

[0115] Animal modeling: female 4-5-week-old BALB / c mice (purchased from Cavens Experimental Animal Company) were taken; B16-F10 cells were planted in the right upper axillary fossa of the mice (inoculation dose 3×10 6 / each); the tumor diameter was monitored every other day, and when the tumor diameter reached 50-100 mm 3 , tumor-bearing mice were used for further in vivo experiments. All animal research experiments were carried out in accordance with the principles formulated by the Ethics Committee of Jiangsu Institute of Nuclear Medicine.

[0116] Example 1: A non-radioactive PD-L1 small molecule inhibitor LG-12

[0117] The present embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-12, which has the structure as shown below:

[0118]

[0119] Example 2: A method for preparing non-radioactive PD-L1 small-molecule inhibitor LG-12

[0120] The present example provides a method for preparing non-radioactive PD-L1 small-molecule inhibitor LG-12 described in Example 1 (see synthesis route below) Figure 1 ), and the specific steps are as follows:

[0121] Step one: Refer to the literature "Dieter Enders; Jeanne Fronert; Tom Bisschops; Florian Boeck. Asymmetric total synthesis of smyrindiol employing an organocatalytical dol key step. Beilstein Journal of Organic Chemistry. 2012, 8, 1112-1117.", "Lv Gaochao; Miao Yinxing; Chen Yinfei; Lu Chunmei; Wang Xiuting; Xie Minhao; Qiu Ling; Lin Jianguo. Promising potential of a 18 F-labelled small-molecular radiotracer to evaluate PD-L1 expression in tumors by PET imaging. Bioorganic Chemistry 2021, 115, 105294." to synthesize compound 6 and compound 7;

[0122] The compound 7 has the following structure:

[0123]

[0124] The compound 6 has the following structure:

[0125]

[0126] Step two: 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; diisopropyl azodicarboxylate (2 mL, 10 mmol) was added dropwise to the solution under ice-bath and nitrogen stirring, and after the dropwise addition was completed, a reaction solution was obtained; the reaction solution was stirred (150 rpm) at room temperature (25 °C) for 16 h, and after the reaction was completed, a reaction product was obtained; the reaction product was rotary evaporated to remove tetrahydrofuran to obtain a solid product; the solid product was first washed with ethyl acetate, and then purified by silica gel column chromatography with a mixture of n-hexane / ethyl acetate (n-hexane: ethyl acetate = 2: 1, v / v) as an eluent to obtain white solid compound 5 (3.1 g, yield: 62%);

[0127] The compound 5 has the following structure:

[0128]

[0129] The 3-bromomethylbenzonitrile has the following structure:

[0130]

[0131] The hydrogen spectrum and carbon spectrum data of compound 5 are as follows (nuclear magnetic resonance hydrogen spectrum and carbon spectrum are shown in Figure 6 and Figure 7 ):

[0132] 1 H NMR (500 MHz, 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 (126 MHz, 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.

[0133] Step three: after mixing compound 5 (2.5 g, 5 mmol), 3-bromomethylbenzonitrile (1.69 g, 7 mmol) and cesium carbonate (4.77 g, 14.6 mmol) in N,N-dimethylformamide (30 mL), stirring (150 rpm) at room temperature (25 °C) for 16 h, the reaction was completed, and the reaction product was obtained; the reaction product was first quenched with water, then extracted with ethyl acetate, dried with anhydrous sodium sulfate, then concentrated by rotary evaporation, and finally purified by silica gel column chromatography with a mixture of n-hexane / ethyl acetate (n-hexane: ethyl acetate = 3:2, v / v) as the eluent to obtain white solid compound 1 (2.6 g, yield: 85%);

[0134] The compound 1 has the following structure:

[0135]

[0136] The hydrogen spectrum and carbon spectrum data of compound 1 are as follows (see Figure 8 and Figure 9 ):

[0137] 1 H NMR (500 MHz, 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 (126 MHz, 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.

[0138] 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 dissolved solution; after adding glacial acetic acid (360 μL) into the dissolved solution, the reaction was stirred (150 rpm) at room temperature (25 °C) for 16 h, and the reaction was completed to obtain a reaction product; the reaction product was first quenched with water, then extracted with ethyl acetate, dried with anhydrous sodium sulfate, then concentrated by rotary evaporation, and finally purified by silica gel column chromatography with a mixture of dichloromethane / methanol (dichloromethane:methanol = 10:1, v / v) as the eluent to obtain white solid compound LG-12 (223 mg, yield: 31%).

[0139] The hydrogen spectrum and carbon spectrum data of compound LG-12 are as follows (see Figure 10 and Figure 11 ):

[0140] 1 H NMR (500 MHz, 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), 7.21-7.12 (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 (126 MHz, 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, 130.2, 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.

[0141] The mass spectrum results of compound LG-12 are (see Figure 3 ): ESI-MS (m / z): 723 [M+H] + .

[0142] Example 3: An Iodine-131 labeled PD-L1 small molecule inhibitor 131I] LG-12

[0143] The present example provides a 131I-labeled PD-L1 small molecule inhibitor 131 I] LG-12, the 131I-labeled PD-L1 small molecule inhibitor 131 I] LG-12 has the following structure:

[0144]

[0145] The present example provides a method for preparing 131I-labeled PD-L1 small molecule inhibitor 131 I] LG-12

[0146] The present example provides a method for preparing 131I-labeled PD-L1 small molecule inhibitor 131 I] LG-12 of Example 3 (see Figure 2 for the synthetic route), and the specific steps are as follows:

[0147] Step one: compound 1 was synthesized according to the method of Example 2.

[0148] Step two: compound 1 (234 mg, 0.38 mmol), tetrakis(triphenylphosphine)palladium (22 mg, 5 mol) and hexabutylditin (217 μL, 0.418 mmol) were dissolved in dry dioxane (5 mL), and the reaction was stirred (150 rpm) at 80 °C under nitrogen protection for 6 h. After the reaction was completed, the reaction product was obtained. The reaction product was filtered, and the filtrate was obtained. The filtrate was first rotary evaporated to remove dioxane, and then purified by silica gel column chromatography using a mixture of n-hexane / ethyl acetate (n-hexane: ethyl acetate = 4:1, v / v) as the eluent to obtain compound 2 (271 mg, yield: 84%) in the form of colorless oil.

[0149] The compound 2 has the following structure:

[0150]

[0151] The hydrogen spectrum and carbon spectrum data of compound 2 are as follows (see Figure 12 and Figure 13 for the nuclear magnetic resonance hydrogen spectrum and carbon spectrum):

[0152] 1H NMR (500 MHz, 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 CNMR (126 MHz, DMSO-d6, δ ppm) 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, 131.5, 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.

[0153] The mass spectrum result of compound 2 is (ESI-MS see Figure 4 ): ESI-MS (m / z): 804 [M+Na] + .

[0154] Step three: compound 2 (390 mg, 0.5 mmol) and 4-methoxy(diacyloxyiodo)benzene (211 mg, 0.5 mmol, CAS: 16308-14-8) were dissolved in acetonitrile (ACN, 10 mL), and then stirred (150 rpm) at room temperature (25 °C) for 16 h under the protection of nitrogen. After the reaction was completed, the reaction product was obtained. The reaction product was first rotary evaporated to remove acetonitrile, then excess ether (20 mL) was added to precipitate the solid, centrifuged, and finally the white solid was collected and vacuum dried to obtain the labeled precursor compound 3 (214 mg, yield: 48%);

[0155] The 4-methoxy(diacyloxyiodo)benzene has the following structure:

[0156]

[0157] The labeling precursor compound 3 has the structure shown below:

[0158]

[0159] The hydrogen spectrum and carbon spectrum data of the labeling precursor compound 3 are as follows (see Figure 14 and Figure 15 ):

[0160] 1 H NMR (500 MHz, 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 (126 MHz, 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.

[0161] The mass spectrum result of the labeling precursor compound 3 is (see Figure 5 ESI-MS (m / z): 724 [M-TsO - ] + .

[0162] Step four: Na131 I solution (740 MBq) to obtain white solid Na 131 I; after adding precursor compound 3 (1.2 mg) dissolved in anhydrous acetonitrile (500 μL) into white solid Na 131 I, the reaction was completed at 90 °C for 60 min, and the reaction product was obtained; 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 eluent to obtain intermediate compound 131 I]1 (500 MBq), which was analyzed using radio-HPLC.

[0163] The intermediate compound 131 I]1 has the following structure:

[0164]

[0165] Step five: nitrogen-dried intermediate compound 131 I]1; the nitrogen-dried intermediate compound 131 I]1, 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 dissolved solution; after adding glacial acetic acid (1.8 μL) into the dissolved solution, the reaction was stirred (150 rpm) at room temperature (25 °C) for 10 h, and the reaction was completed to obtain a reaction product; the reaction product was purified by small silica gel column chromatography using a mixture of dichloromethane / methanol (dichloromethane:methanol = 9:1, v / v) as eluent to obtain iodine-131 labeled PD-L1 small molecule inhibitor 131 I]LG-12, which was analyzed using radio-HPLC.

[0166] The radio-HPLC analysis results of intermediate compound 131 I]1 and iodine-131 labeled PD-L1 small molecule inhibitor 131 I]LG-12 before and after purification are shown in Figure 17 . As shown in Figure 17 , the reaction of precursor compound 3 with Na 131 I in acetonitrile at 90 °C obtained intermediate compound 131 I]1, with a radioconversion rate of 70%, and the radiochemical purity was greater than 98% after purification. The intermediate compound 131 I]1 and Tris were subjected to reductive amination to obtain iodine-131 labeled PD-L1 small molecule inhibitor 131I]LG-12, radioconversion rate was 50%, and radiochemical purity was 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.

[0167] Example 5: A non-radioactive PD-L1 small molecule inhibitor LG-2 ~ LG-11 and LG-13

[0168] The present embodiment provides a non-radioactive PD-L1 small molecule inhibitor LG-2 ~ LG-11 and LG-13, which has the structure as shown below:

[0169]

[0170] Wherein, R is

[0171] Example 6: A method for preparing a non-radioactive PD-L1 small molecule inhibitor LG-2 ~ LG-11 and LG-13

[0172] The present embodiment provides a method for preparing the non-radioactive PD-L1 small molecule inhibitor LG-2 ~ LG-11 and LG-13 described in Example 5, and the specific steps are as follows:

[0173] On the basis of Example 2, tris(hydroxymethyl)aminomethane (Tris, 242 mg, 2 mmol) of step four was replaced with glycine (250 mg, 3.3 mmol), serine (250 mg, 2.4 mmol), glutamic acid (250 mg, 1.7 mmol), aminomethyl phosphonic acid (250 mg, 2.3 mmol), sulfo-propionic acid (250 mg, 1.5 mmol), arginine (250 mg, 1.4 mmol), asparagine (250 mg, 1.9 mmol), proline (250 mg, 1.9 mmol), aspartic acid (250 mg, 1.9 mmol), glucosamine (250 mg, 1.4 mmol), respectively, to obtain 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%), LG-13 (178 mg, yield: 23%).

[0174] Example 7: An Iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-2 ~ 131 I] LG-11 and 131 I] LG-13

[0175] This example 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 I] LG-13 has the structure shown below:

[0176]

[0177] wherein R is

[0178] Experimental Example 1: TR-FRET experiment of non-radioactive PD-L1 small molecule inhibitors LG-2 ~ LG-13

[0179] 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:

[0180] 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 a 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 LPD-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 LPD-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 LPD-L1-Biotin, 5 μL purified water, 5 μL LPD-1-Eu, and 5 μL dye-labeled reagent were mixed to obtain a mixture. Negative group: 5 μL buffer (1×), 5 μL purified water, 5 μL LPD-1-Eu, and 5 μL 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 inhibition rate % = (positive ratio - sample ratio) / (positive ratio - negative ratio) × 100. The analysis results are shown below. Figure 16 See Table 1.

[0181] The experimental results of PD-1 / PD-L1 TR-FRET show that ( Figure 16 (and Table 1), EC5 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 50EC value of LG-9 is 67.83±0.75 μM 50 EC value of LG-10 is 168±11.87 nM 50 EC value of LG-11 is 2.253±0.05 μM 50 EC value of LG-12 is 1.756±0.04 μM 50 EC value of LG-13 is 34.55±3.21 nM 50 It can be seen that LG-12 has high inhibitory activity on PD-1 / PD-L1 interaction, can significantly inhibit tumor growth, and shows good antitumor immunotherapy effect.

[0182] EC values of different small molecule inhibitors 50 Value

[0183]

[0184]

[0185] Experimental Example 2: Iodine-131 labeled PD-L1 small molecule inhibitor 131 I] In-vitro stability experiment of LG-12

[0186] This experimental example provides an in-vitro stability experiment of the iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 of Example 3, and the specific process is as follows:

[0187] Experiment one: the intermediate compound 131 I] 1 (370 KBq) prepared in Example 4 was added into a N,N-dimethylformamide (450 μL) solution to obtain a mixture; the mixture was incubated at 37℃ for 36 h; after the incubation was completed, the incubation solution was taken and subjected to radio-HPLC analysis using Radio-HPLC to investigate the in-vitro stability of the sample, and the analysis results are shown in Figure 18 .

[0188] Experiment two: the iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 (370 KBq) in Example 3 was added into a PBS buffer (pH = 7.4, 0.01 M, 450 μL) to obtain a mixture; the mixture was incubated at 37℃ for 4 h and 12 h; after the incubation was completed, the incubation solution was taken and subjected to radio-HPLC analysis using Radio-HPLC to investigate the in-vitro stability of the sample, and the analysis results are shown in Figure 18 .

[0189] As can be seen from Figure 18 , the intermediate compound 131I]1stable in DMF for 36h, which can meet the requirements of the next reaction; Iodine-131 labeled PD-L1 small molecule inhibitor 131 I]LG-12 still remained stable in PBS buffer for 12h, indicating that both the intermediate and the product have good in vitro stability.

[0190] Experimental Example 3: Iodine-131 labeled PD-L1 small molecule inhibitor 131 I]Cell uptake and biodistribution experiment of LG-12

[0191] This experimental example provides the iodine-131 labeled PD-L1 small molecule inhibitor 131 I]Cell uptake and biodistribution experiment of LG-12, the specific process is as follows:

[0192] B16-F10 cells were inoculated into a six-well plate at a seeding amount of 2.6x10 5 / well with DMEM medium (1500 μL) containing 1% (v / v) penicillin-streptomycin double antibody and 10% (v / v) fetal bovine serum, and then incubated at 37°C in a 5% (v / v) CO2 incubator for 16h; after 16h of incubation, the wells in the six-well plate were divided into two groups, each group having three parallel groups, which were the blocking group and the non-blocking group. Among them, the blocking group: aspirate the culture medium, first add LG-12 (50 μM, 1 mL, solvent is DMEM medium) in the wells of the blocking group, incubate at 37°C in a 5% CO2 incubator for 30 min for blocking, then add 131 I]LG-12 (3.7x10 -2 MBq, 200 μL, solvent is DMEM medium), and incubate at 37°C in a 5% (v / v) CO2 incubator for 1, 2, and 4h, respectively, and then rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3M NaOH to lyse the B16-F10 cells for 10 min to obtain the lysate. The non-blocking group: aspirate the culture medium, directly add 1 mL of DMEM medium and 131 I]LG-12 (3.7x10 -2 MBq, 200 μL, solvent is DMEM medium) in the wells of the non-blocking group, and incubate at 37°C in a 5% (v / v) CO2 incubator for 1, 2, and 4h, respectively, and then rinse the B16-F10 cells in the wells twice with PBS buffer, and finally add 0.3M NaOH to lyse the B16-F10 cells for 10 min to obtain the lysate. Collect the lysate and detect the radioactivity in the cells with a gamma counter (1470 Wizard, Perkins Elmer), and the detection results are shown in Figure 19 .

[0193] From Figure 19It can be seen that 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.

[0194] Experimental Example 4: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 I]LG-12 in vivo imaging experiment

[0195] 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:

[0196] 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 20 A in the middle.

[0197] 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 sacrificed and dissected, and tumors and major organs (heart, liver, spleen, lungs, kidneys, stomach, intestines, bones, muscles, and brain) were collected and weighed; after weighing, the radioactivity of the samples was measured using a gamma counter (1470 Wizard, Perkins Elmer), and the results are shown in […]. Figure 20 B in [the original text]. 131 The biodistribution of I]LG-12 is expressed as a percentage of the dose injected per gram of tissue (%ID / g).

[0198] Radioscintigraphy analysis of tumor and muscle tissue: After anesthetizing tumor-bearing mice (n = 3) with 2% (v / v) isoflurane in oxygen at a flow rate of 2 L / min, the limbs and tail of the tumor-bearing mice were fixed, and the tumor-bearing mice were injected with iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 (5 MBq, dissolved in 100 μL of normal saline); 1 h after injection, the mice were sacrificed and dissected, and the subcutaneous tumor tissue and leg muscle were stripped, washed once with PBS buffer, embedded with a freezing embedding agent at -25°C, and then sectioned into tissue sections (30 μm) on a glass slide using a freezing microtome (CM1950, SLEE / MNT). The glass slide was then laid flat on a phosphor screen for 4 h, and the phosphor screen was scanned using a Cyclone Plus phosphor screen imaging system (C431200, PerkinElmer) to obtain an image. Finally, the image was processed and analyzed using OptiQuant software, and the results are shown in Figure 20 C.

[0199] As shown in A of Figure 20 , B16-F10 tumor-bearing mice were injected with 131 I] LG-12 (11.1 MBq) 30 min later, it was observed by SPECT / CT imaging that 131 I] LG-12 rapidly accumulated in the tumor site. The distribution of 131 I] LG-12 in B16-F10 tumor-bearing mice was further studied. As shown in B of Figure 20 , the uptake of 131 I] LG-12 in tumor tissue was 6.50 ± 1.05% ID / g, which was significantly higher than that in most normal tissues, indicating that 131 I] LG-12 has the ability to target PD-L1 in vivo, but the uptake in non-target tissues such as blood, liver, heart, and intestine is also high, which may be due to the high lipophilicity of 131 I] LG-12. As shown in C of Figure 20 , the activity of 131 I] LG-12 in the tumor was 2.3 times higher than that in the muscle, further demonstrating the targeting of 131 I] LG-12 to PD-L1.

[0200] Experimental Example 5: Biocompatibility experiment of non-radioactive PD-L1 small molecule inhibitor LG-12

[0201] This experimental example provides a biocompatibility experiment of the non-radioactive PD-L1 small molecule inhibitor LG-12 of Example 1, and the specific process is as follows:

[0202] 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. 4 The 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. The plates were then incubated at 37°C and 5% CO2 for 24 h. After h, the culture medium was aspirated, and MTT (diluted to 5 mg / mL with PBS buffer, purchased from Beyotime Biotech, Shanghai) was added to the wells at a rate of 20 μL / well. The wells were incubated at 37℃ in a 5% CO2 incubator for 4 h. After 4 h of incubation, the MTT was aspirated, and DMSO was added to the wells at a rate of 150 μL / well. The wells were shaken for 10 min. After shaking, the absorbance (MD / M5e, VEDENG) of the samples was measured at 490 nm using a microplate reader. The survival rate of B16-F10 tumor cells and T cells was calculated using 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). The experimental results are shown in [Figure number missing]. Figure 21 .

[0203] like Figure 21 As shown, LG-12 has 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.

[0204] Experiment Example 6: Co-culture of T cells / tumor cells with the non-radioactive PD-L1 small molecule inhibitor LG-12

[0205] 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:

[0206] 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 for 24 h in a 5% CO2 incubator. 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 below. Figure 22 In step 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 the plate was incubated at 37°C in a 5% CO2 incubator for 4 h. After 4 h of incubation, the OD values ​​were measured at 450 nm using a microplate reader (MD / M5e, VEDENG). The survival rate of B16-F10 tumor cells was calculated using the formula: Viability = OD value of sample wells / OD value of reference wells (reference wells are those containing 0 μM LG-12). The experimental results are shown in [Figure number missing]. Figure 22A in the middle.

[0207] like Figure 22 As shown in Figure A, 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 treated 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 viability was significantly lower. When LG-12 concentrations were 1:20, the cell survival rate of B16-F10 cells decreased from 0.90±0.06 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 concentrations were 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.

[0208] 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. Figure 22 In the B group, 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 interferon-γ expression level... The expression level of interferon-γ 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 promote the activation of CD8+ T cells by blocking the PD-1 / PD-L1 signaling pathway, increasing interferon-γ secretion.

[0209] Experimental Example 7: In vivo antitumor experiment of LG-12, a non-radioactive small molecule inhibitor of PD-L1

[0210] 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:

[0211] 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 ), calculate tumor volume, and see the test results. Figure 23 After the experiment (day 14), tumor-bearing mice were sacrificed. Before sacrifice, whole blood was collected by enucleation. After standing at room temperature (25℃) for 30 minutes, the blood samples were centrifuged at 1000 rpm for 5 minutes. Serum was collected, and IFN-γ levels were detected using a mouse IFN-γ ELISA kit (KE10001, Proteintech). The results are shown below. Figure 25 In the B group, after sacrifice of tumor-bearing mice, the heart, liver, spleen, lung, kidney, muscle, and tumor were dissected and fixed in 4% (w / v, g / 100 mL) paraformaldehyde solution for 24 h. After fixation, the tissues were dehydrated in 15% (w / v, g / 100 mL) sucrose solution and 30% (w / v, g / 100 mL) sucrose solution for 24 h respectively. The dehydrated tissues were first embedded in cryoemulation medium at -25 °C, and then prepared into tissue sections (6 μm) on glass slides using a cryostat (CM1950, LEICA). The slides were first fixed with fixative (purchased from Wuxi Jiangyuan Industrial Technology & Trade Co., Ltd.) for 10 min, then stained with hematoxylin staining solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 10 min and eosin staining solution (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) for 1 min. Finally, the slides were dehydrated, cleared, and mounted. The H&E staining analysis results are shown in the figure. Figure 24 and Figure 25In section A, tumor tissue from tumor-bearing mice was fixed in 4% (w / v, g / 100 mL) paraformaldehyde for 24 h. After fixation, it was dehydrated with ethanol of different concentrations prepared with n-butanol, successively in 50% (v / v) ethanol for 2 h, 80% ethanol for 3 h, 65% ethanol for 1.5 h, 50% ethanol for 1 h, 30% ethanol for 4 h, 10% ethanol for 4 h, and pure n-butanol for 24 h. After dehydration, the tumor tissue was first immersed in paraffin at 65 °C for 4 h, then embedded in paraffin and sectioned (6 μm). 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-fold using antibody dilution buffer and used as working solutions. All antibodies were purchased from R&D Systems, and the antibody dilution buffer was purchased from BioLegend. The working solutions were incubated at 25°C for 30 min, followed by DAB staining (purchased from Wenzhou Maixin Biotechnology Development Co., Ltd.), and then counterstained with hematoxylin (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). Finally, the slides were dehydrated, cleared, and mounted. Immunohistochemical (IHC) analysis results are shown below. Figure 25 A in the middle.

[0212] like Figure 23 A in Figure 23 As shown in C, 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 finding. Figure 24) and the tumor volume (37±7 mm3) and weight (0.046±0.01 g) of the low-dose (5 mg / kg) treatment group were significantly smaller than those of the blank group (volume 1303±203 mm3 and weight 1.88±0.63 g), while the high-dose (20 mg / kg) group had a poorer anti-tumor effect than the low-dose group, with a tumor volume and weight of 201±44 mm3 and 0.44±0.22 g, respectively. This indicates that anti-tumor immunotherapy based on the small molecule inhibitor LG-12 is not better with a higher dose, and the specific mechanism needs to be further studied.

[0213] After treatment, the tumor tissues of the mice were taken for immunohistochemical analysis to observe the differences in infiltrating lymphocytes (TILs) and PD-L1 expression in the tumor. The results showed that LG-12 significantly increased the CD4 + T cells and CD8 + T cell infiltration levels, and the expression level of PD-L1 was significantly lower than that of the blank group. At the same time, H&E staining of the tumor tissue after treatment showed obvious cell apoptosis. It can be seen that LG-12 has good anti-tumor effect in vivo at a low dose. Similarly, the CD4+, CD8+ T cell content in the tumor tissue of the high-dose group of mice was higher, and the expression of PD-L1 was reduced, but overall lower than the low-dose group, consistent with the anti-tumor results (A) in FIG. 6. Figure 25

[0214] The expression of interferon-γ in the serum of mice was detected using an ELISA kit. The results showed that the expression of interferon-γ in the low-dose treatment group of mice was 153±54 (pg / 100 mL), while the expression of interferon-γ in the serum of the high-dose treatment group of mice was 72±23 (pg / 100 mL), which is consistent with the results of anti-tumor and immunohistochemistry (B) in FIG. 6. Figure 25

[0215] Example 8: Iodine-131 labeled PD-L1 small molecule inhibitor 131 In vitro cloning experiment of iodine-131 labeled PD-L1 small molecule inhibitor

[0216] This experimental example provides an in vitro cloning experiment of the iodine-131 labeled PD-L1 small molecule inhibitor 131 I] LG-12 of Example 3, and the specific process is as follows:

[0217] Cloning experiment: The cloning experiment is used to evaluate the ability of a single tumor cell to proliferate and form a clone after being subjected to radiation treatment. B16-F10 cells were plated at a density of 1.2×10 3 ​​Inoculation amount of hole After inoculation into 24-hole plate added with DMEM culture medium (1 mL) containing 1% (v / v) penicillin-streptomycin double antibody, 10% (v / v) fetal bovine serum, 37 ℃, 5% (v / v) CO2 incubator culture for 16 h; after 16 h of culture, the culture medium was aspirated, and different concentrations (0, 0.148, 0.296, 0.592, 1.184 and 2.368 MBq) of [ 131 I] LG-12 (1 mL, solvent is DMEM culture medium containing 1% penicillin-streptomycin double antibody, 10% fetal bovine serum) were added into the hole, and incubated at 37 ℃, 5% CO2 incubator for 10 h; after 10 h of incubation, the fresh culture medium was replaced, and incubated at 37 ℃, 5% (v / v) CO2 incubator for 9 days; after 9 days of incubation, the B16-F10 cells in the hole were first rinsed twice with PBS buffer, then fixed with methanol for 20 min, then stained with crystal violet (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) for 15 min, then washed with running water to remove crystal violet, and finally dried at 37 ℃ and photographed. The photographing results are shown in Figure 26 .

[0218] Western Blot experiment: In order to test whether [ 131 I] LG-12 can induce ICD in tumor, the characteristics of ICD were searched by treating tumor cells with [ 131 I] LG-12. One of the main characteristics in the process of ICD is damage-associated molecular patterns (DAMPs), such as high mobility group protein 1 (HMGB1) and calreticulin (CRT). The mixture of RIPA lysis buffer (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) and phenylmethylsulfonyl fluoride PMSF (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) (RIPA lysis buffer: phenylmethylsulfonyl fluoride PMSF = 100:1, v / v) was configured; B16-F10 cells were plated in a small culture dish added with DMEM culture medium (1 mL) containing 1% (v / v) penicillin-streptomycin double antibody, 10% (v / v) fetal bovine serum at a seeding amount of 1.5×10 5 cells, and incubated at 37 ℃, 5% (v / v) CO2 incubator for 16 h; after 16 h of culture, the culture medium was aspirated, and [ 131I]LG-12 (1.85 MBq) (1 mL, in DMEM medium containing 1% penicillin-streptomycin and 10% fetal bovine serum) was incubated at 37°C in a 5% CO2 incubator 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 rpm for 15 min), and the supernatant was collected. The proteins in the supernatant were denatured, cooled, and centrifuged at low temperature. The supernatant samples were loaded and electrophoresed (80 V, 30 min; 120 V, 90 min). The protein samples were transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes via transfer (300 mA, 90 min). After membrane deposition, the membrane was first blocked with 5% (w / v, g / 100mL) skim milk powder for 1 hour. Then, it was 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 dilution buffer before use as working solution, HMGB1 antibody was diluted 10000-fold with antibody dilution buffer before use as working solution, and PD-L1 antibody was diluted 500-fold with antibody dilution buffer before use as working solution; all antibodies were purchased from Abcam, and antibody dilution buffers were purchased from BioLegend) at 4°C for 24 hours. Finally, it was incubated with secondary antibodies (CRT and HMGB1 used goat anti-rabbit secondary antibody, and PD-L1 used goat anti-rat secondary antibody; the antibodies were diluted to a concentration of 4000-fold with antibody dilution buffer before use as working solution; anti-rabbit or anti-mouse antibodies were purchased from R&D). The system and antibody dilution buffer were purchased from BioLegend. The working solution was incubated at room temperature (25℃) for 1 hour, and finally, the membrane was developed using an ECL kit and a chemiluminescence imaging system. The development results are shown in [Figure number missing]. Figure 27 A in the middle.

[0219] like Figure 26 As shown, [ 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.

[0220] like Figure 27 As shown in A, B16-F10 cells were subjected to [ 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.

[0221] Experimental Example 9: Iodine-131 labeled PD-L1 small molecule inhibitors [ 131 In vivo antitumor experiments of LG-12

[0222] 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:

[0223] 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 resulting 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 131 I (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, HMGB1 using goat anti-rabbit secondary antibody, the antibody is diluted to a concentration of 4000 times using antibody diluent as working solution, each antibody is purchased from R&D System, and the antibody diluent is purchased from BioLegend) working solution 25℃ incubation for 30min, then using DAB developing solution (purchased from Wenzhou Maynew Biotechnology Development Co., Ltd.) for color development, then using hematoxylin (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) for re-staining, finally dehydration, transparency, and mounting, the results of immunohistochemical IHC analysis are shown in Figure 27 C.

[0224] As shown in B of Figure 27 , the Na 131 I treatment group and the blank group, injection of once[ 131 I]LG-12 can slow down the growth of tumor.

[0225] The secretion expression of HMGB1 protein and CRT protein in tumor tissues of three groups of tumor-bearing mice was detected 48h after the first injection of drug treatment. The results showed that the secretion level of HMGB 1 and CRT in the Na 131 I group increased slightly, but after treatment with[ 131 I]LG-12, the secretion level of the two proteins in the tumor was about 2.5 times that of the blank group, and it can be seen that[ 131 I]LG-12 can induce ICD(C) in vivo and in vitro. Figure 27

[0226] Experimental example 10: in vivo anti-tumor experiment of[ 131 I]LG-12 and LG-12 combined drug

[0227] This experimental example provides an in vivo anti-tumor experiment of[ 131 I]LG-12 and LG-12 combined drug, the specific process is as follows:

[0228] [ 131 I]LG-12 can induce tumor cells to express DMAPs and activate tumor-specific cytotoxic T lymphocytes (CTLs) to kill tumor cells. In order to explore whether[ 131 I]LG-12 and LG-12 combined use can improve the immunotherapy effect on tumor, the in vivo anti-tumor effect of[ 131 I]LG-12 and LG-12 combined use on B16-F10 tumor-bearing mice was studied. BALB / c mice were inoculated with B16-F10 cells for 5 days, and the obtained tumor-bearing mice were randomly divided into three groups, three groups were respectively normal saline group (group A), LG-12 group (group B) and[ 131 I]LG-12 and LG-12 combined drug group (group C) (n=5); among them, 131 ​The mice in the LG-12 and LG-12 combination group were injected with LG-12 (11.1 MBq, dissolved in 100 μL of normal saline) through the tail vein on the first day of the experiment 131 The mice in the LG-12 and LG-12 combination group were injected with LG-12 (11.1 MBq, dissolved in 100 μL of normal saline) through the tail vein on the first day of the experiment 131 The mice in the LG-12 and LG-12 combination group were injected with LG-12 (11.1 MBq, dissolved in 100 μL of normal saline) through the tail vein on the first day of the experiment 2 The tumor size and body weight of the mice were measured every other day during the experiment, and the tumor volume was calculated according to the formula: tumor volume = 1 / 2 (length x width Figure 28 a~b in the results Figure 28 a~b in the results After the experiment (i.e., on the 16th day of the experiment), the tumor-bearing mice were sacrificed. Before the tumor-bearing mice were sacrificed, the eyeballs were enucleated to collect whole blood. After the blood samples were left to stand at room temperature (25°C) for 30 min, they were centrifuged at 1000 r / min for 5 min, and the serum was collected. The content of IFN-γ was detected using a mouse IFN-γ ELISA kit (KE10001, proteintech), and the detection results are shown in c in the results Figure 28 After the tumor-bearing mice were sacrificed, the tumor tissues were fixed in 4% (w / v, g / 100 mL) paraformaldehyde for 24 h. After fixation, the tumor tissues were dehydrated with n-butanol prepared with different concentrations of ethanol. The dehydrated tumor tissues were first immersed in wax at 65°C for 4 h, then paraffin-embedded and sectioned (6 μm). After the glass slides were deparaffinized, they were first subjected to antigen retrieval using sodium citrate antigen retrieval solution (purchased from BBI company), and then blocked with 5% (v / v) goat serum. The blocked glass slides were first incubated with the corresponding primary antibodies (CD4 + , CD8 + , PD-L1, CRT, and HMGB1 antibodies, each of which was diluted 500 times with antibody diluent to serve as a working solution. The CD4 + and CD8 + antibodies were purchased from proteintech, the PD-L1, CRT, and HMGB1 antibodies were purchased from abcam, and the antibody diluent was purchased from BioLegend) working solution at 25°C for 90 min, and then incubated with secondary antibodies (CD4 + , PD-L1 goat anti-mouse secondary antibody, and CD8 +, CRT, HMGB1 using goat anti-rabbit secondary antibody, the antibody is diluted to a concentration of 4000 times using antibody diluent as working solution, each antibody is purchased from R&D System, and the antibody diluent is purchased from BioLegend) working solution 25℃ incubation for 30min, then using DAB developing solution (purchased from Wenzhou Maynew Biotechnology Development Co., Ltd.) color developing, then using hematoxylin (purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.) re-staining, finally dehydration, transparency, mounting, the results of immunohistochemical IHC analysis are shown in Figure 28 d in the figure Figure 28 e in the figure

[0229] As shown in a~b in the figure Figure 28 b in the figure Figure 28 As shown in a~b in the figure The tumor volume of the saline control group grew rapidly, and the tumor volume was 1498.3mm 3 at the end of treatment, while the LG-12 (5mg / kg) treatment significantly delayed the tumor growth (66.7mm 3 ), which is consistent with the results of experimental example 6; in the combined treatment group, the tumor volume of the tumor-bearing mice was effectively inhibited (27mm 3 ), which is significantly different from the LG-12 treatment alone, indicating that 131 I] LG-12 and LG-12 combined treatment achieved good radioimmunotherapy effect.

[0230] The proportion of CD4+T cells, CD8+T cells and PD-L1 level in tumor tissues after 16 days of treatment were detected, and the anti-tumor effect of radioimmunotherapy was further explored. The percentage of CD4 + T cells and CD8 + T cells in the LG-12 treatment group were 1.9 times and 2 times that of the control group, respectively, while the CD4+T and CD8+T cells in the combined treatment group were up-regulated by 2.5 times compared with the control group (d~e in the figure Figure 28 Figure 28 The results showed that 131 I] LG-12 can increase the content of TILs, thereby enhancing the anti-tumor immune response. In addition, the expression level of PD-L1 in the tumor tissue of the combined treatment group was down-regulated by 60% compared with the control group, and down-regulated by 51% compared with the LG-12 treatment group (d~e in the figure Figure 28 Figure 28 In addition, the secretion amount of interferon-γ (285±30pg / 100mL) in the combined treatment group was also significantly higher than that in the LG-12 group (177±31pg / 100mL), which was one of the reasons why the combined treatment had stronger inhibition effect on tumor growth (c in the figure Figure 28

[0231] ​​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.

[0232] 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.

[0233] 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.

[0234] therefore,[ 131 [I] LG-12 and LG-12 in combination enhance the anti-tumor immune response. On the one hand, [ 131I]LG-12 induces ICD in tumors, triggering the release of DMAPs. DMAPs stimulate the maturation of immature dendritic cells, improving the ability of dendritic cells to recognize tumors and present antigens. These processes activate tumor-specific CTLs, increasing the secretion of IFN-γ, thereby enhancing the immunogenicity of tumors. On the other hand, LG-12 blocks the PD-1 / PD-L1 signaling pathway, preventing immune escape of tumor cells. Overall, this strategy enhances the anti-tumor immune response by reshaping the tumor immune microenvironment, including enhancing the presentation of tumor antigens, increasing the sensitivity to anti-tumor immune responses, and increasing the proportion of tumor-infiltrating immune cells.

[0235] Obviously, the above examples are merely illustrative examples for the sake of clarity, and are not limiting to the embodiments. Based on the above description, other different forms of changes or variations can also be made by those of ordinary skill in the art. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

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: 。 2. A non-radioactive PD-L1 small molecule inhibitor, characterized in that, The non-radioactive PD-L1 small molecule inhibitor has the following structure: 。 3. A method of preparing the Iodine-131 labeled PD-L1 small molecule inhibitor of claim 1, characterized in that, The method comprises: dissolving compound 2 and 4-methoxy (diacetoxyiodo) benzene in a solvent, and then performing a reaction under the protection of nitrogen to obtain compound 3; performing a reaction on compound 3 131 to obtain an intermediate compound 131 I]1; dissolving the intermediate compound 131 I]1, tris (hydroxymethyl) aminomethane and sodium cyanoborohydride in a solvent to obtain a dissolved solution; adding glacial acetic acid to the dissolved solution to perform a reaction, and then obtaining the iodine-131 labeled PD-L1 small molecule inhibitor according to claim 1. The compound 2 has the following structure: ； The 4-methoxy (diacetoxyiodo) benzene has the following structure: ； The compound 3 has the following structure: 。 4. The method of claim 3, wherein, The preparation method of the compound 2 comprises the following steps: dissolving compound 1, tetrakis (triphenylphosphine) palladium and hexa-n-butyl distannane in a solvent, and then performing a heating reaction under the protection of nitrogen to obtain the compound 2. The compound 1 has the following structure: 。 5. The method of claim 4, wherein, The preparation method of the compound 1 comprises the following steps: dissolving compound 5, 3-bromomethyl benzonitrile and cesium carbonate in a solvent to perform a reaction, and then obtaining the compound 1. The compound 5 has the following structure: ； The 3-bromomethyl benzonitrile has the following structure: 。 6. The method of claim 5, wherein, The preparation method of the compound 5 comprises the following steps: under ice bath conditions, dissolving compound 7, compound 6 and triphenylphosphine in a solvent to obtain a dissolved solution; under ice bath and nitrogen stirring, adding diisopropyl azodicarboxylate dropwise into the dissolved solution to obtain a reaction solution; and then performing a reaction on the reaction solution to obtain the compound 5. The compound 7 has the following structure: ； The compound 6 has the following structure: 。 7. A method of preparing the non-radioactive PD-L1 small molecule inhibitor of claim 2, wherein, The method comprises the following steps: dissolving compound 1, tris (hydroxymethyl) aminomethane and sodium cyanoborohydride in a solvent to obtain a dissolved solution; adding glacial acetic acid into the dissolved solution to perform a reaction, and then obtaining the non-radioactive PD-L1 small molecule inhibitor of claim 2. The compound 1 has the following structure: 。 8. The method of claim 5, wherein, The preparation method of the compound 1 comprises the following steps: dissolving compound 5, 3-bromomethyl benzonitrile and cesium carbonate in a solvent to perform a reaction, and then obtaining the compound 1. The compound 5 has the following structure: ； The 3-bromomethyl benzonitrile has the following structure: ； The preparation method of the compound 5 comprises the following steps: under ice bath conditions, dissolving compound 7, compound 6 and triphenylphosphine in a solvent to obtain a dissolved solution; under ice bath and nitrogen stirring, adding diisopropyl azodicarboxylate dropwise into the dissolved solution to obtain a reaction solution; and then performing a reaction on the reaction solution to obtain the compound 5. The compound 7 has the following structure: ； The compound 6 has the following structure: 。 9. The iodine-131 labeled PD-L1 small molecule inhibitor of claim 1 or the non-radioactive PD-L1 small molecule inhibitor of claim 2 is used for preparing a drug for preventing and / or treating cancer.

10. A medicament for preventing and / or treating cancer, characterized by, 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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