Application of pyrimidine biphenyl derivative

Abstract

The invention provides application of a pyrimidine biphenyl derivative, the structural general formula of the pyrimidine biphenyl derivative is # imgabs0 #, and the application comprises but is not limited to application in preparation of a PD-1 / PD-L1 small-molecule inhibitor for preventing or treating targeted PD-1 / PD-L1, and application in preparation of a PD-1 / PD-L1 small-molecule inhibitor for preventing or treating targeted PD-1 / PD-L1 small-molecule inhibitor for preventing or treating targeted PD-1 / PD-L1. And / or the application in preparation of a double-target bifunctional molecular drug for preventing or treating KRAS and PD-1 / PD-L1.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to an application of a pyrimidine biphenyl derivative. Background Art

[0002] In the field of biopharmaceuticals, the immune system plays a vital role in maintaining the health of the body, and immune checkpoints are the key link in the precise regulation of immune responses. As a pair of key immune checkpoint molecules, programmed death receptor 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) have become a research hotspot in recent years. PD-1 is mainly expressed on the surface of activated immune cells such as T cells, B cells, and natural killer cells (NK cells), while PD-L1 is widely present on tumor cells, antigen-presenting cells, and a variety of normal tissue cells. After the two are combined, they can transmit inhibitory signals to T cells, just like stepping on the "activation brake" of immune cells, effectively preventing excessive activation of the immune system from causing damage to the body's normal tissues, and playing a core role in maintaining autoimmune tolerance.

[0003] However, tumor cells cleverly use this mechanism to abnormally overexpress PD-L1, which binds to PD-1 on the surface of T cells, inhibits the anti-tumor activity of T cells, and helps tumor cells achieve immune escape. This mechanism plays a key role in promoting the occurrence, development, and metastasis of tumors. According to statistics, high expression of PD-L1 is closely related to poor prognosis in various cancers such as melanoma, lung cancer, and kidney cancer.

[0004] Compared with monoclonal antibody drugs, the development of small molecule inhibitors targeting PD-1 / PD-L1 faces many challenges. First, the protein-protein interaction interface between PD-1 and PD-L1 is relatively flat and wide, with a large contact area. It lacks deep pocket-like binding sites such as those commonly seen in enzyme inhibitor targets, making it difficult for small molecules to accurately and stably anchor and bind, and it is very easy to dissociate in a complex intracellular environment, affecting the inhibitory effect. Secondly, small molecules need to cross the cell membrane to enter the cell to exert their effects, which requires them to have a suitable balance of lipophilicity and hydrophilicity. If the lipophilicity is too strong, it may lead to poor solubility and accumulation of toxicity in the body; conversely, excessive hydrophilicity will hinder cell membrane penetration and fail to effectively reach the target. In addition, there are a large number of endogenous substances in the cell that compete for binding sites. Small molecule inhibitors must not only recognize PD-1 / PD-L1 with high specificity, but also avoid "molecular melee" and accurately block protein interactions in a crowded cell microenvironment, which places extremely high demands on the innovation and accuracy of molecular design, and become a "stumbling block" on the road to the development of small molecule inhibitors.

[0005] Despite the difficulties, researchers are still forging ahead in the field of small molecule inhibitors and have made phased breakthroughs. A class of biphenyl derivative small molecule inhibitors developed by Bristol-Myers Squibb (BMS) shows potential, such as BMS-202, but has not survived the clinical trial stage due to its strong toxicity. If this type of small molecule inhibitor is successfully developed, it will have unique advantages over monoclonal antibody drugs. On the one hand, small molecules can usually be administered orally, and patients have higher compliance, get rid of the inconvenience of intravenous infusion of monoclonal antibody drugs, and reduce medical costs and time costs; on the other hand, the chemical synthesis process of small molecules is relatively mature and easy to mass produce, which is expected to significantly reduce the cost of drug manufacturing, benefit more patients, add new members to the immunotherapy drug family, expand clinical application scenarios, and help the global medical accessibility process.

[0006] In summary, although the development of small molecule inhibitors targeting PD-1 / PD-L1 faces many challenges, its potential advantages in the field of immunotherapy cannot be ignored. Therefore, it is of great significance to design and develop an efficient and stable small molecule inhibitor targeting PD-1 / PD-L1. Summary of the invention

[0007] The applicant of the present invention has discovered through research that a class of pyrimidine biphenyl derivative small molecule inhibitors with clear anti-tumor activity against KRAS also show therapeutic potential for PD-1 / PD-L1, and it has been preliminarily verified in multiple basic activity experiments that they can inhibit PD-1 / PD-L1 signal transduction. Therefore, the first object of the present invention is to provide a use of a pyrimidine biphenyl derivative in the preparation of a drug for preventing or treating the inhibition of PD-1 / PD-L1 signal transduction. The second object of the present invention is to provide a use of a pyrimidine biphenyl derivative in the preparation of a dual-target bifunctional molecular drug for the prevention or treatment of KRAS and PD-1 / PD-L1, providing a new design perspective and reference value for the current research and development of anti-tumor drugs.

[0008] To achieve the above object, the present invention adopts the following technical solution:

[0009] As a first aspect of the present invention, a use of a pyrimidine biphenyl derivative includes but is not limited to:

[0010] Application in the preparation of small molecule inhibitors targeting PD-1 / PD-L1 for prevention or treatment; and / or,

[0011] Application in the preparation of dual-functional molecular drugs for the prevention or treatment of dual targets of KRAS and PD-1 / PD-L1;

[0012] Wherein, the structural formula of the pyrimidine biphenyl derivative is as shown in Formula I:

[0013]

[0014] The R 1 One selected from the group consisting of methyl, deuterated methyl and fluoromethyl;

[0015] The R 2 One selected from the group consisting of hydrogen, deuterium, halogen, alkyl and alkoxy;

[0016] The R 3 , R 4 , R 6 , R 7 , R 9 , R 10 , R 12 and R 13 is independently selected from one of hydrogen, deuterium, halogen, hydroxyl, amine, cyano, sulfonamide, C1-C3 alkyl and C1-C3 alkoxy;

[0017] The R 5 One selected from 4-7 membered cyclic amine, 4-7 membered heterocyclic amine and sulfonyl, wherein the 4-7 membered cyclic amine, 4-7 membered heterocyclic amine and sulfonyl independently contain 1, 2 or 3 R a Substituents;

[0018] The R 8 One selected from hydrogen, deuterium and C1-C6 alkyl;

[0019] The R 11 Selected from -N(R b )C(=O)R c 、-N(R b )S(=O)2R c 、-C(=O)NR b R c and -S(=O)2NR b R c One of;

[0020] Among them, R a One selected from hydrogen, deuterium, halogen, hydroxyl, amine, cyano, sulfonamide, C1-C6 alkyl and C1-C6 alkoxy;

[0021] Rb is selected from one of hydrogen, deuterium and C1-C6 alkyl;

[0022] R c One selected from C1-C8 alkyl, phenyl and 5-6 membered heteroaryl, wherein C1-C8 alkyl, phenyl and 5-6 membered heteroaryl independently contain 1, 2 or 3 R a Substituent.

[0023] According to the present invention, the biphenyl derivative is used in the preparation of an anti-tumor drug for preventing or treating the inhibition of PD-1 / PD-L1 signal transduction.

[0024] According to the present invention, the pyrimidine biphenyl derivative is a PM-1 compound, and its structural formula is shown in Formula II:

[0025]

[0026] The beneficial effect of the present invention is that the pyrimidine biphenyl derivatives have a good inhibitory effect on PD-1 / PD-L1, and can be used in the preparation of drugs for preventing and treating the inhibition of PD-1 / PD-L1 signal transduction, small molecule inhibitors targeting PD-1 / PD-L1, and dual-functional molecular drugs with dual targets of KRAS and PD-1 / PD-L1. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 A in the figure is a comparison of the SERS spectra of the silver magnetic probe mixture after being treated with gradient concentrations of XL-1063 (0-500 μg / mL). Figure 1 B in FIG. 1 is a histogram drawn at the location where the SERS intensity is maximum.

[0029] Figure 2 This is the result of the in vivo inhibition experiment of XL-1063 on BxPC-3 cell line. Figure 2 a in the figure is the tumor tissue photograph obtained from each group; Figure 2 b in the figure is the curve of tumor volume change over time in each group; Figure 2 c in the figure is the volume of tumor tissue in each group; Figure 2 The d in the figure is the curve of animal weight change over time; Figure 2 e in is the body weight of animals in each group.

[0030] Figure 3 The results of the in vivo inhibition experiment of XL-1063 on MIApaca-2 cell line; Figure 3 A in the figure is the tumor tissue photos obtained from each group; Figure 3 B in the figure is the curve of the volume change of tumor tissue in each group over time; Figure 3 C in the figure is the volume statistics of tumor tissues in each group; Figure 3 D in the figure is the curve of weight change of animals in each group over time; Figure 3 E in the figure is the statistical chart of the weight of animals in each group; Figure 3 F in the figure is the statistical chart of tumor tissue weight in each group.

[0031] Figure 4 H&E and immunohistochemical analysis of BxPC-3 tumor tissue sections.

[0032] Figure 5 H&E and immunohistochemical analysis of MIApaca-2 tumor tissue sections.

[0033] Figure 6 Diagram showing the grouping setup for the ELISA experiment for PM-1 drug.

[0034] Figure 7 The ELISA system is used to verify the binding inhibition ability of the small molecules screened; among them, the figure legends: A is the blank control group of r-huPD+r-huPD-L1; B is r-huPD-1+r-huPD-L1+BMS-202; C is r-huPD-1+r-huPD-L1+PM-1.

[0035] Figure 8 This is a schematic diagram of the PD-1 / PD-L1 molecule blocking experiment.

[0036] Fig. 9 This is the blocking activity dose-effect curve of PM-1 and positive drug BMS-202.

[0037] Fig.10 It is the killing rate of active tumor cells in the Jurkat+HuH cell co-culture system.

[0038] Fig.11 It is the killing rate of active tumor cells in the Jurkat+Hela cell line co-culture system. DETAILED DESCRIPTION

[0039] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0040] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0041] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0042] The following will combine the examples and experimental data to describe the application of a pyrimidine biphenyl derivative as shown in Formula II. It mainly includes the activity verification of drugs targeting PD-1 / PD-L1, PD-1 / PD-L1 blocking reporter gene experiments, and the binding of PD-1 and PD-L1 under the tumor microenvironment. The preparation method of the pyrimidine biphenyl derivative of the present invention can be prepared by referring to the Chinese patent document CN118994031A.

[0043]

[0044]

[0045] Experimental Example 1

[0046] To evaluate the selective inhibitory ability of the novel pyrimidine biphenyl derivatives in an in vitro human pancreatic cancer cell model, we performed inhibition experiments in MIA paca-2 and BXPC-3 tumor cells.

[0047] Cells used in the experiment: human pancreatic ductal adenocarcinoma (PDAC) cell line (MIApaca-2 cells) that depends on the oncogenic Kras gene for survival and a cell line expressing the wild-type Kras gene (BxPC-3 cells).

[0048] Experimental method: MIA paca-2 cells and BxPC-3 cells in the logarithmic growth phase were prepared into 1×10 4 pc·mL -1 100 μL of cell suspension was inoculated into a 96-well plate in each well, and cultured at 37°C for 48 hours with 5% CO2 until the bottom was completely covered. The supernatant was removed, and 200 μL of gradient concentrations of drug-containing culture medium (the compound storage solution dissolved in DMSO was diluted to the required concentration with complete culture medium, where the final concentration of DMSO was 0.1% and the final concentration of serum was 2%) was added to each well. Three replicate wells were set for each concentration, and blank control wells (no cells, solvent control group) and negative control wells (cells and equal volume of solvent) were set up, and three replicate wells were set up. After adding the drug, the culture was continued for 48 hours. After terminating the culture, the fresh culture medium was replaced (the culture medium was removed, and the cells were washed twice with the culture medium, and then new culture medium was added) to remove the influence of the drug. 10% CCK-8 solution was added to each well, and the color was developed at 37°C for 1 to 4 hours. During this period, the absorbance at 450nm of each well was detected every half an hour. The inhibition rate of the compound for 48 hours was calculated according to the following formula, and the half inhibition concentration (IC50) was calculated.

[0049] Cell viability (%) = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] × 100

[0050] A (drug added): absorbance of wells with cells, CCK-8 solution, and drug solution

[0051] A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells

[0052] A(0 drug addition): absorbance of the well with cells, CCK-8 solution and no drug solution

[0053] The specific experimental results are as follows:

[0054] The half inhibitory concentration (IC50) value of the pyrimidine biphenyl derivative represented by formula II on MIA PaCa-2 or BxPC-3 cells is less than 10 μM.

[0055] Although the half-maximal inhibitory concentration (IC50) value of the pyrimidine biphenyl derivative shown in formula II on MIA PaCa-2 or BxPC-3 cells is higher than 10 μM, it is lower than 100 μM and also has a certain inhibitory effect.

[0056] The pyrimidine biphenyl derivatives shown in formula II have better effects on MIA PaCa-2 cells than on BxPC-3 cells.

[0057] Experimental Example 2

[0058] In order to verify the ability of the compound XL-1063 (compound 2.1) with the strongest cell inhibitory ability to inhibit protein interaction, an immunoaffinity inhibition potency evaluation experiment was performed:

[0059] Experimental method: XL-1063 solution was diluted with buffer PBS to obtain the corresponding concentration gradient (0.5μg / mL, 5μg / mL, 50μg / mL, 500μg / mL). Raman detection was performed in parallel. 150μL of silver probe (unlabeled 4-MBA) was added to each well, and different concentrations of small molecule compound XL-1063 were added accordingly. Then 50μL of magnetic probe was added to each well, reacted for 1h, magnetically separated and washed 3 times, and resuspended with PBS buffer. 2μL of 4-MBA (1.0mmol / L) was added to each well for 1 hour, magnetically separated and washed 3 times, resuspended with PBS buffer and SERS detection was performed in situ after enrichment in an external magnetic field. During data processing, three batches of detection were performed for each small molecule, and each detection was measured in parallel for more than three times. After processing by NGSLabSpec software, the final spectrum data of each well was processed and plotted.

[0060] The specific process is as follows:

[0061] (1) Blank control: immunoaffinity structures of Kras4B and PDEδ.

[0062] The Raman probe was prepared before use. The silver probe and the magnetic probe were mixed in a 96-well plate at a volume ratio of 150 μL:50 μL and an equal amount of PBS buffer was added. After incubation for 60 minutes, in situ cleaning and magnetic aggregation were performed, and 2 μL 4-MBA (1.0 mM) was added to react for 1 hour. The sample was magnetically separated and washed 3 times, resuspended with PBS buffer and enriched in an external magnetic field for in situ SERS detection.

[0063] (2) Experimental group: evaluation of small molecule immunoaffinity inhibitory efficacy.

[0064] Accurately pipette 150 μL of silver probe and 50 μL of magnetic probe, add different concentrations of XL-1063 at the same time, incubate for 60 minutes, incubate the signal molecules and wash according to the above operation, and perform Raman signal detection in a 96-well plate.

[0065] The exposure time was set to 1 second and the number of exposures was set to 10 times and the average was taken.

[0066] The specific experimental results are as follows Figure 1 As shown, Figure 1 A in the figure is a comparison of the SERS spectra of the silver magnetic probe mixture after being treated with gradient concentrations of XL-1063 (0-500 μg / mL). Figure 1 B in the figure is a histogram at the point where the SERS intensity is maximum. It can be seen from the figure that the Raman signal gradually weakens as the concentration of XL-1063 increases, confirming that the small molecule XL-1063 can regularly inhibit the protein interaction between Kras4B and PDEδ, and is indeed a potential small molecule inhibitor.

[0067] Experimental Example 3

[0068] In order to verify the ability of XL-1063, the compound with the strongest cell inhibitory ability, to inhibit protein interaction, an immunoaffinity inhibition potency evaluation experiment was conducted. The experimental method is as follows:

[0069] The inhibitor solution was diluted with buffer PBS to obtain the corresponding concentration gradient (0.5μg / mL, 5μg / mL, 50μg / mL, 500μg / mL) of compound XL-1063 solution. Raman detection was performed in parallel. 150μL of silver probe (unlabeled 4-MBA) was added to each well, and different concentrations of small molecule compound XL-1063 were added accordingly. Then 50μL of magnetic probe was added to each well, reacted for 1h, magnetically separated and washed 3 times, and resuspended with PBS buffer. 2μL of 4-MBA (1.0mmol / L) was added to each well to react for 1 hour, magnetically separated and washed 3 times, resuspended with PBS buffer and SERS detection was performed in situ after enrichment in an external magnetic field. During data processing, three batches of detection were performed for each small molecule, and each detection was measured in parallel for more than three times. After processing with NGSLabSpec software, the final spectrum data of each well was processed and obtained. Figure 1 , Figure 1 a in the figure is a comparison of the SERS spectra of the silver magnetic probe mixture after being treated with gradient concentrations of XL-1063 (0-500 μg / mL). Figure 1 The b in the figure is the histogram at the location where the SERS intensity is maximum.

[0070] The specific process is as follows:

[0071] (1) Blank control: immunoaffinity structures of Kras4B and PDEδ.

[0072] The Raman probe was prepared before use. The silver probe and the magnetic probe were mixed in a 96-well plate at a volume ratio of 150 μL:50 μL and an equal amount of PBS buffer was added. After incubation for 60 minutes, in situ cleaning and magnetic aggregation were performed. 2 μL 4-MBA (1.0 mmol / L) was added to react for 1 hour, magnetically separated and washed 3 times, resuspended with PBS buffer and enriched in an external magnetic field for in situ SERS detection.

[0073] (2) Experimental group: evaluation of small molecule immunoaffinity inhibitory efficacy.

[0074] Accurately pipette 150 μL of silver probe and 50 μL of magnetic probe, add different concentrations of XL-1063 at the same time, incubate for 60 minutes, incubate the signal molecules and wash according to the above operation, and perform Raman signal detection in a 96-well plate.

[0075] The exposure time was set to 1 second and the number of exposures was set to 10 times and the average was taken.

[0076] The specific experimental results are as follows:

[0077] The Raman signal gradually weakened as the concentration of XL-1063 increased, confirming that the small molecule XL-1063 can regularly inhibit the protein interaction between Kras4B and PDEδ, and is indeed a potential small molecule inhibitor.

[0078] Experimental Example 4

[0079] Cells used in the experiment: human pancreatic ductal adenocarcinoma (PDAC) cell line (MIApaca-2) that depends on the oncogenic Kras gene for survival, and a cell line expressing the wild-type Kras gene (BxPC-3 cells).

[0080] To evaluate the antitumor activity of XL-1063, we established nude mouse models with human BXPC-3 and MIA paca-2 tumor cell xenografts.

[0081] Experimental method: Tumor cells were injected subcutaneously on the back of nude mice and the growth rate of the tumor was monitored. When the tumor size reached 125 mm 3After the size was measured, the BXPC-3 and MIA paca-2 model groups were randomly divided into five groups, with seven mice in each BXPC-3 (control group) and ten mice in each MIA paca-2 (experimental group). The detailed grouping is as follows:

[0082] BXPC-3 control group

[0083] (a) Model control group;

[0084] (b) Positive control group (15 mg / kg Deltarasin) QD ip;

[0085] (c) Model + low-dose drug (7.5 mg / kg XL-1063) QD ip;

[0086] (d) Model + medium dose drug (15 mg / kg XL-1063) QD ip;

[0087] (e) Model + high dose drug (20 mg / kg XL-1063) QD ip

[0088] MIA paca-2 experimental group

[0089] (a) Model control group;

[0090] (b) Positive control group (15 mg / kg Deltarasin) QD ip;

[0091] (c) Model + low-dose drug (3.75 mg / kg XL-1063) QD ip;

[0092] (d) Model + medium dose drug (7.5 mg / kg XL-1063) QD ip;

[0093] (e) Model + high dose drug (15 mg / kg XL-1063) QD ip

[0094] Mice were treated for 14 days (MIA Paca-2) and 21 days (BXPC-3). They were weighed and tumor size was measured every three to four days. Tumors were measured in two dimensions using a vernier caliper, and tumor volume was estimated based on the measurements using the following formula: tumor volume (TV) (mm 3 ) = [length (mm) × width (mm)2] / 2.

[0095] When the tumor volume reached 1000 cubic millimeters, the treatment of mice was stopped. The mice were euthanized the next day after drug withdrawal, the tumor was removed, the mice and the tumor were weighed, photos were taken, and the tumor inhibition rate and body weight change were calculated.

[0096] The drug was administered once daily (QD) by intraperitoneal injection in the right side of the body. Three dose regimens of XL-1063 were tested in both the experimental and control groups.

[0097] The tumor growth inhibition of tumor volume (TGItv) was calculated as follows: TGItv = [1-(average TV in the treatment group during the experiment-average TV at the beginning of the treatment group / average TV in the control group during the experiment-average TV at the beginning of the control group)] × 100%

[0098] The tumor growth inhibition of tumor weight (TGItw) was calculated as follows: TGItw = [1-(average tumor weight of the control group-average tumor weight of the drug-treated group / average tumor weight of the control group)] × 100%

[0099] All data results are presented as mean ± standard error. Statistical analysis was performed using Excel 2019. Tumor volume and tumor mass among groups were compared using Student's t test. P < 0.05 was considered statistically significant. Graphpad Prism 8.0 software was used for plotting.

[0100] The excised tumors were stained with hematoxylin and eosin, and immunohistochemically stained for the cell proliferation-related index MKi-67 (Ki-67) and the epithelial differentiation-related marker cytokeratin 19 (CK19).

[0101] The specific experimental results are as follows Figure 2 , Figure 3 As shown, the appearance of tumors in each group was observed at the end of the experiment ( Figure 2 a and Figure 3 In A), the tumor volume of the experimental group after XL-1063 treatment was significantly lower than that of the control group, while there was no significant difference in the control group, which preliminarily indicated that XL-1063 had higher selectivity and better anti-Kras-dependent PDAC tumor activity.

[0102] Table 1 The efficacy of different doses of XL-1063 on BXPC-3 model mice

[0103]

[0104] Table 2 The efficacy of different doses of XL-1063 on MIA paca-2 model mice

[0105]

[0106] (a: compared with the control group)

[0107] As shown in Table 1 and Table 2: For the control group, the tumor inhibition rate of the positive drug Deltarasin was higher than that of XL-1063 at the same concentration at the end of treatment. For the experimental group, the tumor inhibition rate of the positive drug Deltarasin was much lower than that of XL-1063 at the same concentration at the end of treatment, and the low-dose XL-1063 still had an excellent tumor inhibition rate. Only in the experimental group, the therapeutic effect of high-dose XL-1063 was significantly different from that of the model control (p<0.05), indicating that XL-1063 has a good therapeutic effect on the corresponding PDAC.

[0108] The mice in the positive control group treated with deltarasin had a significant decrease in body weight ( Figure 2 d) in. Figure 3 The results of the in vivo inhibition experiment of XL-1063 on MIA paca-2 cell line; Figure 3 A in the figure is the tumor tissue photos obtained from each group; Figure 3 B in the figure is the curve of the volume change of tumor tissue in each group over time; Figure 3 C in the figure is the volume statistics of tumor tissues in each group;

[0109] Figure 3 D in the figure is the curve of weight change of animals in each group over time; Figure 3 E in the figure is the statistical chart of the weight of animals in each group; Figure 3 F in the figure is a statistical graph of tumor tissue weight in each group. It can be seen from the figure that compared with the model control group, the tumor growth rate and tumor volume of the MIAPaca-2 group of mice treated with XL-1063 were significantly reduced. The results of the study indicate that inhibiting the PDEδ-Kras4B interaction by small molecules affects the spatial organization of Kras-dependent tumors, providing a new opportunity to inhibit oncogenic RAS signals and thus inhibit tumor growth.

[0110] The results of hematoxylin and eosin staining and immunohistochemical analysis showed that compared with the insensitive BxPC-3 tumor model group ( Figure 4 ), the proportion of necrotic tissue in MIA paca-2 tumor tissue treated with positive drugs and XL-1063 increased, the number of tumor cells decreased, the expression of Ki-67 and CK 19 decreased significantly, and the number and intensity of Ki-67 staining related to proliferation were significantly lower than those in the model control and the positive control group; CK19 was strongly and diffusely expressed in the model control, while it was weakly expressed in MIA paca-2 tumor tissue treated with XL-1063, and was significantly weaker than the positive control group. It proves that the proliferation and migration abilities of tumors are inhibited, and the inhibitory effect of the XL-1063 group is stronger than that of the positive drug group ( Figure 5 ). In insensitive tumor tissues, the situation is slightly different. The positive drug group showed stronger inhibition ability, proving that the positive drug has poor selectivity, and XL-1063 is a candidate small molecule inhibitor with high selectivity and lethality.

[0111] Example 5 Inhibition Binding Experiment of PM-1 Compound

[0112] ELISA is a reliable method widely used in in vitro protein interaction verification, and it performs well in the field of binding inhibition screening. After preliminary exploration, the present invention determined the appropriate amount of coating material in the direct ELISA method, and further implemented the inhibition binding experiment of the target drug at equal concentrations. By monitoring the trend of the photometric value, the present invention aims to verify whether the inhibitory effect of the small molecule drug previously obtained can be confirmed by the ELISA method, and preliminarily evaluate its inhibitory effect.

[0113] The verification steps are as follows:

[0114] (1) First, use the coating solution to dilute the protein at the optimal concentration conditions previously found, and coat it first. Then, according to the experimental grouping, add 100 μL to each well of the ELISA capture plate and incubate it overnight at 4°C.

[0115] (2) The next day, remove the solution in the wells and tap on absorbent paper to remove any residue. Then, add 300 μL of washing solution to each well and perform shaking washing. Each wash lasts for 3 minutes, for a total of 3 washes.

[0116] (3) After washing, prepare fresh 5% blocking solution using PBS. Add 300 μL of blocking solution to each well and incubate at 37°C for 2 hours. After blocking, no additional washing steps are required.

[0117] (4) Within 30 minutes before the end of blocking, use blocking solution to dilute the remaining protein solution to be bound to the optimal concentration. According to the experimental grouping, add the prepared solution to the corresponding wells. For example, the wells coated with r-huPD-1 protein should be added with r-huPD-L1 protein, and so on. Add 100 μL to each well. At the same time, according to the group setting, DMSO is added to the drug blank group, and then incubated at 37°C for 1 hour.

[0118] (5) Following the washing method of step (2), wash again three times, each wash lasting 3 minutes.

[0119] (6) According to the label instructions of the recombinant protein, add rabbit anti-human antibody with HRP (diluted to 1 μg / mL in blocking solution) to the incubation wells, 100 μL per well, and then incubate at 37°C for 45 minutes. For the incubation wells coated with r-huPD-1 as the substrate, add His tag antibody (diluted to 1 μg / mL in blocking solution) and incubate for 45 minutes, then wash, add goat anti-mouse HRP antibody (diluted to 1 μg / mL in blocking solution) again, 100 μL per well, and continue incubating at 37°C for 45 minutes.

[0120] (7) Following the washing method of step (2), wash again three times, each wash lasting 3 minutes.

[0121] (8) After washing, add 100 μL of TMB substrate solution to each well for color development and place in the dark at room temperature for 20 minutes.

[0122] (9) Finally, add 100 μL of dilute sulfuric acid to each well to terminate the color development reaction. Within 30 minutes, use an ELISA reader to detect the OD value at a wavelength of A450 nm. Data processing;

[0123] The intensity of the fluorescent chemiluminescence value measured by the microplate reader will be affected by factors such as experimental conditions, time differences or repeated experiments, resulting in large fluctuations in the data. In order to ensure the accuracy of the ELISA experimental data and the repeatability of the experiment, we have taken the following measures: at least three replica wells are set up in each well for each experiment, and their average values ​​are calculated to improve the reliability of the test data. In addition, during the data processing process, we will deduct the OD value of the empty wells of each experiment and the influence of the well plate itself on the test results to obtain the actual OD value difference. Subsequently, we use these OD values ​​to calculate the inhibition rate of small molecule inhibitors. The specific calculation formula is as follows:

[0124]

[0125] The results are as follows Figure 7 shown.

[0126] ELISA results show that PM-1 compound molecules and positive drug BMS-202 both exhibit certain binding inhibition ability under the ELISA system, see Figure 7 According to the calculation formula, the inhibition rate of small molecule inhibitors was obtained. Both small molecule compounds had certain inhibition ability. The inhibition rate of BMS-202 reached 39.06%, while the inhibition rate of compound PM-1 was 22.7%. There was no significant statistical difference between the inhibition rates of the two. In the ELISA system, the inhibition binding ability of PM-1 was comparable to that of BMS-202.

[0127] Example 6 PD-1 / PD-L1 blocking test of PM-1 compound

[0128] At present, small molecule drugs targeting PD-1 / PD-L1 targets are still in the research and development exploration stage. Therefore, many mature activity verification systems suitable for small molecule drugs have not yet been established for reference and application. However, it is worth noting that a variety of therapeutic antibody drugs have successfully used reporter cell lines to conduct T cell function reactivation experiments. These monoclonal antibody drugs successfully restored the inhibitory signal during T cell activation by effectively blocking the binding of PD-1 and PD-L1. Despite this, there are still few reports of small molecule compound inhibitors that have been successfully verified and marketed using such cell reporter systems. In view of this, this example attempts to explore the evaluation of the blocking function activity of small molecule compounds on PD-1 / PD-L1 targets through PD-1 / PD-L1 blocking reporter gene experiments, in order to provide new ideas and methods for research in this field.

[0129] The PD-1 / PD-L1 blocking test method is an experimental method that uses the principle of bioluminescence at the cellular level to evaluate the blocking activity of antibodies and small molecule inhibitors on the interaction between PD-1 and PD-L1. This method involves two specific cell types: one is Jurkat-NFAT-Luc2 cells expressing PD-1, and the other is 293T-OS8 cells expressing PD-L1. When these two cells are co-cultured, the binding of PD-1 and PD-L1 inhibits the activation of the T cell receptor (TCR) signaling pathway, thereby weakening the luminescent signal mediated by NFAT. The principle is detailed in Figure 8 However, when PD-1 / PD-L1 specific antibodies or small molecule inhibitors are added, they can effectively block the interaction between PD-1 and PD-L1, thereby relieving the inhibition of TCR signaling and enhancing the NFAT-mediated luminescence signal. In order to quantify the change in this luminescence signal, the experiment used the One-StepTM Luciferase Assay System kit for precise measurement.

[0130] The steps for PD-1 / PD-L1 blocking test are as follows:

[0131] (1) First, 293T-PD-L1 target cells were collected by centrifugation and then resuspended in their dedicated complete culture medium.

[0132] (2) Next, the target cell density was adjusted to an appropriate level and inoculated into a 96-well test plate. The test plate was then placed in a cell culture incubator (set at 37°C and 5% CO2) for about 16 hours.

[0133] (3) During this period, use the experimental buffer (composed of 99% 1640 medium and 1% FBS) to prepare double concentrations of control and test sample working solutions.

[0134] (4) Subsequently, the Jurkat-NFAT-Luc2-PD-1 effector cells were collected by centrifugation and resuspended in the assay buffer while adjusting the cell density to the desired level.

[0135] (5) After the incubation time is over, remove the 96-well experimental plate from the cell culture incubator and carefully remove the culture medium.

[0136] (6) Next, the control substance and the test sample working solution were added to the experimental plate in sequence, and after incubation with the target cells for 2 hours, the effector cell suspension was added.

[0137] (7) Then, the experimental plate was placed back into the cell culture incubator (set temperature at 37°C and CO2 concentration at 5%) and incubated for about 6 hours.

[0138] (8) During this period, prepare the working solution of Fire-LumiTM luciferase detection reagent.

[0139] (9) After the incubation is completed, remove the 96-well test plate from the cell culture incubator and add the working solution of the One-StepTM Luciferase Assay System reagent into it.

[0140] (10) Finally, the chemiluminescence value was read using an ELISA reader and the data was recorded. Subsequently, the data was processed and analyzed using GraphPad Prism software.

[0141] like Fig. 9 And as shown in Table 3.

[0142] Table 3 Fitting parameters of PM-1 compound blocking activity dose-effect curve

[0143]

[0144] Fig. 9 The results in Table 3 show that the blocking activity of PM-1 and BMS-202 was measured. PM-1 still did not show the upper platform of the test when it was close to the highest concentration. The overall test results were not stable, and the EC50 reference value was ~578.6μM; while BMS-202 did not show a plateau for many times, and high concentrations easily caused cell death. This cell blocking activity platform may not be suitable for the activity detection of small molecule compounds, because the absorption degree and poor specificity of the compounds are far less stable than monoclonal antibody drugs. However, the PM-1 compound still produced a better activity effect than BMS-202.

[0145] Example 7 Verification of the regulatory ability of PM-1 compound in the tumor microenvironment

[0146] PD-1 is mainly expressed on the surface of immune cells such as activated T lymphocytes and B lymphocytes, while tumor necrosis factor can significantly enhance the expression level of PD-1 on the cell membrane. In contrast, PD-L1 is widely expressed on the membrane surface of various tumor cells. When PD-1 and PD-L1 bind to ligands as receptors, the tumor immune escape mechanism is triggered. However, once an inhibitor intervenes and blocks the interaction between the two, it can stimulate an immune response, thereby restoring the normal anti-tumor immune process. In order to simulate the binding of PD-1 and PD-L1 in the tumor microenvironment, we tried to co-culture cells expressing these two molecules respectively. This approach aims to verify whether the previous small molecule compounds have the ability to regulate the tumor microenvironment, thereby achieving the killing effect on tumor cells.

[0147] Jurkat cell line:

[0148] Human acute T lymphoblastic leukemia cells, grown in suspension, 90% RPMI 1640 + FBS 10%;

[0149] 293 cells: human kidney epithelial cells, adherent growth, 90% DMEM + 10% FBS;

[0150] HepG2 cell line: human hepatoma cells, adherent growth, 90% DMEM + 10% FBS;

[0151] Hela cell line: human cervical cancer cells, adherent growth, 90% IMDM + 10% FBS;

[0152] (1) Cell recovery process: First, preheat a 37°C water bath and ensure that fetal bovine serum and culture medium have been thawed at 37°C. Quickly remove the cell cryopreservation tube from liquid nitrogen or -80°C refrigerator, place it in 37°C warm water and gently shake to thaw (avoid water from seeping into the tube) until the cells are completely thawed. In a cell clean bench, which needs to be exposed to ultraviolet light for more than 30 minutes in advance, use a pipette to transfer the thawed cells to a clean 15mL centrifuge tube, and add at least 10 times the volume of fresh culture medium. Mix well and centrifuge at 1000rpm for 5 minutes. Remove the supernatant, resuspend the cells with fresh culture medium containing 10% fetal bovine serum, inoculate them in a sterile culture bottle, and culture overnight at 37°C and 5% CO2. Check the cell status the next day and replace the culture medium as appropriate.

[0153] (2) Cell freezing steps: After the experiment, for adherent cells, first remove the old culture medium, add 2mL of trypsin digestion solution, and digest at 37°C until the cells begin to loosen. Then add 2mL of fetal bovine serum to terminate the digestion, blow evenly and transfer to a clean 15mL centrifuge tube, centrifuge (1000rpm, 5 minutes). Remove the supernatant, resuspend the cells with cell freezing solution, transfer to a cell freezing tube, and finally store in a -80°C refrigerator or liquid nitrogen. For suspended cells, transfer directly to a clean 15mL centrifuge tube, resuspend with cell freezing solution after centrifugation, and freeze.

[0154] (3) Jurkat cell line culture: Select Jurkat cells with good growth status and inoculate them into 6-well experimental plates, with 800,000 cells in each well. Add PHA reagent according to the instructions and culture them in an incubator for 24 hours.

[0155] (4) Treatment of HepG2 cell line: Select HepG2 cells with good growth status and inoculate them into 96-well experimental plates, with 50,000 cells in each well. After static culture for 4-5 hours until the cells are completely attached to the wall, add 1 mg / mL of r-huIFN-γ reagent to each well to make the final concentration of 10 μg / mL, and incubate in an incubator overnight.

[0156] (5) Co-culture experiment: The next day, the culture medium in the 96-well experimental plate was removed, and Jurkat cell lines were added at a ratio of 1:3, and the serum ratio was adjusted to 2%. Then, different concentrations of compounds were added according to Table 3, and the cells were cultured in an incubator for 36 hours.

[0157] (6) Cell washing: After 36 hours, remove the cell suspension and gently wash the well plate several times with culture medium preheated at 37°C to ensure that the Jurkat cells are thoroughly washed away while maintaining the good adhesion of the HepG2 cells.

[0158] (7) CCK8 detection: Add CCK8 reagent to the well at a ratio of 100:1 and let stand in the incubator for 20-40 minutes. Use an ELISA reader to detect at a wavelength of 450nm, and use Graph Pad Prism to analyze the results. The inhibition rate (or cell death rate) of small molecule compounds can be calculated by the formula, where OD control group 2 is a blank control group (containing only the same number of active HepG2 cells) and OD control group 1 is a negative control group (containing only the same number of active Jurkat cells).

[0159] Table 4 Experimental design and grouping of cell co-culture system

[0160]

[0161] The experimental results are as follows Fig.10 and Fig.11 shown.

[0162] The results showed that as the drug concentration gradually increased, the cell death rate showed a steady upward trend, which means that the inhibitory effect of small molecule compounds gradually increased.

[0163] contrast Fig.10 and Fig.11 It can be clearly seen that drugs show different sensitivities in different co-culture systems, and this finding provides valuable clues for determining the potential indications of small molecule inhibitors. Fig.10 The overall inhibition rate was relatively low, which may be attributed to the failure of small molecule compounds to fully act on cells or the insensitivity of the cell line to the PD-1 / PD-L1 interaction. Therefore, in the co-culture environment of tumor cells and Jurkat cell lines, small molecule drugs can regulate the tumor microenvironment to a certain extent, thereby having a killing effect on tumor cells.

[0164] In conclusion, PM-1 compound and BMS-202 showed comparable anti-tumor killing ability in the co-culture system, which deserves further in-depth study.

[0165] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of a pyrimidine biphenyl derivative, characterized in that: The applications include but are not limited to: Application in the preparation of small molecule inhibitors targeting PD-1 / PD-L1 for prevention or treatment; and / or, Application in the preparation of dual-functional molecular drugs for the prevention or treatment of dual targets of KRAS and PD-1 / PD-L1; Wherein, the structural formula of the pyrimidine biphenyl derivative is as shown in Formula I: The R 1 One selected from the group consisting of methyl, deuterated methyl and fluoromethyl; The R 2 One selected from the group consisting of hydrogen, deuterium, halogen, alkyl and alkoxy; The R 3 , R 4 , R 6 , R 7 , R 9 , R 10 , R 12 and R 13 is independently selected from one of hydrogen, deuterium, halogen, hydroxyl, amine, cyano, sulfonamide, C1-C3 alkyl and C1-C3 alkoxy; The R 5 One selected from 4-7 membered cyclic amine, 4-7 membered heterocyclic amine and sulfonyl, wherein the 4-7 membered cyclic amine, 4-7 membered heterocyclic amine and sulfonyl independently contain 1, 2 or 3 R a Substituents; The R 8 One selected from hydrogen, deuterium and C1-C6 alkyl; The R 11 Selected from -N(R b )C(=O)R c 、-N(R b )S(=O)2R c 、-C(=O)NR b R c and -S(=O)2NR b R c One of; Among them, R a One selected from hydrogen, deuterium, halogen, hydroxyl, amine, cyano, sulfonamide, C1-C6 alkyl and C1-C6 alkoxy; Rb is selected from one of hydrogen, deuterium and C1-C6 alkyl; R c One selected from C1-C8 alkyl, phenyl and 5-6 membered heteroaryl, wherein C1-C8 alkyl, phenyl and 5-6 membered heteroaryl independently contain 1, 2 or 3 R a Substituent.

2. The use according to claim 1, characterized in that The biphenyl derivative is used in the preparation of an anti-tumor drug for preventing or treating the inhibition of PD-1 / PD-L1 signal transduction.

3. The use according to claim 1 or 2, characterized in that: The pyrimidine biphenyl derivative is a PM-1 compound, and its structural formula is shown in Formula II:

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