CD97 and GSDME interaction inhibitor with anti-tumor activity and application of CD97 and GSDME interaction inhibitor

Abstract

The invention is suitable for the technical field of medicines, and provides a CD97 and GSDME interaction inhibitor with anti-tumor activity and application of the CD97 and GSDME interaction inhibitor. The compound screened by the invention improves the sensitivity of NK cells to kill tumor cells by inhibiting the interaction process of GSDME and CD97 in the tumor cells, so that the immune anti-tumor effect is induced. Meanwhile, the compound improves the sensitivity of tumor cells to pyroptosis by blocking the protective effect of CD97 in the tumor cells on GSDME protein shearing. Therefore, the compound can be used for treatment of GSDME and CD97 double-positive malignant tumors, combined chemotherapy, immunotherapy and the like, the sensitivity of the malignant tumors to chemotherapy and immunotherapy is improved, and the overall lifetime of malignant tumor patients is further prolonged. Specifically, the invention relates to a compound with a structure as shown in formula (I), pharmaceutically acceptable salt, solvent compound or deuterated compound # imgabs0 #, and pharmaceutically acceptable salt, solvent compound or deuterated compound thereof

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a CD97 and GSDME interaction inhibitor with anti-tumor activity and application thereof. Background Art

[0002] At present, conventional treatments for malignant tumors include surgical treatment, radiotherapy, chemotherapy, and biological regulation therapy. Among them, chemotherapy, as a systemic treatment, plays an important role in the treatment of malignant tumors that cannot be replaced by surgery and radiotherapy. In recent years, the emergence of new chemotherapy drugs has greatly improved the prognosis of cancer patients, but the toxicity of chemotherapy drugs and the drug resistance of tumor cells are becoming two major problems that plague chemotherapy.

[0003] Gasdermin E (GSDME for short), encoded by the DFNA5 gene, was initially identified as a genetic cause of non-syndromic hearing loss. In recent years, GSDME has been identified as a pore-forming protein precursor that can be cleaved by apoptosis-related Caspase-3 or granzyme B (GZMB) secreted by immune cells, releasing an N-terminal pore-forming domain that binds to the membrane to form a pore with an inner diameter of 10-15 nanometers (nm), thereby inducing tumor cell pyroptosis. Tumor cell pyroptosis not only inhibits the growth of the tumor itself, but also causes the intratumoral infiltrating CD8 + The proportion of T and NK (Natural Killer Cell) cells increases, thereby promoting the "cold-hot conversion" of tumors. Therefore, the tumor treatment strategy targeting GSDME will become a new direction for the treatment of malignant tumors. Despite this, GSDME-positive malignant tumors do not show high sensitivity to chemotherapy and immunotherapy.

[0004] CD97 is a member of the epidermal growth factor-seven transmembrane receptor (EGF-TM7) family and belongs to the class B G-protein-coupled receptor (GPCR). Studies have shown that CD97 can participate in the differentiation, migration, invasion and metastasis of tumors and is highly expressed in many cancers, including pancreatic cancer, cervical cancer, colon cancer, thyroid cancer and oral cancer, as well as leukemia. For this reason, the present invention proposes a CD97 and GSDME interaction inhibitor with anti-tumor activity and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a CD97 and GSDME interaction inhibitor with anti-tumor activity and its application, aiming to solve the problems raised in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A compound, a pharmaceutically acceptable salt, a solvate or a deuterated product thereof, having a structure as shown in formula (I):

[0008]

[0009] in:

[0010] R 1 is selected from the group consisting of methyl, deuterated methyl and fluoromethyl;

[0011] R 2 is selected from hydrogen, deuterium, halogen, C1-C3 alkyl and C1-C3 alkoxy;

[0012] R 3 and R 4 are independently selected from cycloalkyl, aryl and heteroaryl, R 3 and R 4 The cycloalkyl, aryl and heteroaryl groups are optionally substituted with 1, 2, 3, 4 or 5 R;

[0013] R is selected from hydrogen, deuterium, halogen, hydroxy, amino, trifluoromethyl, cyano, ester, carbonyl, acyl, aminoacyl, amide, sulfonyl, aminosulfonyl, sulfonamido, alkyl, alkoxy, aryl, furanyl, thienyl, pyridyl, oxazolyl and pyrazolyl.

[0014] Furthermore, R 2 Selected from hydrogen and deuterium.

[0015] Furthermore, R 3 and R 4 is selected from aryl and heteroaryl.

[0016] A use of the above-mentioned compound, its pharmaceutically acceptable salt, solvent compound or deuterated product as a CD97 and GSDME interaction inhibitor with anti-tumor activity in the preparation of a drug for treating cancer.

[0017] Furthermore, the cancer includes malignant tumors with dual positive expression of GSDME and CD97, the amino acid sequence of GSDME is shown as SEQ ID NO.1, and the amino acid sequence of CD97 is shown as SEQ ID NO.2.

[0018] Furthermore, the malignant tumor is lung cancer.

[0019] Furthermore, the malignant tumor is breast cancer.

[0020] Furthermore, the malignant tumor is colorectal cancer.

[0021] Furthermore, the malignant tumor is melanoma.

[0022] The following terms are used throughout this document and are defined as follows:

[0023] The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable and can retain the biological effectiveness of the free base without other side effects. Such salts include: acid addition salts formed with inorganic acids or organic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.; organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, trifluoroacetic acid, formic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.; or salts formed when the acidic protons present on the parent compound are replaced by metal ions, such as alkali metal ions or alkaline earth metal ions, such as sodium salts, potassium salts, calcium salts and magnesium salts, etc. Or a coordination compound formed with an organic base, such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. The pharmaceutically acceptable salt of the present invention can be synthesized by conventional chemical methods from a parent compound containing an acid radical or a base. In general, the preparation method of the salt is: in water or an organic solvent or a mixture of the two, these compounds in the form of free acid or base are reacted with a stoichiometric amount of an appropriate base or acid to prepare. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. In addition to the form of salts, the compounds provided by the present invention also exist in the form of prodrugs. The prodrugs of the compounds described herein are easily chemically changed under physiological conditions to be converted into the compounds of the present invention. In addition, drug precursors can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0024] The term "prodrug" refers to a compound obtained by chemically modifying the compound of the present invention, which is inactive or less active in vitro, and releases active compounds through enzymatic or non-enzymatic conversion in vivo to exert its pharmacological effect. When referring to a specific salt, pharmaceutical composition, composition, excipient, etc. as "pharmaceutically acceptable", it means that the salt, pharmaceutical composition, composition, excipient, etc. is generally non-toxic, safe, and suitable for use by a subject, preferably a mammalian subject, and more preferably a human subject.

[0025] The term "subject" refers to any animal that is about to or has been administered the compound or pharmaceutical composition according to the embodiments of the present invention, preferably a mammal, and most preferably a human. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., with humans being the most preferred.

[0026] The term "solvate" refers to a substance formed by combining a compound of the present invention with a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include water, ethanol, acetic acid, etc. Solvates include stoichiometric solvents and non-stoichiometric solvents, preferably hydrates. Certain compounds of the present invention may exist in unsolvated or solvated forms, including hydrate forms. Generally speaking, solvated forms are comparable to unsolvated forms and are included within the scope of the present invention.

[0027] When referring to certain elements such as hydrogen or H, it is intended to include all isotopes of that element. For example, if the R group is defined to include hydrogen or H, it also includes deuterium and tritium, and thus, isotopically labeled compounds are within the scope of the present invention.

[0028] The term "treatment" or "treating" refers to the improvement, prevention or reversal of a disease or condition or at least one identifiable symptom thereof. In another embodiment, "treatment" or "treating" refers to the improvement, prevention or reversal of at least one measurable physical parameter of the disease or condition being treated, and the disease or condition may not be identified in a mammal. However, in another embodiment, "treatment" or "treating" refers to slowing the progression of a disease or condition, either physically, such as the stabilization of identifiable symptoms, or physiologically, such as the stabilization of physical parameters, or both. In another embodiment, "treatment" or "treating" refers to delaying the onset of a disease or condition. In certain embodiments, the compounds of the present invention are administered as a preventive measure. As used herein, "prevention" or "preventing" refers to reducing the risk of acquiring a given disease or condition. In a preferred mode of the embodiment, the specified compound is administered to a subject, such as a subject with a family history or predisposition to cancer or autoimmune disease, as a preventive measure.

[0029] The term "halogen" refers to fluorine, chlorine, bromine and iodine. The term "alkyl" refers to a straight or branched saturated hydrocarbon group. In some embodiments, the alkyl group is a C1-C3 alkyl group; in other embodiments, the alkyl group is a C1-C6 alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl (including isopropyl and n-propyl), butyl (including n-butyl, isobutyl), pentyl (including n-pentyl, isopentyl and neopentyl), and hexyl. The alkyl group can be monovalent (such as methyl), divalent (such as methylene), polyvalent (such as methine), and can be monosubstituted (such as -CH2F) or polysubstituted (-CF3).

[0030] The term "alkoxy" refers to a group having an -O-alkyl structure, wherein alkyl is as defined above. Specific examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, n-pentoxy, and the like.

[0031] The term "alkenyl" refers to a straight or branched hydrocarbon group containing one or more carbon-carbon double bonds. In some embodiments, the alkenyl is a C2-C4 alkenyl; in other embodiments, the alkenyl is a C2-C6 alkenyl; in other embodiments, the alkenyl is a C2-C8 alkenyl.

[0032] The term "ring" refers to substituted or unsubstituted cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl. The ring includes monocyclic, bicyclic, polycyclic. The number of atoms on the ring is defined as the number of ring members.

[0033] The term "hetero" refers to heteroatoms or heteroatoms (atomic groups containing heteroatoms), that is, atoms other than carbon (C) and hydrogen (H) and atomic groups containing these heteroatoms, such as oxygen (O), nitrogen (N), sulfur (S), etc.

[0034] The term "cycloalkyl" refers to a cyclic alkyl group including a monocyclic, bicyclic or tricyclic ring system, wherein the bicyclic or tricyclic ring system includes a spirocyclic, a bridged ring, or a paracyclic ring; the alkyl group may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0035] The term "cycloalkenyl" refers to a cyclic alkenyl group containing one or more unsaturated carbon-carbon double bonds, wherein the bicyclic or tricyclic ring system includes spirocyclic, bridged, and fused rings; the alkyl group may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0036] The term "cycloalkynyl" refers to a cyclic alkyl group containing one or more carbon-carbon triple bonds, including monocyclic, bicyclic, and polycyclic systems, wherein the bicyclic or tricyclic system includes spirocyclic, fused, and bridged rings. The alkyl group can be monovalent, divalent, or polyvalent, and can also be monosubstituted or polysubstituted.

[0037] The term "heterocycloalkyl" refers to cyclized heteroalkyl groups, including monocyclic, bicyclic, and polycyclic ring systems, wherein bicyclic or tricyclic ring systems include spiro, fused, and bridged rings.

[0038] The term "heterocycloalkenyl" refers to cyclized heteroalkenyl groups, including monocyclic, bicyclic, and polycyclic ring systems, wherein bicyclic or tricyclic ring systems include spiro, fused, and bridged rings.

[0039] The term "heterocycloalkynyl" refers to cyclized heterocycloalkynyl groups, including monocyclic, bicyclic, and polycyclic ring systems, wherein bicyclic or tricyclic ring systems include spiro, fused, and bridged rings.

[0040] The term "aromatic ring" or "aryl group" refers to a polyunsaturated carbon ring system, including a monocyclic, bicyclic, or polycyclic system, wherein at least one ring is aromatic and may be monovalent, divalent, or polyvalent, and may be mono- or poly-substituted.

[0041] The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or not substituted, and unless otherwise specified, the type and number of the substituents may be arbitrary on the basis of chemical achievable.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention discloses a compound represented by the general formula (I), and its pharmaceutically acceptable salt, solvent compound or deuterated product for use in the preparation of a drug for treating cancer. The compound improves the sensitivity of NK cells to killing tumor cells by inhibiting the interaction between GSDME and CD97 in tumor cells, thereby inducing an immune anti-tumor effect. At the same time, the compound improves the sensitivity of tumor cells to pyroptosis by blocking the protective effect of CD97 in tumor cells on GSDME protein shearing. Therefore, the compound can be used for the treatment of GSDME and CD97 double-positive malignant tumors, as well as combined chemotherapy, immunotherapy, etc., to improve the sensitivity of malignant tumors to chemotherapy and immunotherapy, thereby prolonging the overall survival of patients with malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The figure shows the preparation process of two Raman probes, the establishment of a screening platform, and the screening of inhibitors from a combinatorial compound library; wherein, a is a schematic diagram of the preparation process of two Raman probes and the establishment of a screening platform; b is a schematic diagram of the screening process of inhibitors from a combinatorial compound library (20 compounds, 7 μM each).

[0045] Figure 2 The graph shows the effect of the compounds on the sensitivity of tumor cells to NK cells, and the data are expressed as mean ± SEM.

[0046] Figure 3 The diagram shows the model for establishing the screening system and the Elisa test result for IFN-γ release; wherein, a is the model for establishing the screening system; and b is the Elisa test result for IFN-γ release.

[0047] Figure 4The data of the effect of knocking down CD97 molecules on the immune killing sensitivity of tumor cells; a is immunoblotting, the effect of short interfering RNA (siRNA) on the expression of ADGRE5 gene (encoding CD97 protein) in NCI-H446, NCI-H1688 and A375 cells; Ctrl, disrupted siRNA; KD1, CD97-specific siRNA1; KD2, CD97-specific siRNA2; b is the effect of Ctrl, KD1 or KD2 induced by expanded human NK cells (effector cell / target cell (E / T) ratio of 5:1) in NCI-H446, NCI-H1688 and A375 cells. Cell lysis of 1688 or A375 tumor cells, 4 h, N = 5 donors per group; c is the percentage of tumor cell lysis induced by NK cells under different treatment conditions; d is the percentage of CD107a expression on the surface of NK cells under different treatment conditions; e is the microscopic image of NCI-H446 cells (Ctrl, KD1 or KD2) co-incubated with or without primary human NK cells for 16 h, arrows indicate pyroptotic vacuoles, and the number of live tumor cells is quantified (n = 3 donors per group), scale bar is 50 μm; f is the GSDME induced by primary human NK cells when CD97 is knocked down (KD) or pressed. - / - or GSDME + / + Cell lysis of tumor cells (E / T ratio = 5:1; 16 h; N = 5 donors per group). All data are presented as mean ± SEM. One-way ANOVA (dh) was performed. Ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0048] Figure 5 The results of in vivo treatment with siRNA targeting CD97 are shown in Figure 1 ; wherein, a is a graph showing the growth curve of B16 tumor in vivo;

[0049] b is the in vivo CT26 tumor growth curve; c, left, is an immunoblot, showing that mouse GSDME (mGSDME)

[0050] Ectopic expression of GSDME in 4T1 cells, treated with Chol-scramble or Chol-siCD97 in c (right)

[0051] Growth of empty vector (EV) or mGSDME 4T1 tumors in BALB / c mice (n=7 per group).

[0052] Figure 6The results of the interaction between CD97 and GSDME are shown in Figure 1. The left figure in a shows that NCI-H446 cells were treated with scramble (Ctrl), cd97-specific siRNA1 (KD1) or cd97-specific siRNA1 (KD2), and then stimulated or not stimulated with primary human NK cells for 16 h, NT, N-terminal GSDME cleavage products, and full-length GSDME; the right figure in a shows the quantitative determination of the ratio of NT to FL; the left figure in b shows that recombinant Caspase-3 (rCASP3) cleaved GSDME, and rCASP3 (0, 5, 25×10 -2 IU) was added to the lysate of NCI-H446 cells transfected with CD97 expression construct (CD97) or empty vector (EV); the right panel in b is the quantification of NT / FL ratio; c is immunoblotting, CD97-KD or Ctrl cells were pretreated with Caspase-3 inhibitor (zDEVD, 100 μM) After 1 h of incubation, NCI-H446 cells were stimulated with primary human NK cells for 16 h, and the GSDME in the cell lysates was detected; d is CD97 knockdown (KD) or Ctrl, primary human NK cells induced cell lysis of NCI-H446 tumor cells in the presence of DMSO, zDEVD (100 μM, pretreatment of tumor cells for 1 h) or GZMB inhibitor (100 μM, pretreatment of NK cells for 1 h); e and f are immunoblot (IP) experiments with IgG or anti-GSDME antibody (e) to pull down NCI-H446 tumor cell lysates, CD97-his (rCD97-his) and GAPDH (rGAPDH) were mixed and stimulated with IgG or Pull down with anti-his antibody (f); g is the design of different deletion mutants based on different domains of GSDME, centered on the GSDME cleavage site; h is the ability of GSDME deletion mutants to interact with GSDME detected by IP (left figure in h), and their binding affinity was quantified by measuring the amount of bound GSDME (right figure in h); I is the immunoblot of rCASP3 or recombinant granzyme B (rGZMB) incubated with recombinant GSDME (rGSDME) in vitro for 1 h, showing the quantitative results, in which 1.25 μg of rGSDME was incubated with the specified doses of rCASP3 (0, 3.125, 6.25, 12.5, 25, 50×10 -2 IU) (left panel in I) or rGZMB (0, 0.75, 1.5, 3, 6 ng / μl) (right panel in I) were incubated with recombinant CD97 (rCD97, 2.5 μg). The data shown are representative of 3 independent experiments.

[0053] Figure 7Characterization diagram of silver nanoparticles in Example 5; wherein, a is the UV-visible spectrum of silver nanoparticles (AgNPs) (diluted three times); b is the transmission electron microscope (TEM) image of AgNPs.

[0054] Figure 8 This is an electron microscope characterization image of the silver magnetic nanoparticle complex in Example 5.

[0055] Fig. 9 The SERS screening result diagram and characteristic peak 1587cm in Example 5 -1 The corresponding intensity bar graph; a is the SERS screening result graph; b is the characteristic peak 1587cm -1 Histogram of corresponding intensities.

[0056] Fig.10 The SERS detection results after the gradient concentration WXLL-5471 treatment in Example 5 and the characteristic peak 1587cm -1 Scatter plot of corresponding intensity; a is the SERS detection result after treatment with gradient concentration WXLL-5471; b is the characteristic peak 1587cm -1 Scatter plot of corresponding intensities.

[0057] Fig.11 The results of the compound inhibiting the interaction between CD97 and GSDME; a is the treatment of 293T cell lysate expressing Flag-GSDME with WXLL-5471 (0.5, 1, 5 μM) for 18 h, and immunoprecipitation with anti-flag antibody; b is the pretreatment of DSMO or WXLL-5471 (5 μM) for 18 h, and human primary NK cells induce NCI-H446 cell lysis (E / T ratio of 5:1, 16 h; N = 5 donors per group); c is the pretreatment of NCI-H446 with DSMO or WXLL-5471 (5 μM) for 18 h, without stimulation or with primary human NK cells for 16 h (left figure in c). The ratio of NT to FL was quantitatively determined (right figure in c). Data are expressed as mean ± SEM.

[0058] Fig.12 The weight changes and blood test results of mice in the compound and control groups during treatment; a is the weight change of mice in the compound and control groups during treatment; b is the blood routine test results of mice in the compound and control groups for 7 consecutive days; c is the biochemical parameter results of mice in the compound and control groups for 7 consecutive days. White blood cells; hemoglobin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; lactate dehydrogenase; CK, creatine kinase; ALP, alkaline phosphatase; BUN, urea nitrogen. All data are expressed as mean ± SEM. Unpaired t test was performed. Ns, p>0.05; *, p<0.05.

[0059] Fig.13 The results of the in vivo therapeutic efficacy of the compound and its effect on the immune microenvironment are shown in Figure 1. Among them, a is the growth of CT26 and B16 tumors in BALB / c and C57BL / 6 mice after intraperitoneal injection of normal saline or 100 mg / kg WXLL-5471 (8 mice in each group); b is the mGSDME in C57BL / 6 mice after treatment with normal saline or 100 mg / kg WXLL-5471 WT 、mGSDME D267A / D270A and mGSDME - / - Figure 3 B16 tumor growth (5 mice in each group); c is a mass spectrometry flow T-SNE diagram depicting the composition of intratumoral immune cells in B16 tumors treated with saline or WXLL-5471 for 7 days (n = 5 mice in each group); d is a volcano plot showing the broken line changes in immune cell types and adjusted p-values; e is a radar chart depicting the expression of CD8+T (left) and TIL-NK (right) markers GZMB, Expression levels of CD69, Ki-67, IFN-γ, CD107a, and PD1; f is the comprehensive immune score of the six markers in each group e; g is the expression of PD1 in TIL-NK and CD8+T in B16 tumors; hi are the tumor volume (h) and survival rate (i) of B16 tumors treated with combination therapy (WXLL-5471 and anti-PD-1) or monotherapy (WXLL-5471 or anti-PD-1) (n = 10 mice per group). WXLL-5471, once a day; anti-PD-1, twice a week. Data are expressed as mean ± SD. The area under the curve of the tumor growth curve in a, b, and g was analyzed by two-tailed Student's t test and one-way ANOVA, respectively, and the Holm-Sidak correction was performed for type I errors. Data were analyzed by one-way ANOVA (f, g, h) or log-rank (Mantel-Cox) test (i). ns, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. DETAILED DESCRIPTION

[0060] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0061] Example 1: Screening CD97 molecules using horizon G-PCR siRNA library;

[0062] like Figure 3 As shown in a, the screening system was first established: the GSDME-positive cell line NCI-H446 was plated into a 96-well plate, and the horizon G-PCR siRNA library (396 candidate genes) was transfected 24 hours later. NaiveNK was added at a 5:1 effector-target ratio 48 hours after transfection. After the above system was incubated overnight, the supernatant was taken and the release of IFN-γ was detected by Elisa to indirectly reflect the sensitivity of tumor cells to NK cell killing through the degree of NK cell activation. By this screening system, the genes FFAR1, FFAR2, FZD3, CXCR4, PTGER2 and ADGRE5, which significantly upregulated the release of IFN-γ after knockdown, were screened out. On this basis, NK cell killing experiments were further performed to verify that knocking down ADGRE5, that is, CD97 protein, can significantly enhance the sensitivity of SCLC cells to NK cell killing ( Figure 3 (b)

[0063] Example 2: Knockdown of CD97 can enhance the sensitivity of GSDME-positive tumor cells to immune killing;

[0064] CD97 was knocked down (CD97-KD) using siRNA in three GSDME-expressing tumor cell lines, NCI-H446, NCI-H1688, and A375 ( Figure 4 (a). After 4 h of incubation of transfected cells with donor-expanded NK cells, the sensitivity of tumor cells to NK cell killing was assessed by calcein release. In each case, knockout of CD97 rendered tumor cells more susceptible to NK cell-mediated cytotoxicity ( Figure 4 In contrast, ectopic expression of CD97 in NCI-H446 tumor cells restored their sensitivity to CD97-KD-induced NK cytotoxicity ( Figure 4 A large number of pyroptotic bodies were observed in CD97-KD tumor cells co-incubated with NK cells ( Figure 4 To determine whether NK cell-induced killing is mediated by GSDME-mediated pyroptosis when CD97 expression is attenuated, we constructed GSDME-deficient NCI-H446 and A375 cell models and found that NK cells were responsive to CD97-deficient GSDME. + / + Tumor cells showed strong cytotoxicity but were resistant to GSDME - / - No toxicity to tumor cells ( Figure 4 Middle f) These findings suggest that CD97 knockout sensitizes tumor cells to NK cell killing through a mechanism that requires GSDME.

[0065] Example 3. Treatment with cholesterol-modified CD97 siRNA can control the growth of malignant tumors in vivo;

[0066] In C57BL / 6 mice, 2*10 5 When the subcutaneous tumor grew to 5*5mm, cholesterol-modified CD97 siRNA (Chol-siCD97) or irrelevant sequence siRNA (Chol-scramble) was injected into the tumor and the growth of subcutaneous tumor in mice was recorded ( Figure 5 Then, CT26 and 4T1 mouse tumor models were used ( Figure 5 In b and c), the tumor growth of mice was recorded after the above treatment, confirming that cholesterol-modified CD97 siRNA treatment can control the growth of malignant tumors in vivo.

[0067] Example 4: CD97 can bind to the GSDME cleavage site to protect it from Caspase-3 and GZMB-mediated cleavage;

[0068] Using human primary NK cells to stimulate, western blot experiments were used to detect the shearing of GSDME, and it was found that GSDME protein was more susceptible to shearing after CD97 was knocked down in malignant tumor cells ( Figure 6 After overexpressing CD97, the whole cell lysate of GSDME-positive tumors was treated with recombinant Caspase-3 purified protein at 37°C for 30 minutes, and the shearing of GSDME was detected by western blot. It was found that the shearing rate of GSDME in malignant tumor cells in the CD97 overexpression group was significantly reduced ( Figure 6 This effect of cleavage activity was rescued after the tumor cells were pretreated with the Caspase-3 inhibitor zDEVD for 30 min in advance ( Figure 6 (c). At the same time, after NK cells were pretreated with the GZMB inhibitor zIETD-fmk 30 minutes in advance, they were also rescued ( Figure 6 d). Further endogenous immunoprecipitation ( Figure 6 e) and in vitro purified proteins directly interact ( Figure 6 f) Experiments have found that CD97 protein interacts directly with GSDME protein. Based on the different domains of GSDME, different deletion mutants were designed with the GSDME cleavage site as the center ( Figure 6 g), after being transferred into 293T cells, the pull-down of the interacting protein CD97 was detected by immunoprecipitation technology, and it was found that the GSDME cleavage site region 250-290 amino acid deletion mutant (△250-290) affected the binding of CD97 to it, suggesting that the binding site of CD97 and GSDME is located in the cleavage site region of GSDME ( Figure 6h). Further in vitro cleavage experiments were performed to add CD97 protein to the recombinant Caspase-3 and GZMB cleavage system, which significantly reduced its cleavage efficiency on GSDME ( Figure 6 Middle I).

[0069] Example 5: Screening of inhibitors of the interaction between CD97 and GSDME using surface enhanced Raman spectroscopy;

[0070] 1. Preparation of silver nanoparticles (AgNPs): Take a 250mL three-necked flask and soak it in an acid cylinder overnight. Take it out the next day, rinse it, and then wash it with pure water three times to ensure that the wall is pure and free of impurities. Put it in an oven to dry and cool to room temperature. During this period, accurately weigh 0.018g of silver nitrate powder and carefully pour it into the three-necked flask. Add 100mL of ultrapure water, shake it well, connect the condenser and set the temperature of the electric heating jacket to 100℃. Stir and heat the solution to a slight boil, then quickly add 2mL of 1% sodium citrate solution. Observe the color change of the solution: from light yellow to dark yellow to gray-green. Observe that the color no longer changes, cool it down slightly to 90℃, continue magnetic stirring, and maintain for 40 minutes to obtain a silver nanoparticle dispersion system. Use a UV spectrophotometer to measure the absorbance curve of silver nanoparticles: dilute the silver nanoparticles 3 times and observe that the maximum absorption wavelength range (420-480nm) is correct, and there are no other impurity peaks ( Figure 7 From a), we can preliminarily judge that the synthesized particle size is uniform. Transmission electron microscopy image ( Figure 7 The particle size in b) is about 50nm, which is relatively uniform and well dispersed. Using the formula: A = kbc (A = Amax, k = 3x10 11 M -1 cm -1 , b = 1 cm Calculate the concentration of silver nanoparticles. Store at 4℃ for future use.

[0071] 2. Preparation of magnetic nanoparticles: Commercial carboxyl magnetic beads (500 nm) produced by Shanghai Aladdin Company were prepared at a concentration of 0.5 mg / mL. The beads were rinsed with magnetic bead preservation solution and magnetic separation was repeated 3 times. The beads were resuspended in preservation solution to prevent aggregation of the beads and stored at 4°C for future use.

[0072] 3. Synthesize two Raman probes related to CD97-GSDME interaction inhibitor screening; the specific process is as follows Figure 1 As shown:

[0073] (1) Preparation of AgNPs@GSDME silver probe: 1 mL of silver nanoparticles AgNPs (0.25 nM) and 20 μL of Tween 20 were mixed in a 1.5 mL EP tube for 20 min to prevent aggregation during the surface modification of AgNPs. 10 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (2.5 mM) and 10 μL of N-hydroxysuccinimide (NHS) (2.5 mM) were added, reacted for 1 h, centrifuged at 4000 g for 7 min to remove excess activator, and resuspended. Then 2 μL of GSDME (1 mg / mL) and 25 μL of 4-MBA (1 mM, if a 96-well plate is used, 4-MBA is not added in this step) were added to the solution and reacted at room temperature for 1 h. Unreacted molecules were removed by centrifugation (4000 g, 7 min) and resuspended. The prepared AgNPs@GSDME was stored at 4°C for later use.

[0074] (2) Preparation of MNs@CD97 magnetic probe: Take 500μL 0.5mg / mL carboxyl magnetic beads, add 5μL Tween 20, mix for 30min, add 10μL 100mM EDC / NHS, activate for 1h, remove excess activator by magnetic separation, wash the magnetic beads again with PBS buffer, and resuspend. Add 2μL CD97 (1mg / mL) to the magnetic bead solution and react at room temperature for 1h. Wash the magnetic beads by magnetic separation 3 times to remove non-specifically bound proteins, then resuspend in PBS buffer solution to obtain MNs@CD97 and store at 4°C for later use.

[0075] 4. Establishment of a screening platform for CD97-GSDME interaction inhibitors;

[0076] A high-throughput targeted drug screening platform (SERScreen) was established using surface-enhanced Raman spectroscopy (SERS) as the sensing principle to screen CD97-GSDME interaction inhibitors.

[0077] Negative control: Evaluation of immunoaffinity structure; Raman probes are prepared immediately, silver probes and magnetic probes are mixed in a volume ratio of 3:1, and samples are taken after incubation for 60 minutes for immediate Raman signal detection and mapping. This signal is a negative control. Electron microscopy image ( Figure 8 ) shows that after incubation and washing three times, a large number of silver probes (small balls) are attached to the surface of the magnetic probe (large ball) in the field of view, proving that the probe preparation is successful. The two form a silver magnetic probe complex under the protein-protein interaction. After magnetic aggregation driven by a magnetic field, Raman detection of the probe aggregation site in a standard PCR tube (exposure time 1s, exposure times 10 times and average value) can detect the characteristic peak of the enhanced 4-MBA signal molecule.

[0078] 5. Using SERScreen, high-throughput screening of small molecule combinatorial compound libraries was performed for CD97 and GSDME interaction targets;

[0079] High-throughput screening is carried out in a well plate, taking a 96-well plate as an example: 150 μL AgNPs@GSDME (unlabeled 4-MBA) is added to each well, and a different small molecule compound (7 μM) is added to each well. Then, 50 μL MNs@CD97 is added to each well, reacted for 1 hour, magnetically separated and washed 3 times, and resuspended with PBS buffer. 4 μL 4-MBA (1 mM) is added to each well for 1 hour of reaction, magnetically separated and washed 3 times, and magnetically aggregated again, and SERS detection is performed at the probe aggregation point (633 nm, exposure time 1 s, exposure times are 10 times and the average is taken). During data processing, 3 batches of tests are performed for each small molecule, and each test is measured in parallel for more than 3 times. After removing the impurities and lowering the baseline using NGSLabSpec software, the final spectrum data of each well is processed and plotted ( Fig. 9 ).

[0080] The specific process is as follows:

[0081] (1) Negative control: Evaluation of immunoaffinity structure: Raman probes were prepared immediately before use. Silver probes and magnetic probes were mixed in a 96-well plate at a volume ratio of 150 μL:50 μL. After incubation for 60 min, samples were taken and Raman signal detection was performed immediately.

[0082] (2) Experimental group: Evaluation of immunoaffinity inhibition potency; Accurately pipette 150 μL of silver probe and 50 μL of magnetic probe, and add the compound for screening at the same time. After incubation for 60 minutes, perform Raman signal detection on a 96-well plate, set the exposure time to 1 second, and take the average value of 10 exposures to determine whether the compound has an inhibitory binding effect. If the Raman signal becomes very weak, it may be an inhibitor. From the compound library, select small molecules with potential for subsequent experiments, and finally screen to obtain new small molecule inhibitors. The small molecule compound to be screened was added to each group at a final concentration of 7 μM, and the final groups and numbers were (1:1, 2:67, 3:66, 4:69, 5:71, 6:90, 7:128, 8:267, 9:1071, 10:1428, 11:779, 12:1544, 13:145, 14:163, 15:550, 16:571, 17:1365, 18:1615, 19:1695, 20:1808, 21: negative control: immunoaffinity structure), and Raman detection was performed after incubation with the probe for 60 minutes, and the small molecule number that significantly reduced the Raman signal was recorded. The screening results show that the small molecule numbered 71 (WXLL-5471) has a better inhibitory effect ( Fig. 9In (a) and (b), there are potential small molecule inhibitors targeting the CD97-GSDME interaction target.

[0083] (3) Quantitative detection of candidate small molecule WXLL-5471: A quantitative experiment was performed on the screened inhibitor WXLL-5471. The inhibitor was diluted with buffer to the corresponding concentration gradient ( Fig.10 In a). Raman detection was performed in parallel, and the detection method was the same as above, and the corresponding concentration gradient scatter plot was drawn ( Fig.10 b). The SERS signal was evaluated to decrease regularly when the concentration of the small molecule increased, proving that its inhibitory effect on the CD97-GSDME interaction target increased step by step, confirming that the small molecule is a potential inhibitor.

[0084] Example 6: Compound WXLL-5471 can enhance the shearing of GSDME protein in tumor cells during immune killing;

[0085] Flag-GSDME was transferred into 293T cells, and after pretreatment with different concentrations of compound WXLL-5471 for 18 hours, pull-down enrichment was performed using Flag magnetic beads. Finally, the enrichment of CD97 protein was detected by western blot. The results showed that the higher the concentration of WXLL-7451 compound, the lower the efficiency of CD97 enrichment ( Fig.11 In a), it can be seen that the compound WXLL-5471 has a significant inhibitory effect on the interaction between CD97 and GSDME. After pre-treatment of GSDME-positive malignant tumor cells with 5 μM compound WXLL-5471 or DMSO for 18 hours, calcein was added to stain the malignant tumor cells, and human primary NK cells were added at a ratio of 5:1. After incubation for 16 hours, the degree of calcein release was detected ( Fig.11 b). After the above conditions, the malignant tumor cells were sorted for further western blot detection to detect the shearing of GSDME. The results showed that after the compound WXLL-7451 was treated, the shearing of GSDME in the malignant tumor cells under the immune stimulation of NK cells increased significantly ( Fig.11 (c).

[0086] Example 7: In vivo experiments confirm the safety of compound WXLL-5471;

[0087] The body weight of C57BL / 6 mice was recorded one day before intraperitoneal injection of saline or WXLL-5471, which was designated as day 0. The mice were then injected and monitored for 8 consecutive days (10 mice in each group). The changes in body weight are shown in Table 1. Fig.12Middle a; After B16 tumor-bearing mice were intraperitoneally injected with normal saline or WXLL-5471 for 8 consecutive days, orbital blood samples were collected to evaluate the blood routine and biochemical parameters of the mice (8 mice in each group for blood routine and 7 mice in each group for biochemical parameters). The results of blood routine and biochemical parameters are shown in Table 1. Fig.12 As shown in b and c, the results in the figure show that there was no obvious weight loss, hematological toxicity, or damage to liver, kidney, and myocardial function.

[0088] Example 8: In vitro experiments confirmed that compound WXLL-5471 can significantly enhance the immune killing sensitivity of malignant tumors;

[0089] NCI-H446 (left) and B16 (right) were pretreated with DSMO or WXLL-5471 (5.0 μM) to induce cell lysis for 18 h (E / T ratio of human NK was 5:1, and E / T ratio of mouse NK was 20:1; N = 5 donors / mice per group). In vitro killing experiments verified that the compound WXLL-5471 can significantly enhance the immune killing sensitivity of malignant tumor cells ( Figure 2 ).

[0090] Example 9: The therapeutic efficacy of compound WXLL-5471 in treating malignant tumors in vivo;

[0091] To evaluate the therapeutic effect of WXLL-5471 in vivo, we injected 100 mg / kg intraperitoneally into BALB / c mice bearing CT26 tumors and C57 mice bearing B16 tumors. On the 4th day after subcutaneous injection of tumor cells, we began to monitor the growth of subcutaneous tumors by daily intraperitoneal injection of WXLL-5471 or saline. The results showed that WXLL-5471 had a significant inhibitory effect on tumor growth ( Fig.13 However, this therapeutic effect was not observed in mGSDME D267A / D270A and mGSDME- / - Not present in B16 tumors ( Fig.13 b), indicating that the in vivo antitumor effect of WXLL-5471 depends on the cleavage activity of GSDME in the tumor. After 7 consecutive days of administration of WXLL-5471 or saline, we used mass cytometry to compare the differences in the number and activity of individual immune cells in the B16 tumor microenvironment (n = 4 mice). The mass cytometry t-SNE plot depicts the composition of intratumoral immune cells in B16 tumors treated with saline or WXLL-5471 for 7 days (n = 5 mice per group) ( Fig.13 c), the volcano plot shows the line changes and adjusted p-values ​​of immune cell types ( Fig.13 d), the results in the figure show that WXLL-5471 treatment leads to a significant increase in the number of infiltrating NK cells ( Fig.13 c and d), the activity of infiltrating NK and CD8+T cells was significantly upregulated, and the expressions of CD69, GZMB, CD107a, IFN-γ and Ki-67 were upregulated ( Fig.13 e and f). PD1 / PDL1 immune checkpoint therapy is a key therapeutic approach for the treatment of various malignancies, including melanoma. Exploring effective combined treatment strategies is expected to improve the clinical efficacy of immunotherapy for malignant tumors. Therefore, we preliminarily studied the effect of WXLL-5471 treatment on PD1 expression in B16 tumor-infiltrating immune cells. WXLL-5471 treatment significantly increased PD1 expression in CD8+T cells ( Fig.13 On this basis, WXLL-5471 combined with anti-PD1 antibody more effectively controlled the growth of B16 tumors in mice ( Fig.13 h), and significantly prolonged the overall survival of mice ( Fig.13 i).

[0092] Example 10: Synthesis of novel pyrimidine derivatives;

[0093] (A) Synthesis route 1 of aromatic amide substituted pyrimidine biphenyl compound 5:

[0094]

[0095] Synthesis route 2 of aromatic amide substituted pyrimidine biphenyl compound 5:

[0096]

[0097] The synthesis process of compound 2 is as follows: add bis-pinacol borate (7.626 g, 30 mmol) and 1,4-dioxane (40 mL) into a 250 mL three-necked flask, add tetrakistriphenylphosphine palladium (1.156 g, 1 mmol), potassium acetate (5.888 g, 60 mmol) and p-bromobenzene 1 (20 mmol) under nitrogen protection, stir and heat to reflux, monitor the reaction after 8 h, wait until the raw materials react completely, cool the reaction solution to room temperature, add 2,4-dichloro -5-methylpyrimidine or 2,4-dichloropyrimidine (30 mmol) and saturated sodium carbonate solution (24 mL), stirred and heated to reflux overnight, after monitoring the completion of the reaction of the intermediate product, water (100 mL) was added, extracted with ethyl acetate (3×50 mL), the organic phases were combined, dried over anhydrous sodium sulfate, the solvent was evaporated, and separated by column chromatography [V (petroleum ether): V (ethyl acetate) = 10: 1-4: 1], and then recrystallized from [V (petroleum ether): V (ethyl acetate) = 3: 1] to obtain the target product 2.

[0098]

[0099] The above formula is 2-chloro-5-methyl-4-(4-nitrophenyl)pyrimidine 2.1: mp: 118–120°C; 73%; 1 H NMR (300MHz, CDCl3) δ8.60(d,J=0.7Hz,1H),8.44–8.34(m,2H),7.89–7.79(m,2H),2.41(d,J=0.7Hz,3H); ES-MS m / z 250.1[M+H + ].

[0100]

[0101] The above formula is 2-chloro-4-(4-nitrophenyl)pyrimidine 2.2: mp: 54–55°C; 54%; ES-MS m / z 236.1 [M+H + ].

[0102]

[0103] The above formula is 2-chloro-5-methyl-4-phenylpyrimidine 2.3: mp: 123–125°C; 38%; ES-MS m / z 205.1 [M+H + ].

[0104]

[0105] The above formula is 2-chloro-4-(4-methoxyphenyl)-5-methylpyrimidine 2.4: mp: 93–94°C; 94%; ES-MS m / z 235.1 [M+H + ].

[0106]

[0107] The above formula is 4-(2-chloro-5-methylpyrimidin-4-yl)benzonitrile 2.5: mp: 152–154°C; 83%; ES-MS m / z 230.0 [M+H + ].

[0108]

[0109] The above formula is N-(4-(2-chloro-5-methylpyrimidin-4-yl)phenyl)-3,3-dimethylbutanamide 2.6: mp: 159–161°C; 72%; 1 H NMR (300MHz, CDCl3) δ8.48(s,1H),7.66(d,J=1.1Hz,4H),2.40(s,3H),2.27(s,2H),1.12(s,9H); ES-MS m / z 318.0[M+H + ].

[0110] Among them, the synthesis process of compound 3 is as follows: compound 2 (11 mmol) is added to a 150 mL thick-walled pressure bottle, and then 50 mL of 7 mol / L ammonia methanol solution is added, and the temperature is raised to 110°C for reaction. After the raw materials react completely, the reaction solution is evaporated to dryness, 100 mL of water is added, the aqueous phase is extracted with ethyl acetate (3×50 mL), the organic phases are combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent is evaporated, and separated by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1-1:1] to obtain the target product 3.

[0111]

[0112] The above formula is 2-amino-5-methyl-4-phenylpyrimidine 3.1: mp: 174–178°C; 65%; ES-MS m / z 186.1 [M+H + ].

[0113]

[0114] The above formula is 4-(4-methoxyphenyl)-5-methylpyrimidin-2-amine 3.2: mp: 136–138°C; 65%; 1 H NMR (300MHz, CDCl3) δ8.16(s,1H),7.65–7.43(m,2H),7.02–6.93(m,2H),5.03(s,2H),3.86(s,3H),2.22(s,3H); ES-MS m / z 216.1[M+H + ].

[0115] Wherein, the synthetic route 1 of compound 4 is as follows:

[0116]

[0117] The process of the above-mentioned synthetic route 1 is as follows: compound 3 (14 mmol) is weighed and added to a 250 mL round-bottom flask, 70 mL of dichloromethane is added to dissolve it, and benzoic acid (17.5 mmol), EDCI (4.026 g, 21 mmol), and DMAP (855 mg, 7 mmol) are added in sequence, and the reaction is stirred at room temperature for 96 h. After the reaction stops, 350 mL of water is added, the aqueous phase is extracted with dichloromethane (3×175 mL), the organic phases are combined, washed twice with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent is evaporated, and the target product 4 is obtained by column chromatography separation [V (petroleum ether): V (ethyl acetate) = 4:1-1:1].

[0118]

[0119] The above formula is N-(5-methyl-4-phenylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.1: mp: 158–159°C; 53%; 1 H NMR (300MHz, CDCl3) δ8.98(s,1H),8.60(s,1H),8.35(s,2H),8.05(s,1H),7.65–7.53(m,2H),7.52–7.42(m,3H),2.38(s,3H); 13 C NMR (75MHz, CDCl3) δ166.5,163.1,160.3,155.7,137.2,136.7,132.5,132.1,129.9,128.8,128.6,128.2,125.5,124.8,124.4,121.2,16.7; ES-MS m / z 426.1[M+H + ].

[0120]

[0121] The above formula is N-(4-(4-methoxyphenyl)-5-methylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.2: mp: 141–142°C; 64%; 1 H NMR (300MHz, CDCl3) δ9.02(s,1H),8.55(s,1H),8.35(s,2H),8.05(s,1H),7.58(d,J=8.7Hz,2H),6.98(d,J=8.7Hz,2H),3.87(s,3H),2.40(s,3H); 13 CNMR (75MHz, CDCl3) δ165.8,163.2,161.1,160.2,155.7,136.9,132.5,132. 0,130.6,129.5,128.1,125.5,124.8,123.9,121.2,114.0,55.5,17.1; ES-MS m / z 456.1[M+H + ].

[0122] Among them, the second synthetic route of compound 4 is as follows:

[0123]

[0124] The process of the above-mentioned synthetic route 2 is as follows: weigh the corresponding benzamide (10mmol), compound 2 (12mmol), Pd2(dba)3 (458mg, 0.5mmol), Xantphos (868mg, 1.5mmol) and Cs2CO3 (4.561g, 14mmol) in a 250mL three-necked flask, place it under positive nitrogen pressure, and perform three vacuum nitrogen replacement cycles under high vacuum. Add 1,4-dioxane (100mL) under positive nitrogen pressure and react overnight at reflux temperature. After the reaction stops, add 350mL of water, extract the aqueous phase with ethyl acetate (3×175mL), combine the organic phases, wash twice with saturated sodium chloride solution, add anhydrous sodium sulfate to dry, evaporate the solvent, and separate by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1-1:1] to obtain the target product 4.

[0125]

[0126] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.3: mp: 191–192°C; 83%; 1 H NMR (300MHz, CDCl3) δ8.81(s,1H),8.67(s,1H),8.37–8.34(m,4H),8.09(s,1H),7.82(d,J=9.0Hz,2H),2.41(s,3H); 13 C NMR (75MHz, CDCl3) δ164.0,163.3,161.0,155.9,148.5,143.3,136.5,132.6,132.2,130.1,128.2,125.8,124.7,124.4,123.8,121.1,16.6; ES-MS m / z 471.1[M+H + ].

[0127]

[0128] The above formula is N-(4-(4-nitrophenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.4: mp: 181–183°C; 76%; 1 H NMR (300MHz, CDCl3) δ8.95(s,1H),8.84(d,J=5.2Hz,1H),8.40(s,2H),8.39–8.33(m,2H),8.30–8.22(m,2H),8.11(s,1H),7.61(d,J=5.2Hz,1H); 13C NMR (75MHz, CDCl3) δ163.3,160.0,157.9,149.8,141.6,136.6,132.9,132.4,128.4,128.2,126.0,124.7,124.3,121.1,113.6; ES-MS m / z 457.0[M+H + ].

[0129]

[0130] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidin-2-yl)-3-(trifluoromethyl)benzamide 4.5: mp: 195–196°C; 75%; 1 H NMR (300MHz, CDCl3) δ8.72(s,1H),8.66(s,1H),8.36(d,J=8.5Hz,2H),8.19(s,1 H),8.14(d,J=7.9Hz,1H),7.89–7.79(m,3H),7.66(t,J=7.9Hz,1H),2.40(s,3H); 13 C NMR (75MHz, CDCl3) δ164.0,163.9,161.09,161.07,156.1,148.5,143.5,135.2,131.7,1 31.5, 131.3, 131.0, 130.1, 129.6, 129.1, 125.5, 124.6, 124.1, 123.8, 121.9, 16.6; ES-MS m / z 403.1[M+H + ].

[0131]

[0132] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidin-2-yl)-4-(trifluoromethyl)benzamide 4.6: mp: 187–188°C; 83%; 1 H NMR (300MHz, CDCl3) δ8.70(s,1H),8.65(s,1H),8.39–8.32(m,2H),8.06(d,J=8.5Hz,2H),7.86–7.79(m,2H),7.77(d,J=8.6Hz,2H),2.39(s,3H); 13C NMR (75MHz, CDCl3) δ164.1,163.9,161.1,156.0,148.5,143.5,137.7,134.4,133.9,130.1,128.1,126.0,124.1,123.8,121.8,16.6; ES-MS m / z 403.1[M+H + ].

[0133]

[0134] The above formula is N-(4-(4-cyanophenyl)-5-methylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.7: mp: 214–216°C; 69%; 1 H NMR (300MHz, CDCl3) δ8.91(s,1H),8.65(s,1H),8.37(s,2H),8.08(s,1H),7.87–7.77(m,2H),7.78–7.69(m,2H),2.39(s,3H); 13 C NMR (75MHz, CDCl3) δ164.2,163.2,161.0,155.8,141.6,136.6,132.7,132.4 ,132.2,129.7,128.2,125.8,124.7,124.4,121.1,118.3,113.7,16.6; ES-MS m / z 451.1[M+H + ].

[0135] The synthesis process of compound 5 is as follows: weigh compound 4 (3 mmol), iron powder (672 mg, 12 mmol), ammonium chloride (481 mg, 9 mmol), 30 mL of ethanol, and 7.5 mL of water, add them into a 100 mL round-bottom flask in sequence, heat to reflux under stirring, monitor the reaction after 4 hours, wait for the reaction of the raw materials to be complete, filter the reaction solution with diatomaceous earth, rinse the filter cake with 400 mL of ethanol, and evaporate the filtrate. Add 300 mL of water, extract the aqueous phase with ethyl acetate (3×150 mL), combine the organic phases, wash twice with saturated sodium chloride solution, add anhydrous sodium sulfate to dry, evaporate the solvent, and separate by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1] to obtain the target product 5.

[0136]

[0137] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 5.1 (also known as WXLL-5471): mp: 108–110°C; 75%; 1HNMR (300MHz, CDCl3) δ9.28(s,1H),8.51(s,1H),8.30(s,2H),8.02(s,1H),7.42(d,J=8.5Hz,2H),6.68(d,J=8.5Hz,2H),3.95(s,2H),2.39(s,3H); 13 C NMR (75MHz, CDCl3) δ165.9,163.2,160.2,155.8,148.4,136.9,132.3,131.8,130.6,128.1,126.6,125.3,124.8,123.6,121.2,114.4,17.2; ES-MS m / z 441.1[M+H + ].

[0138]

[0139] The above formula is N-(4-(4-aminophenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 5.2: mp: 145–146°C; 67%; 1 H NMR (300MHz, CDCl3) δ8.85 (s, 1H), 8.59 (d, J = 5.5Hz, 1H), 8.37 (s, 2H), 8.07 (s, 1H) ,7.87(d,J=8.6Hz,2H),7.38(d,J=5.4Hz,1H),6.72(d,J=8.6Hz,2H),4.06(s,2H); 13 CNMR (75MHz, CDCl3) δ167.3,165.5,158.3,157.5,150.1,137.2,132.5,132.2,132.1,129.0,128.2,125.4,124.8,121.2,114.9,111.5; ES-MS m / z 427.1[M+H + ].

[0140]

[0141] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-3-(trifluoromethyl)benzamide 5.3: mp: 114–116°C; 61%; 1H NMR (300MHz, CDCl3) δ8.71(s,1H),8.51(s,1H),8.18(s,1H),8.11(d,J=7.2Hz,1H),7.82(d,J=7.2Hz ,1H),7.62(t,J=7.2Hz,1H),7.53(d,J=8.2Hz,2H),6.75(d,J=8.2Hz,2H),3.92(s,2H),2.40(s,3H); 13 C NMR (75MHz, CDCl3) δ165.9,164.2,160.0,155.8,148.3,135.6,131.5,130.9,130.7,129.4,128.6,127.1,124.7,123.3,114.5,17.3; ES-MS m / z 373.1[M+H + ].

[0142]

[0143] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-4-(trifluoromethyl)benzamide 5.4: mp: 161–163°C; 72%; 1 H NMR (300MHz, CDCl3) δ8.50(s,1H),8.02(d,J=8.6Hz,2H),7.74(d,J=8.6Hz,2H),7.52(d,J=8.5Hz,2H),6.74(d,J=8.4Hz,2H),2.40(s,3H); 13 C NMR (75MHz, CDCl3) δ165.9,164.4,160.1,155.8,148.4,138.2,133.4,130.7,128.1,127.1,125.8,123.3,121.9,114.5,17.3; ES-MS m / z 373.1[M+H + ].

[0144] (B) The synthetic route of bis-aromatic amide substituted pyrimidine biphenyl compound 6 is as follows:

[0145]

[0146] The synthesis process of compound 6 is as follows: compound 5.1 (0.3 mmol) is weighed and added into a 50 mL round-bottom flask, 5 mL of dichloromethane is added to dissolve it, and R 6COOH (0.375mmol), EDCI (83mg, 0.45mmol), DMAP (18mg, 0.15mmol), stirred at room temperature for 9h. After the reaction stopped, 10mL of water was added, the aqueous phase was extracted with dichloromethane (3×5mL), the organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent was evaporated, and the target product 6 was obtained by column chromatography separation [V (petroleum ether): V (ethyl acetate) = 2:1-1:1].

[0147]

[0148] The above formula is N-(4-(2-(3,5-bis(trifluoromethyl)benzamido)-5-methylpyrimidin-4-yl)phenyl)nicotinamide 6.1: mp: 231–232°C; 75%; 1 H NMR (300MHz, CDCl3) δ9.38(s,1H),9.14(s,1H),8.68(d,J=4.6Hz,1H),8.59(s,1H),8.53(s,1H),8.38(s,2H),8 .22(d,J=8.4Hz,1H),8.06(s,1H),7.74(d,J=8.4Hz,2H),7.57(d,J=8.3Hz,2H),7.47–7.36(m,1H),2.38(s,3H); 13 C NMR (75MHz, DMSO-d6) δ164.3,162.8,160.2,155.9,152.2,148.7,140.1,136.7,135.5,132.6, 130.6,130.5,130.2,129.7,129.0,125.3,124.9,123.8,123.5,121.3,119.6,16.5; ES-MSm / z 546.1[M+H + ].

[0149]

[0150] The above formula is N-(4-(4-(3,3-dimethylbutyramido)phenyl)-5-methylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.2: mp: 121–122°C; 70%; 1 H NMR (300MHz, CDCl3) δ9.04(s,1H),8.56(s,1H),8.38(s,2H),8.06(s,1H),7.65(d,J=8 .4Hz,2H),7.59(d,J=8.6Hz,2H),7.28(s,1H),2.39(s,3H),2.26(s,2H),1.12(s,9H);13 C NMR (75MHz, CDCl3) δ170.6,165.6,163.1,160.3,160.1,155.7,148.4,139.6,136.9,136.7,132.4,132.0,130 .6,129.8,128.1,126.7,125.5,124.8,124.2,123.7,121.2,119.5,114.4,51.7,31.5,29.9,17.3,16.9; ES-MS m / z539.2[M+H + ].

[0151]

[0152] The above formula is N-(5-methyl-4-(4-(3-(o-tolyl)propionamido)phenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.3: mp: 107–109°C; 61%; 1 H NMR (300MHz, CDCl3) δ9.43(s,1H),8.52(s,1H),8.40(s,2H),8.04(s,1H),7.73–7. 44(m,4H),7.13(s,4H),3.03(s,2H),2.62(s,2H),2.35(s,3H),2.31(s,3H); ES-MS m / z 587.0[M+H + ].

[0153]

[0154] The above formula is N-(5-methyl-4-(4-(3-(m-tolyl)propionamido)phenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.4: mp: 108–110°C; 75%; 1 H NMR (300MHz, CDCl3) δ9.05(s,1H),8.55(s,1H),8.38(s,2H),8.06(s,1H),7.56(s,4H),7.31(s,1H),7.19(t,J=7. 7Hz,1H),7.05(s,2H),7.03(s,1H),3.02(t,J=7.4Hz,2H),2.68(t,J=7.5Hz,2H),2.38(s,3H),2.32(s,3H); ES-MS m / z 587.0[M+H + ].

[0155]

[0156] The above formula is N-(4-(4-formamidophenyl)-5-methylpyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.5: mp: 237–239°C; 70%; 1 HNMR (300MHz, CDCl3) δ10.19(s,1H),8.99(d,J=11.1Hz,1H),8.59(s,1H),8.52(s,2H),8 .34(s,1H),7.93(s,1H),7.46(d,J=8.4Hz,2H),6.86(d,J=8.4Hz,2H),2.33(s,3H); ES-MS m / z 469.0[M+H + ].

[0157] (C) The synthetic route of amine-substituted pyrimidine biphenyl compound 8 is as follows:

[0158]

[0159] The synthesis process of compound 7 is as follows: compound 2.1 (249 mg, 1 mmol) is weighed and added to a 50 mL round-bottom flask, 10 mL of 1,4-dioxane is added to dissolve it, and TsOH·H2O (304 mg, 1.6 mmol) and amine (4 mmol) are added in sequence, the temperature is raised to reflux temperature to start the reaction, and after TLC monitoring shows that there is no raw material, the reaction stops, the solvent is evaporated, 30 mL of water is added, and the mixture is extracted with dichloromethane (3×20 mL), the organic phases are combined, dried over anhydrous sodium sulfate, and the organic phases are evaporated under reduced pressure. The target product 7 is obtained by column chromatography separation [V (petroleum ether): V (ethyl acetate): V (acetone) = 5:1:1].

[0160]

[0161] The above formula is N-(3,5-bis(trifluoromethyl)phenyl)-5-methyl-4-(4-nitrophenyl)pyrimidin-2-amine 7.1: mp: 180–182°C; 68%; 1 HNMR (300MHz, CDCl3) δ8.48(s,1H),8.42–8.34(m,2H),8.21(s,2H),7.90–7.82(m,2H),7.57(s,1H),7.51(s,1H),2.36(s,3H); ES-MS m / z443.1[M+H + ].

[0162]

[0163] The above formula is N-(3,5-bis(trifluoromethyl)benzyl)-5-methyl-4-(4-nitrophenyl)pyrimidin-2-amine 7.2: mp: 158–160°C; 52%; 1 ES-MS m / z 457.1[M+H + ].

[0164]

[0165] The above formula is 5-methyl-N-(3-(morpholinesulfonyl)phenyl)-4-(4-nitrophenyl)pyrimidin-2-amine 7.3: mp: 196–198°C; 90%; 1 H NMR (300MHz, DMSO-d6) δ10.12(s,1H),8.57(s,1H),8.44(t,J=1.9Hz,1H),8.41–8.32(m,2H),8.02–7.9 1(m,3H),7.55(t,J=8.0Hz,1H),7.29–7.23(m,1H),3.62–3.54(m,4H),2.87–2.79(m,4H),2.26(s,3H); 13 C NMR (75MHz, CDCl3) δ162.3,160.5,158.0,147.6,144.0,141.4,134.6,130.0,129.4,123.2,122.5,119.1,116.6,65.1,45.8,15.4; ES-MS m / z 456.1[M+H + ].

[0166] The synthesis process of compound 8 is as follows: weigh compound 7 (3mmol), iron powder (672mg, 12mmol), ammonium chloride (481mg, 9mmol), 30mL of ethanol, and 7.5mL of water, add them into a 100mL round-bottom flask in sequence, heat to reflux under stirring, monitor the reaction by TLC, and filter the reaction solution through diatomaceous earth after the reaction of the raw materials is complete, rinse the filter cake with 400mL of ethanol, and evaporate the filtrate. Add 300mL of water, extract the aqueous phase with ethyl acetate (3×150mL), combine the organic phases, wash twice with saturated sodium chloride solution, dry with anhydrous sodium sulfate, evaporate the solvent, and separate by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1] to obtain the target product 8.

[0167]

[0168] The above formula is 4-(4-aminophenyl)-N-(3,5-bis(trifluoromethyl)phenyl)-5-methylpyrimidin-2-amine 8.1: mp: 189–190°C; 80%; 1 H NMR (300MHz, CDCl3) δ8.31(s,1H),8.25(s,2H),7.77(s,1H),7.63(d,J=8.7Hz,2H),7.47(s,1H),6.78(d,J=8.7Hz,2H),2.39(s,3H); 13 C NMR (75MHz, CDCl3) δ165.3,159.9,157.9,148.3,141.9,132.3,131.8,130.8,127.8,125.4,121.8,119.7,118.1,114.5,17.3; ES-MS m / z 413.1[M+H + ].

[0169]

[0170] The above formula is 4-(4-aminophenyl)-N-(3,5-di(trifluoromethyl)benzyl)-5-methylpyrimidin-2-amine 8.2: mp: 132–134°C; 86%; 1 H NMR (300MHz, CDCl3) δ8.11(s,1H),7.83(s,2H),7.75(s,1H),7.41(d,J=8.6Hz ,2H),6.70(d,J=8.6Hz,2H),5.83(s,1H),4.71(d,J=6.2Hz,2H),2.24(s,3H); 13 CNMR (75MHz, CDCl3) δ165.6,160.9,159.8,147.7,143.3,131.8,131.3,130.4,128.6,127.7,125.4,121.7,120.9,117.1,114.4,44.8,16.8; ES-MS m / z 427.1[M+H + ].

[0171]

[0172] The above formula is 4-(4-aminophenyl)-5-methyl-N-(3-(morpholinesulfonyl)phenyl)pyrimidin-2-amine 8.3: mp: 196–198°C; 81%; 1H NMR(300MHz, DMSO-d6)δ9.83(s,1H),8.61(s,1H),8.34(s,1H),7.94–7.80(m,1H),7.64–7.46(m,3H),7.32 ES-MS m / z 426.2[M+H + ]. 13 C NMR (75MHz, DMSO-d6)164.0,159.5,158.0,150.1,142.0,134.6,130.2,129.2,124.7,122.2,119.1,117.8,116.1,112.8,65.1,45.8,16.8; ES-MS m / z 426.4[M+H + ].

[0173] (D) The synthetic route of N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-N-methyl-3,5-bis(trifluoromethyl)benzamide 10 is as follows:

[0174]

[0175] The synthesis process of compound 9 is as follows: compound 4.3 (235 mg, 0.5 mmol) is weighed and added to a 50 mL round-bottom flask, 5.5 mL of N,N-dimethylformamide is added to dissolve it, and iodomethane (142 mg, 1 mmol) and cesium carbonate (163 mg, 0.5 mmol) are added in sequence to react under room temperature with stirring. After the reaction of the raw materials is complete as monitored by TLC, the reaction is stopped, 15 mL of water is added, the aqueous phase is extracted with ethyl acetate (3×7.5 mL), the organic phases are combined, washed twice with a saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent is evaporated, and the target product 9 is obtained by column chromatography separation [V (petroleum ether): V (ethyl acetate) = 1:1].

[0176] N-Methyl-N-(5-methyl-4-(4-nitrophenyl)pyrimidin-2-yl)-3,5-bis(trifluoromethyl)benzamide 9 (Compound 9): mp: 109–111 °C; 87%; 1 H NMR (300MHz, CDCl3) δ8.48(s,1H),8.25–8.19(m,2H),7.82(s,1H),7.80(s,2H),7.29–7.22(m,2H),3.74(s,3H),2.32(s,3H); ES-MS m / z 485.1[M+H +].

[0177] The synthesis process of compound 10 is as follows: weigh compound 9 (1.452 g, 3 mmol), iron powder (672 mg, 12 mmol), ammonium chloride (481 mg, 9 mmol), 30 mL of ethanol, and 7.5 mL of water, add them into a 100 mL round-bottom flask in sequence, heat to reflux under stirring, monitor the reaction after 4 hours, wait for the reaction of the raw materials to be complete, filter the reaction solution with diatomaceous earth, rinse the filter cake with 400 mL of ethanol, and evaporate the filtrate. Add 300 mL of water, extract the aqueous phase with ethyl acetate (3×150 mL), combine the organic phases, wash twice with saturated sodium chloride solution, add anhydrous sodium sulfate to dry, evaporate the solvent, and separate by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1] to obtain the target product 10.

[0178] N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-N-methyl-3,5-bis(trifluoromethyl)benzamide 10: mp: 106–107 °C; 71%; 1 H NMR (300MHz, CDCl3) δ8.33(s,1H),7.80(s,2H),6.95–6.87(m,2H),6.62–6.54(m,2H),3.71(s,3H),2.34(s,3H); 13 C NMR (75MHz, CDCl3) δ169.1,164.8,159.9,159.4,148.5,140.4,131.8,131.4,130.4,128.1,126.5,124.9,123.6,123.0,114.2,34.5,17.3; ES-MS m / z 455.1[M+H + ].

[0179] (E) The synthetic route of 4-(4-aminophenyl)-5-methylpyrimidin-2-amine 11 is as follows:

[0180]

[0181] The synthesis process of compound 11 is as follows: weigh compound 3.1 (690 mg, 3 mmol), iron powder (672 mg, 12 mmol), ammonium chloride (481 mg, 9 mmol), 30 mL of ethanol, and 7.5 mL of water, add them into a 100 mL round-bottom flask in sequence, heat to reflux under stirring, monitor the reaction after 4 hours, wait for the reaction of the raw materials to be complete, filter the reaction solution with diatomaceous earth, rinse the filter cake with 400 mL of ethanol, and evaporate the filtrate. Add 300 mL of water, extract the aqueous phase with ethyl acetate (3×150 mL), combine the organic phases, wash twice with saturated sodium chloride solution, add anhydrous sodium sulfate to dry, evaporate the solvent, and separate by column chromatography [V (petroleum ether): V (ethyl acetate) = 2:1] to obtain the target product 11.

[0182] 4-(4-Aminophenyl)-5-methylpyrimidin-2-amine 11: mp: 188–190°C; 72%; 1 HNMR (300MHz, CDCl3) δ8.12(s,1H),7.54–7.37(m,2H),6.79–6.59(m,2H),5.04(s,2H),3.50(s,2H),2.22(s,3H); ES-MS m / z 201.1[M+H + ].

[0183] The synthetic route of (F) N-(4-(2-((3-(N-benzylsulfonamido)phenyl)amino)-5-methylpyrimidin-4-yl)phenyl)-3,3-dimethylbutyramide 12 is as follows:

[0184]

[0185] The synthesis process of compound 12 is as follows: N-benzyl-3-aminobenzenesulfonamide (236 mg, 0.9 mmol), compound 2.6 (300 mg, 0.945 mmol), Pd2(dba)3 (82 mg, 0.09 mmol), XPhos (43 mg, 0.09 mmol) and K2CO3 (373 mg, 2.7 mmol) were weighed in a 25 mL three-necked flask, placed under positive nitrogen pressure, and vacuumed and nitrogen replaced for 3 times under high vacuum. Tert-butyl alcohol (9 mL) was added under positive nitrogen pressure, and the reaction was carried out at reflux temperature overnight. After the reaction stopped, 50 mL of water was added, the aqueous phase was extracted with ethyl acetate (3×25 mL), the organic phases were combined, washed twice with saturated sodium chloride solution, dried over anhydrous sodium sulfate, the solvent was evaporated, and the target product 12 was obtained by column chromatography separation [V (dichloromethane): V (methanol) = 100:1].

[0186] N-(4-(2-((3-(N-benzylsulfonamido)phenyl)amino)-5-methylpyrimidin-4-yl)phenyl)-3,3-dimethylbutanamide 12: mp: 225–226°C; 60%; 1 H NMR (300MHz, DMSO-d6) δ10.02(s,1H),9.91(s,1H),8.59(t,J=1.8Hz,1H),8.45(s,1H),8.07(t,J=6.4Hz,1H),7.91–7.86(m,1H),7.7 6(s,4H),7.47(t,J=8.0Hz,1H),7.38–7.31(m,1H),7.29–7.19(m,5H),3.99(d,J=6.3Hz,2H),2.30(s,3H),2.23(s,2H),1.04(s,9H); 13 C NMR (75MHz, DMSO-d6) δ170.3,163.7,163.5,160.1,158.3,141.6,141.1,140.3,137.8,132.4,12 9.6,129.3,128.2,127.5,127.1,121.5,118.5,118.3,115.9,49.6,46.2,30.9,29.6,16.3; ES-MS m / z 544.2[M+H + ].

[0187] Example 11: Analysis of the effect of compounds on enhancing the sensitivity of tumor cells to NK cell killing;

[0188] Experimental method: 5000 NCI-H446 cells were counted and inoculated into 96-well plates. 5 μM of the following compounds were added for overnight culture. Then human primary NK cells (effector-target ratio 5:1) were added and incubated for 16 hours. The cytotoxic activity of NK cells against NCI-H446 cells in different treatment groups was detected by calcein release assay.

[0189] The specific experimental results are as follows:

[0190] The following compounds have the effect of enhancing the sensitivity of tumor cells to NK cell killing, and the killing ratio of NCI-H446 cells is greater than that of the negative control: compound 4.1, compound 4.2, compound 4.3, compound 4.7, compound 5.1, compound 5.3, compound 5.4, compound 6.2, compound 6.3, compound 6.4, compound 6.5, compound 8.1, compound 8.3, and compound 12.

[0191] The following compounds had no effect on enhancing the sensitivity of tumor cells to NK cell killing, and their killing ratios to NCI-H446 cells were lower than that of the negative control: Compound 5.2, Compound 6.1, Compound 8.2, Compound 10, and Compound 11.

Claims

1. A compound, a pharmaceutically acceptable salt, a solvate or a deuterated substance thereof, characterized in that: The structure is shown in formula (I): in: R 1 is selected from the group consisting of methyl, deuterated methyl and fluoromethyl; R 2 is selected from hydrogen, deuterium, halogen, C1-C3 alkyl and C1-C3 alkoxy; R 3 and R 4 are independently selected from cycloalkyl, aryl and heteroaryl, R 3 and R 4 The cycloalkyl, aryl and heteroaryl groups are optionally substituted with 1, 2, 3, 4 or 5 R; R is selected from hydrogen, deuterium, halogen, hydroxy, amino, trifluoromethyl, cyano, ester, carbonyl, acyl, aminoacyl, amide, sulfonyl, aminosulfonyl, sulfonamido, alkyl, alkoxy, aryl, furanyl, thienyl, pyridyl, oxazolyl and pyrazolyl.

2. The compound according to claim 1, its pharmaceutically acceptable salt, solvate or deuterated substance, characterized in that: R 2 Selected from hydrogen and deuterium.

3. The compound according to claim 1, or its pharmaceutically acceptable salt, solvate or deuterated substance, characterized in that: R 3 and R 4 is selected from aryl and heteroaryl.

4. Use of the compound according to any one of claims 1 to 3, its pharmaceutically acceptable salt, solvate or deuterated product as an inhibitor of CD97 and GSDME interaction with anti-tumor activity in the preparation of a drug for treating cancer.

5. The use according to claim 4, characterized in that: The cancer includes malignant tumors with dual positive expression of GSDME and CD97, the amino acid sequence of GSDME is shown as SEQ ID NO.1, and the amino acid sequence of CD97 is shown as SEQ ID NO.

2.

6. The use according to claim 5, characterized in that: The malignant tumor is lung cancer.

7. The use according to claim 5, characterized in that: The malignant tumor is breast cancer.

8. The use according to claim 5, characterized in that: The malignant tumor is colorectal cancer.

9. The use according to claim 5, characterized in that: The malignant tumor is melanoma.

Images