CN114605482B

CN114605482B - Small molecular compound with PD-1/PD-L1 inhibiting activity and application thereof

Info

Publication number: CN114605482B
Application number: CN202210383222.0A
Authority: CN
Inventors: 陆茵; 邹伟; 韦忠红; 王爱云; 陈文星; 赵杨; 万雪婷; 李晓曼; 吴媛媛; 余苏云
Original assignee: Nanjing University of Chinese Medicine
Current assignee: Nanjing University of Chinese Medicine
Priority date: 2022-04-13
Filing date: 2022-04-13
Publication date: 2023-09-22
Anticipated expiration: 2042-04-13
Also published as: CN114605482A

Abstract

The invention relates to a small molecular compound with PD-1/PD-L1 inhibiting activity and application thereof, wherein the small molecular compound has better affinity with PD-L1protein, can block interaction of PD-1/PD-L1, can promote T activation in vivo and in vitro, and can enhance immune response of T cells, thereby blocking immune escape of tumor cells. In addition, the small molecular compound has no obvious toxic or side effect, can be used for treating colon cancer, melanoma, lung cancer, breast cancer and the like, and has wide development prospect and clinical application value.

Description

Small molecular compound with PD-1/PD-L1 inhibiting activity and application thereof

Technical Field

The invention belongs to the field of tumor immunotherapy, and particularly relates to a natural product-derived small molecule compound with PD-1/PD-L1 inhibition activity and application thereof.

Background

Inhibitors of targeted immune checkpoints innovate the means of clinical treatment of tumors, and the apoptosis receptor 1 (PD-1) and its ligand (PD-L1) are honored as the most successful targets to date in the field of tumor immunotherapy. Compared with traditional chemotherapy, the traditional Chinese medicine has the remarkable advantages of remarkable and durable curative effect, wide anti-tumor spectrum and the like. Particularly in advanced cancer, such as melanoma and lung cancer, the survival time of many patients is obviously prolonged, and some patients are even completely relieved, thus bringing new hope for tumor treatment.

PD-1 belongs to a CD28 superfamily member and is an important immunosuppressive receptor, and is mainly expressed on the surface of immune-related lymphocytes, such as T cells, B cells, myeloid cells and the like. PD-L1 is a transmembrane protein that is normally expressed on the surface of antigen presenting cells and tumor cells. PD-1 can be specifically combined with PD-L1 to promote tyrosine in ITSM structural domain of PD-1 to be phosphorylated, so that downstream protein kinase Syk and PI3K are dephosphorylated, activation of signal paths such as AKT, ERK and the like is further inhibited, and finally, the effect of negatively regulating T cell activity is exerted, so that tumor cells can generate immune escape. Thus, blocking the PD-1/PD-L1 pathway will enhance T cell responses to tumor cells.

The existing targeted PD-1/PD-L1 clinically used medicines are monoclonal antibodies (mAbs), and the mAb-based targeted preparation has the defect of biomacromolecules: only for fast growing tumors, not for tumors in slow-progressing stages; the oral bioavailability is low, poor permeability leads to uneven distribution in tumor tissues, and the cell membrane cannot be penetrated to act on the PD-1/PD-L1 intracellular domain; (macromolecular drugs are difficult to clear rapidly, longer in vivo half-life is easy to induce fatal autoimmune attacks such as colonitis, hepatitis, myocarditis and the like, monoclonal antibodies are difficult to prepare, expensive in price, inconvenient to store and transport and limit the application in clinic, and small-molecule compounds have remarkable advantages in solving the problems that the immunogenicity side effect is small, the small-molecule compounds can be directly combined with a plurality of functional domains in protein cells, outside cells and on membranes, the pharmacodynamic and pharmacokinetic (PK/PD) process is easy to accurately control, adverse events related to drug-induced immunity are avoided, the preparation method is more suitable for oral administration, the production management cost is low, and the clinical treatment in future is facilitated.

In the recent development of small molecule compounds at home and abroad, small molecule compounds directly aiming at PD-1/PD-L1 interaction, including sulfonamides, biphenyls, heterocycles and the like, have been widely studied in pre-clinical models of cancers in vitro and in vivo. However, to date, no PD-1/PD-L1 small molecule compound has been approved for the market. Therefore, in order to meet the clinical unmet demand, development of small molecule compounds having novel backbones is desired to be able to be applied clinically. The natural product has wide source, is produced in the evolution process of millions of years, has unique structure, various biological activities and good biocompatibility, and can be matched with the space requirements of various targets of human bodies, so that the natural product is a treasury for blocking the discovery of PD-1/PD-L1 small molecule drugs.

The project is based on the crystal structure (5J 89) of the humanized PD-L1protein, combines with computer virtual screening to find the active small molecule (2R, 3R,4S,5S, 6R) -2- [ (E) -3-phenylprop-2-yloxy ] -6- [ [ (2S, 3R,4S, 5S) -3,4, 5-trihydroxyan-2-yl ] oxyethyl ] oxy-3, 4, 5-diol of the targeting PD-L1 immune check point with a brand-new framework structure, further proves that the targeting PD-L1 immune check point can be combined with the PD-L1protein, blocks the interaction of PD-L1 and PD-1, shows good tumor growth inhibiting effect in vivo, and does not find obvious toxic and side effects, and can be used as an immune check point small molecule compound with potential application value.

Disclosure of Invention

The invention aims at analyzing a composite crystal structure (5J 89) formed by PD-L1protein and a small molecular compound BMS-202 thereof, and taking the composite crystal structure as a template and utilizingThe software performs high-throughput virtual screening, and screens out a dominant small molecular compound which can be combined with PD-L1protein to play an anti-tumor activity from a natural product monomer compound library. In vivo and in vitro experiments prove that the small molecular compound can block the interaction of PD-1 and PD-L1, promote T cell activation, enhance the killing function of T cells on tumor cells, further effectively inhibit tumor growth and bring new hope for immune checkpoint treatment.

The small molecular compound with PD-1/PD-L1 inhibitory activity related by the invention, (2R, 3R,4S,5S, 6R) -2- [ (E) -3-phenylprop-2-enoxy ] -6 [ (2S, 3R,4S, 5S) -3,4, 5-trihydroxyan-2-yl ] oxyethyl ] oxy-3, 4,5-triol has the following structural formula:

the invention provides any application of the PD-1/PD-L1 small molecular compound or the inhibitor pharmaceutical composition, such as the application of the PD-1/PD-L1 small molecular compound or the inhibitor pharmaceutical composition in preparing medicines for enhancing the effect of antitumor medicines, the application of the PD-L1 small molecular compound or the inhibitor pharmaceutical composition in preparing medicines for enhancing the blocking effect on PD-L1 sites and the like.

The small molecular compound can be used as an active ingredient and a pharmaceutically acceptable carrier and/or excipient thereof to prepare antitumor small molecular medicines in any dosage form suitable for human or animals, such as oral pharmaceutical preparations, injections and the like.

The small molecule compound has a wide anti-tumor spectrum, and is suitable for various tumors including colon cancer, melanoma, lung cancer, breast cancer and the like.

The small molecular compound has no toxic or side effect on organisms.

Drawings

FIG. 1 is a graph showing MST identification of the affinity of small molecule compounds to PD-L1 proteins;

FIG. 2 is a graph of HTRF corroborating the blocking effect of small molecule compounds on PD-1/PD-L1;

FIG. 3 is a graph depicting the effect of IF on binding of a small molecule compound to hPD-L1B 16F10 cell surface rhPD-1 Fc;

FIG. 4 is a graph showing the effect of crystal violet staining on detection of small molecule compounds on T cell mediated killing effects of A375 melanoma cells;

FIG. 5 is a graph of the effect of a small molecule compound on tumor-bearing growth of hPD-L1 MC38, hPD-L1B 16F10 evaluated in humanized PD-L1 mice;

FIG. 6 is a graph showing the effect of flow-through detection of small molecule compounds on tumor-infiltrating T lymphocyte function in hPD-L1 MC38 colon cancer mice;

FIG. 7 is a graph showing the effect of H & E on mice on viscera.

Detailed Description

The invention provides a potential PD-L1 immune checkpoint small molecule compound for tumor immunotherapy, which is described in further detail below in connection with the specific embodiments of the invention:

the applicant takes a compound crystal structure 5J89 formed by hPD-L1 protein and BMS-202 small molecular compound as a template, usesThe software realizes the high-flux virtual screening of the Chinese natural product monomer compound library, through HTVS coarse screening, SP standard precision screening and XP ultra-high precision screening, experimental verification is carried out according to the binding energy selection dominant structure of the hit structure, and finally the small molecular compound (small molecular compound, no. 9294) has better activity of blocking PD-1/PD-L1 binding, and shows obvious effect of inhibiting tumor growth in vivo. The applicant entrusts to the synthesis of the small molecule compound by the Duremifene biotechnology limited company and applies the compound to in-vitro and in-vivo experimental verification.

Example 1: identification of affinity of small molecule Compounds to PD-L1 proteins by micro-thermophoresis (MST)

1.1 Experimental drugs and Primary reagent consumables

Small molecule compounds (Chengdoremifene biotechnology Co., ltd., lot RFS-L02411802020), DMSO (Sigma, lot D4540), PD-L1protein (Novoprotein, accession number C315), PBST (manufactured by labs), RED-tris-NTA His Tag 2nd Generation dye (Nano template, cat# NT-L118), MO NT.115 capillary (Nano template, cat# MO-K022).

1.2 major instrumentation

Electronic balance (Sidoris, germany), MST microphoresis apparatus (Nano template).

1.3 Experimental methods

First screening for dye concentration binding to PD-L1 protein: preparing 16 PCR centrifuge tubes, adding 10 mu L of PBST into the 2 nd-16 th centrifuge tubes, adding 20uL of 4 mu M His-Tagged PD-L1protein into the 1 st centrifuge tube, transferring 10 mu L of protein solution into the 1 st centrifuge tube to mix into the 2nd centrifuge tube, lightly blowing 4-6 times, transferring 10 mu L of protein solution from the 2nd centrifuge tube to mix into the 3 rd centrifuge tube, removing 10 mu L of protein solution from the 16 th centrifuge tube, adding 10 mu L of 50nM dye into the 1-16 th centrifuge tube, mixing and lightly blowing. Kd value is less than or equal to 10nM, 100 μL of 100nM dye, 100 μL of 200nM His-Tagged PD-L1protein are prepared, 90 μL of PD-L1protein and 90 μL of dye are uniformly mixed, incubated for 30min at room temperature, centrifuged at 12000rpm/min for 10min at 4 ℃, and transferred to a new centrifuge tube. Finally, drug and protein binding assays are performed: preparing small molecular compounds with the final concentration of 0.4mM, preparing 16 PCR centrifuge tubes, adding 20 mu L of small molecular compounds into a No. 1 centrifuge tube, adding 10 mu L of PBST into a No. 2-16 centrifuge tube, transferring 10 mu L of liquid into a No. 1 centrifuge tube to mix into a No. 2 centrifuge tube, lightly blowing 4-6 times, transferring 10 mu L of liquid from a No. 2 centrifuge tube to mix into a No. 3 centrifuge tube, removing 10 mu L of liquid from a No. 4-16 centrifuge tube, adding 10 mu L of 200nM His-Tagged PD-L1protein into a No. 1-16 centrifuge tube, mixing and lightly blowing uniformly, and finally sucking the mixed liquid into a Monolith NT.115pico micro-thermophoresis instrument by using a capillary tube for detection, as shown in FIG. 1.

1.4 conclusion of results

The results show that: as the concentration of the small molecule compound increases, MST fluorescent signals are amplified, and the affinity Kd value of the small molecule compound and PD-L1 is 1.1 mu M.

Conclusion: the small molecule compound has better combination with PD-L1 protein.

Example 2: homogeneous Time Resolved Fluorescence (HTRF) corroborating the blocking effect of small molecule compounds on PD-1/PD-L1

2.1 Experimental drugs and Primary reagent consumables

Small molecule compounds (Chengdoremifene biotechnology Co., ltd., lot RFS-L02411802020), DMSO (Sigma, lot D4540), PD-1/PD-L1 Binding kit (Cisbio, cat# 64PD1 PEG), 384-microwell plates (Perkinelmer, cat# 6008280).

2.2 major instrumentation

Electronic balance (Sidoris, germany) and enzyme labelling instrument (Perkinelmer).

2.3 Experimental methods

Preparing a small molecular compound mother solution with the concentration of 10mM by using DMSO, taking out the PD-1/PD-L1 binding detection kit, balancing to room temperature, and gradually diluting the small molecular compound by using PPI Eu detection buffer solution to obtain the final concentrations of 250, 50, 10, 2, 0.4, 0.08, 0.016 and 0.0032 mu M respectively. The experiment is provided with a Buffer control group, a negative control group, a positive control group and small molecule compound sample groups with different concentrations, each group is provided with 3 compound holes, and all hole systems are 20 mu L. In 384-microwell plates, sample groups were added with 2. Mu.L of the above concentrations of small molecule compound, 4. Mu.L of Tag1-PD-L1, 4. Mu.L of Tag2-PD-1, 5. Mu.L of Anti-Tag1 Eu Cryptate reagent and 5. Mu.LAnti-Tag 2 xl665 Anti-ibody, respectively; the Buffer control group was added with only 20 μl PPI Eu detection Buffer; the negative control group was not added with compound and Tag2-PD-1, and 6. Mu.L of PPI Eu detection buffer was added; the positive control group was not added with compound, and 2. Mu.L of PPI Eu detection buffer was added. After the solutions are uniformly mixed, the room temperature light is cut off for 1 hour, and the use is carried outThe microplate reader reads fluorescence values at 665nm and 620nm wavelengths, see FIG. 2.

2.4 conclusion of results

The results show that: IC for inhibiting PD-1/PD-L1 protein binding by small molecule compound ₅₀ 4.448. Mu.M.

Conclusion: the protein level proves that the small molecular compound can inhibit the combination of PD-1/PD-L1 proteins.

Example 3: immunofluorescence (IF) examination of the effect of small molecule compounds on the binding of hPD-L1B 16F10 cell surface rhPD-1Fc

3.1 cells

hPD-L1B 16F10 (constructed by the present laboratory).

3.2 Experimental drugs and Primary reagent consumables

Small molecule compounds (Chengdoremi-biosciences, inc., lot number RFS-L02411802020), DMSO (Sigma, lot number D4540), RPMI-1640 (Gibco, cat number 31800022), FBS (MULYICELL, cat number 073150003), PBS (laboratory self-made), trypsin (BIOFROX, cat number 1004GR 100), recombinant human PD-1Fc chimeric proteins (R & D system, cat number 1086-PD-050), 4% paraformaldehyde universal fixative (Biosharp, cat number 1810898), BSA (Biosharp; cat number 738328), PD-1 monoclonal antibodies (Proteintech, cat number 66220-1-Ig), goat anti-mouse fluorescent secondary antibodies (Abcam, ab 150115), hoechst (cloud days, cat number 33258), anti-fluorescent sealing tablets (Bifluorescent days, cat number P0126), 6 (NEST, cat number 703001), cell plates (NEST, cat number 801009).

3.3 major instrumentation

Electronic balance (Sidoris, germany), pure water meter (Nanjing Han Long laboratory apparatus Co., ltd.), ultra clean bench (ESCO BIOTECH), CO ₂ Incubator (Thermo Scientific), inverted light microscope (ZEISS).

3.4 Experimental methods

Placing the cell climbing sheet in 6-well plate, collecting hPD-L1B 16F10 cells growing logarithmically, digesting with pancreatin, inoculating into 6-well plate, and inoculating into 1×10 cells per well ⁵ And (3) interfering with each cell by adding 0, 6, 12 and 24 mu M small molecular compound for 24 hours after the cell is attached, wherein each concentration is provided with 3 compound holes, and then 5 mu g/mL recombinant human PD-1Fc chimeric protein is added into each hole at room temperatureIncubate for 30min, discard medium, wash 3 times with PBS, fix with 4% paraformaldehyde, block with 1.5% bsa, incubate with primary antibody, hoechst stain, anti-fluorescence quencher seal and observe and shoot using an inverted fluorescence microscope, see fig. 3.

3.5 conclusion of results

The results show that: compared with the control group, the binding of the rhPD-1Fc protein on the surface of the cell membrane of hPD-L1B 16F10 in the small molecule compound treatment group with different concentrations is obviously reduced, and the fluorescence intensity is obviously reduced.

Conclusion: the blocking effect of the small molecule compound on the PD-1/PD-L1 protein is proved on the cell level.

Example 4: crystal violet staining to detect effect of small molecule compounds on T cell mediated killing effect of A375 melanoma cells

4.1 cells

A375/PD-L1 ^hi (constructed by the present laboratory), jurkat.

4.2 Experimental drugs and Primary reagent consumables

Small molecule compounds (Chenoporen biosciences, inc., lot number RFS-L02411802020), DMSO (Sigma, lot number D4540), RPMI-DMEM (Gibco, cat# 12100-046), FBS (MULYICELL, cat# 073150003), PBS (laboratory self-made), trypsin (BIOFROX, cat# 1004GR 100), CD3 antibodies (BioLegend, cat# 300402), CD28 antibodies (BioLegend, cat# 302902), crystal violet staining solution (Biyun, cat# C0121), 12 well plates (NEST, cat# 712001).

4.3 major instrumentation

Electronic balance (Sidoris, germany), pure water meter (Nanjing Han Long laboratory apparatus Co., ltd.), ultra clean bench (ESCO BIOTECH), CO ₂ Incubator (Thermo Scientific), all microscope (Leica).

4.4 Experimental methods

Pretreatment of A375/PD-L1 with small molecule Compounds at 0, 6, 12, 24. Mu.M, respectively ^hi Cells were plated in 12 well plates with 3 wells per concentration for 24h, trypsinized and counted, inoculated in 2X 10 wells ⁴ Cells, adherent overnight. Using 100ng/mL CD3 antibody and 100ng/mL CD28 antibody activated Jurkat cells and was associated with pretreated A375/PD-L1 ^hi Cells were individually targeted at an effective target ratio of 10: 1. 20:1 co-culture for 72h. Residual survival A375/PD-L1 ^hi Cells were stained with crystal violet, photographed with a microscope and quantified by scanning gray scale, see table 1 and fig. 4.

4.5 conclusion of results

TABLE 1 Crystal Violet staining to detect Effect of small molecule Compounds on T cell mediated killing of A375 melanoma cells

Note that: * Indicating that P <0.05, the difference was statistically significant compared to the Control group.

The results show that: crystal violet staining results showed that activated Jurkat cells with a 10:1 or 20:1 effective target ratio to a375/PD-L1 ^hi The melanoma cells are co-cultured, and the pretreatment of the small molecule compound effectively reduces the number of surviving melanoma cells.

Conclusion: the small molecule compounds are capable of enhancing T cell mediated tumor killing effects.

Example 5: humanized PD-L1 mice evaluate the Effect of small molecule Compounds on tumor-bearing growth of hPD-L1 MC38, hPD-L1B 16F10

5.1 cells

hPD-L1 MC38 (constructed by the present laboratory), hPD-L1B 16F10 (constructed by the present laboratory).

5.2 animals

SPF-grade male C57BL/6/JGpt-Pdl1 ^tm1(hPDL1) 56 mice per Gpt, weight 18-22 g, provided by Jiangsu Jiuyaokang biotechnology Co., ltd., production license number: SCXK 2018-0008.

5.3 Experimental drugs and Primary reagent consumables

Small molecule compound (Chengdoremifant Biotechnology Co., ltd., lot RFS-L02411802020), CMC-Na (Sigma-Aldrich, cat# C4888), PD-L1 mab (BioXcell, cat# BE 0285), PBS (manufactured by labs).

5.4 Main instruments

Electronic scale (mertrer tolidol), vernier caliper (zebra equipment company).

5.5 animal model establishment

hPD-L1 MC38 colon cancer cells and hPD-L1B 16F10 melanoma cells were resuspended in PBS to adjust the cell concentration to 1X 10 ⁶ /mL. Inoculating 200 μl of cell suspension into right inguinal region of humanized PD-L1 mouse, and until tumor volume reaches 50-100mm ³ At this time, model mice were randomly divided into 4 groups (n=7) including: negative control (Vehicle, 0.3% CMC-Na), positive control (PD-L1 Ab,200 ug/min), small molecule compound treated group (25 mg/kg,100 mg/kg). PD-L1 Ab group was intraperitoneally injected once every 3 days, and the other groups were administered by intragastric administration once daily for 3 weeks, and the body weight was weighed once every 3 days during the administration, and the tumor volume was measured and calculated (tumor volume=1/2×a×b ² A is the major diameter of the tumor and b is the minor diameter of the tumor), and a tumor growth curve is plotted. Mice were sacrificed 21 days on treatment, tumor tissue and organ tissue were dissected and weighed and saved as needed for subsequent experiments, see table 2 and fig. 5.

5.7 evaluation index

Tumor growth curve, tumor volume, tumor weight.

5.8 statistical methods

Experimental data were analyzed using SPSS software (version 20.00), all data expressed in SD or SEM. Group difference analysis was performed using Prism 9.0 (Graph Pad Software Inc, san Diego, CA). The difference of P <0.05 is statistically significant.

5.9 conclusion of results

TABLE 2 humanized PD-L1 mice evaluation of Small molecule Compounds effects on tumor-bearing growth of hPD-L1 MC38, hPD-L1B 16F10

Note that: * Indicating that P <0.05, the difference was statistically significant compared to the veccle group.

The results show that: in a hPD-L1 MC38 colon cancer subcutaneous transplantation tumor model, 25mg/kg and 100mg/kg of small molecular compounds are respectively and continuously administered to mice by gastric lavage for 21 days, so that the growth of hPD-L1 MC38 tumors can be obviously inhibited, the tumor weight is obviously lower than that of a Vehicle group, and the drug effect is equivalent to that of a PD-L1 monoclonal antibody group. In the model of hPD-L1B 16F10 melanoma subcutaneous transplantation tumor, 25mg/kg and 100mg/kg of small molecular compounds have remarkable inhibition effect on hPD-L1B 16F10 tumors.

Conclusion: the small molecule compound shows good effect of inhibiting tumor growth in a colon cancer and melanoma mouse model.

Example 6: flow detection of influence of small molecular compounds on tumor infiltration T lymphocyte function of hPD-L1 MC38 colon cancer mice

6.1 Experimental drugs and Primary reagent consumables

FBS (MULYICELL, cat# 073150003), EDTA (Sigma-Aldrich, cat# 798681), PBS (laboratory self-made), RPMI-1640 (Gibco, cat# 31800022), collagenase IV (Sigma-Aldrich, cat# C4-22), DNase I (Soilebao, D8071), red blood cell lysate (Soilebao, cat# R1010), CD3 streaming antibody (BioLegend, 100204), CD8 streaming antibody (Proteintech, APC-65069), IFNγ streaming antibody (Invitrogen, cat# 12-7311-82), granzyme B streaming antibody (Invitrogen, cat# 12-8898-80), perforin streaming antibody (Invitrogen, cat# 12-9392-82).

6.2 Main instruments

CytoFLEX flow cytometer (Beckman).

6.3 Experimental methods

Placing the removed hPD-L1 MC38 tumor tissues in a dish, washing 5 tumor tissues in each group by PBS, transferring the tumor tissues into 2mL of digestive juice, shearing the tumor tissues by scissors, placing the tumor and the digestive juice into a 15mL EP tube together, adding 5mL of digestive juice, digesting the tumor tissues by a shaker at 37 ℃ for 45min, and blowing the tumor tissues by a gun every 10min in the digestion process. After digestion was complete, digestion was terminated by adding a pre-chilled equal volume 1640 of complete medium and filtered through a 70 μm nylon sun screen into a new 15mL EP tube. Centrifuging at 4 ℃ at 1000rpm/min for 5min, discarding the supernatant, adding 3mL of erythrocyte lysate to lyse erythrocytes, centrifuging again, discarding the supernatant, and washing once by using FACS-EDTA to obtain tumor-infiltrating lymphocytes. Each sample was equally divided into 3 aliquots using PBS, 100 μl of cell suspension was added to each glass flow tube, and appropriate amounts of FITC anti-mouse CD3, APC anti-mouse CD8, and PE anti-mouse ifnγ, PE anti-mouse Granzyme B, PE anti-mouse performin were added according to the instructions, incubated on ice for 45min, PBS was washed 2 times, and cells were resuspended using FACS buffer for detection using flow cytometry, see table 3 and fig. 6.

6.4 conclusion of results

TABLE 3 flow assay of the Effect of small molecule Compounds on tumor-infiltrating T lymphocyte function in hPD-L1 MC38 colon cancer mice

The results show that: the activated T cells can secrete pro-inflammatory cytokines, release Granzyme B and Perforin, so that tumor cells are killed, and the IFN-gamma, granzyme B and the proportion of Perforin positive CD8+T cells infiltrated in tumor tissues of hPD-L1 MC38 colon cancer mice can be obviously increased by 25mg/kg and 100mg/kg of small molecular compounds.

Conclusion: the small molecular compound can obviously enhance the function of hPD-L1 MC38 colon cancer mouse tumor infiltration CD8+ T lymphocytes.

Example 7: evaluation of the Effect of small molecule Compounds on the organs of mice by H & E

7.1 Experimental drugs and Primary reagent consumables

4% paraformaldehyde universal tissue fixative (Biosharp, cat# 1810898), paraffin (national pharmaceutical systems and chemicals, cat# 60019562), xylene (national pharmaceutical systems and chemicals, cat# 20170801), absolute ethanol (Nanjing systems and chemicals, cat# 180711446K), hematoxylin and eosin staining kit (Biyun, cat# C0105), neutral resin gel (Biosharp, cat# 88671250), glass slide (CITOGLAS, cat# 18038), cover slip (CITOGLAS, cat# 10212450C).

7.2 Main instruments

Tissue embedding machine (Leica), paraffin microtome (Leica), multifunctional pathology imager (PE).

7.3 Experimental methods

Taking 4% paraformaldehyde fixed organ tissue blocks (liver, lung and spleen) of the mice, embedding conventional paraffin, slicing into 4 mu m slices, dewaxing the slices by using xylene, and washing the slices by using ethanol of various levels until the slices are washed with water: xylene (I) 5 min- & gt xylene (II) 5 min- & gt 100% ethanol 2 min- & gt 95% ethanol 1 min- & gt 80% ethanol 1 min- & gt 75% ethanol 1 min- & gt distilled water washing 2min, hematoxylin dyeing 5min, tap water washing, hydrochloric acid ethanol differentiation 30s, tap water soaking 15min, eosin solution 2min, conventional dehydration, transparency and sealing: 95% ethanol (I) min, 95% ethanol (II) 1min, 100% ethanol (I) 1min, 100% ethanol (II) 1min, xylene (I) 1min, xylene (II) 1min and neutral resin sealing. The pathology was observed under a pathology imager and analyzed by photographing, see fig. 7.

7.4 conclusion of results

The results show that: the H & E staining results showed that the organs tissues of mice in the groups to which 25mg/kg and 100mg/kg of the small molecule compound were administered were not abnormal compared with those of normal mice (liver, lung, spleen).

Conclusion: the small molecular compound has no obvious toxic or side effect on the organs of mice.

Claims

1. The application of a small molecular compound with PD-1/PD-L1 inhibiting activity in preparing an anti-tumor medicament, wherein the tumor is one of colon cancer and melanoma, and the structure is as follows:

。

2. the use of a small molecular compound having an inhibitory activity against PD-1/PD-L1 as claimed in claim 1, wherein the molecular weight of the small molecular compound is 428.43Da.

3. The use of a small molecule compound having PD-1/PD-L1 inhibitory activity according to claim 1 for the manufacture of an antitumor agent, wherein said antitumor effect is PD-1/PD-L1 immunobinding.

4. The use of a small molecular compound with PD-1/PD-L1 inhibiting activity according to claim 1 for the preparation of an antitumor drug, wherein the small molecular compound is formulated with pharmaceutically acceptable excipients into a pharmaceutically acceptable dosage form.