CD97-GSDME interaction inhibitors with antitumor activity and their use

Abstract

An inhibitor of the interaction between CD97 and GSDME having antitumor activity, and its use. [Solution] A compound represented by formula (I), its pharmaceutically acceptable salt, solvate, or deuteride is provided. JPEG2026069453000042.jpg3673 The compound of the present invention inhibits the interaction process between GSDME and CD97 in tumor cells, thereby improving the sensitivity of NK cells to killing tumor cells and inducing an immunoantitumor effect. Furthermore, the compound improves the sensitivity of tumor cells to pyrotosis by blocking the protective effect of CD97 against GSDME protein cleavage within tumor cells. Therefore, the compound can be used in the treatment of GSDME and CD97-double-positive malignant tumors, as well as in combination with chemotherapy and immunotherapy, improving the sensitivity of malignant tumors to chemotherapy and immunotherapy, and further extending the overall survival of malignant tumor patients.

Description

[Technical Field]

[0001] This invention belongs to the field of pharmaceutical technology, and more particularly to an inhibitor of the interaction between CD97 and GSDME having antitumor activity, and its use. [Background technology]

[0002] Malignant tumors are currently one of the diseases that pose the most serious threat to human health. According to data from the World Health Organization's Institute for Cancer Research, in 2012, there were over 14 million new cases of malignant tumors worldwide, and approximately 8 million deaths were attributed to malignant tumors, making it one of the leading causes of death. Malignant tumors have become a major public health problem in China, surpassing cerebrovascular and cardiovascular diseases since 2010 to become the leading cause of death in China. With the accelerating aging of the population and the transformation of the environment and lifestyles, it is expected that the incidence and death rates from malignant tumors in China will continue to rise in the coming decades, posing a serious threat to the health of the people and imposing a serious disease burden on society. Currently, the usual treatment methods for malignant tumors include surgical treatment, radiation therapy, chemotherapy, and biomodulatory therapy. Chemotherapy, as a systemic treatment tool, holds an important position in the treatment of malignant tumors that cannot be replaced by surgery or radiation therapy. In recent years, the emergence of new chemotherapy drugs has significantly improved the prognosis for cancer patients. However, the toxicity of chemotherapy drugs and drug resistance in tumor cells remain two major challenges that make chemotherapy difficult.

[0003] Gasdermin E (abbreviated as GSDME) is encoded by the DFNA5 gene and was first identified as a genetic cause of non-syndromic hearing loss. Recently, GSDME has been recognized as a pore-forming protein precursor. It is cleaved by Caspase-3, which is associated with apoptosis, or by granzyme B (GZMB) secreted from immune cells, releasing a pore-forming domain N-terminus containing one amino terminus. The N-terminus binds to the membrane, forming a pore with an inner diameter of 10-15 nanometers (nm), inducing pyroptosis of tumor cells. Pyroptosis of tumor cells not only suppresses the growth of the tumor itself, but also inhibits intratumoral infiltration of CD8 +By increasing the proportion of T and NK (Natural Killer Cell) cells, it is possible to promote the "thermal-cold conversion" of tumors. Therefore, tumor treatment strategies targeting GSDME represent a new direction in malignant tumor treatment. However, GSDME-positive malignancies do not show high sensitivity to chemotherapy and immunotherapy.

[0004] CD97 is a member of the epidermal growth factor-seven transmembrane (EGF-TM7) family and belongs to the class B G protein-coupled receptors (GPCRs). Studies have shown that CD97 is involved in tumor differentiation, metastasis, invasion, and remission processes, and can be highly expressed in many cancers, including pancreatic cancer, cervical cancer, colon cancer, thyroid cancer, oral cancer, and leukemia. Therefore, the present invention provides an inhibitor of the interaction between CD97 and GSDME having antitumor activity and its use. [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide an interaction inhibitor of CD97 and GSDME having antitumor activity and its use in order to solve the problems presented in the background art described above. [Means for solving the problem]

[0006] The object of the present invention is achieved by the following technical means.

[0007] A compound, its pharmaceutically acceptable salt, solvate, or deuteride is defined as having a structure represented by formula (I),

[0008] [ka]

[0009] During the ceremony, R 1 is selected from a methyl group, a deuterated methyl group, and a fluoromethyl group, R 2 is selected from hydrogen, deuterium, a halogen, a C1-C3 alkyl group, and a C1-C3 alkoxy group, R 3 and R 4 are independently selected from a cycloalkyl group, an aryl group, and a heteroaryl group, and the cycloalkyl group, aryl group, and heteroaryl group of R 3 and R 4 are optionally substituted with 1, 2, 3, 4, or 5 Rs, R is selected from the group consisting of hydrogen, deuterium, a halogen, a hydroxyl group, an amino group, a trifluoromethyl group, a cyano group, an ester group, a carbonyl group, an acyl group, an aminoacyl group, an amide group, a sulfonyl group, an aminosulfonyl group, a sulfonamide group, an alkyl group, an alkoxy group, an aryl group, a furyl group, a thienyl group, a pyridyl group, an oxazolyl group, and a pyrazolyl group.

[0010] Furthermore, R 2 is selected from hydrogen and deuterium.

[0011] Furthermore, R 3 and R[[ID=3a]] 4 are selected from an aryl group and a heteroaryl group.

[0012] Use of the compound, its pharmaceutically acceptable salt, solvate or deuteride as described above in the manufacture of a cancer therapeutic agent as an inhibitor of the interaction between CD97 and GSDME having antitumor activity.

[0013] Furthermore, the cancer includes a malignant tumor with dual positive expression of GSDME and CD97, the amino acid sequence of GSDME is shown in SEQ ID NO:1, and the amino acid sequence of CD97 is shown in SEQ ID NO:2.

[0014] Furthermore, the malignant tumor is lung cancer.

[0015] Furthermore, the malignant tumor is breast cancer.

[0016] Furthermore, the aforementioned malignant tumor is colorectal cancer.

[0017] Furthermore, the aforementioned malignant tumor is malignant melanoma.

[0018] The following terms are used throughout this specification and are defined as follows:

[0019] The term "pharmaceutically acceptable salt" refers to a salt of the compound of the present invention that is pharmaceutically acceptable, maintains the biological efficacy of the free base, and has no other side effects. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid, or organic acids such as acetic acid, propionic acid, caproic acid, cyclopentylpropionic 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, and muconic acid; or salts in which the acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion or an alkaline earth metal ion, such as sodium salts, potassium salts, calcium salts, and magnesium salts; or coordination compounds formed with organic bases such as ethanolamine, diethanolamine, triethanolamine, and N-methylglucosamine. The pharmaceutically acceptable salts of the present invention can be synthesized from a parent compound containing an acid group or a base by conventional chemical methods. Generally, the salts are produced by reacting these compounds in the form of a free acid or base with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. In addition to the salt form, the compounds provided in the present invention also exist in prodrug form. The prodrugs of the compounds described herein are readily chemically modified under physiological conditions, thereby converting into the compounds of the present invention. The prodrugs may also be converted into the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0020] The term "prodrug" refers to a compound obtained after the chemical structure of the present invention has been modified, which is inactive or has low activity in vitro, and releases an active compound in vivo by enzymatic or non-enzymatic conversion to exert a pharmacokinetic effect. When referring to "pharmaceutically acceptable" for a particular salt, pharmaceutical composition, composition, excipient, etc., it means that the salt, pharmaceutical composition, composition, excipient, etc., is generally non-toxic and safe and suitable for use in subjects, preferably mammalian subjects, more preferably human subjects.

[0021] The term "subject" refers to any animal, preferably a mammal, most preferably a human, that is to be administered or has been administered the embodiment of the present invention, the compound, or the pharmaceutical composition. The term "mammal" as used herein includes all mammals. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0022] The term "solvate" refers to a substance formed when a compound of the present invention combines with a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include water, ethanol, acetic acid, and the like. Solvates include stoichiometric amounts of solvates and non-stoichiometric amounts of solvates, and are preferably hydrates. Some compounds of the present invention may exist in non-solvated or solvated forms, including hydrate forms. Generally, solvated forms correspond to non-solvated forms, and both are included within the scope of the present invention.

[0023] When referring to a specific element such as hydrogen or H, it is assumed that all isotopes of that element are included. For example, if the R group is defined as containing hydrogen or H, then deuterium and tritium are also included, and therefore isotope-labeled compounds are within the scope of this invention.

[0024] The terms “treatment” or “treatment” refer to the improvement, prevention, or recovery of a disease or condition or at least one identifiable symptom thereof. In other embodiments, “treatment” or “treatment” refer to the improvement, prevention, or recovery of at least one measurable physical parameter of a disease or condition being treated, which may not be recognized in mammals. However, in other embodiments, “treatment” or “treatment” means slowing the progression of a disease or condition, or stabilizing physically identifiable symptoms, or stabilizing physiological physical parameters, or both. In other embodiments, “treatment” or “treatment” refers to delaying the onset of a disease or condition. In some embodiments, the compounds of the present invention are administered as a precautionary measure. As used herein, “prevention” or “prevention” refers to reducing the risk of developing a given disease or condition. In preferred embodiments of the examples, a given compound is administered as a precautionary measure to a subject, for example, a subject with a family history or predisposition to cancer or autoimmune disease.

[0025] The term "halogen" refers to fluorine, chlorine, bromine, and iodine. The term "alkyl group" refers to a linear or branched saturated hydrocarbon group. In some embodiments, the alkyl group is a C1-C3 alkyl group, and 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 groups), butyl (including n-butyl and isobutyl groups), pentyl (including n-pentyl, isopentyl, and neopentyl groups), and hexyl groups. The alkyl group may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine), and may be monosubstituted (e.g., -CH2F) or polysubstituted (-CF3).

[0026] The term "alkoxy group" refers to a group having an -O-alkyl structure, and the definition of an alkyl group is as described above. Specifically, examples include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, isobutoxy, and n-pentyloxy groups.

[0027] The term "alkenyl group" refers to a linear or branched hydrocarbon group containing one or more carbon-carbon double bonds. In some embodiments, the alkenyl group is a C2-C4 alkenyl group; in other embodiments, the alkenyl group is a C2-C6 alkenyl group; and in other embodiments, the alkenyl group is a C2-C8 alkenyl group.

[0028] The term "ring" refers to substituted or unsubstituted cycloalkyl groups, cycloalkenyl groups, cycloalkynyl groups, aryl groups, heterocycloalkyl groups, heterocycloalkenyl groups, heterocycloalkynyl groups, and heteroaryl groups. These rings include monocyclic, bicyclic, and polycyclic structures. The number of atoms in a ring is defined as the number of members in the ring.

[0029] The term "hetero" refers to heteroatoms or heteroatomic groups (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), and sulfur (S).

[0030] The term "cycloalkyl group" refers to a cyclic alkyl group including monocyclic, bicyclic, or tricyclic ring systems, where bicyclic or tricyclic ring systems include spiro rings, crosslinking rings, and fused rings, and the alkyl group may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0031] The term "cycloalkenyl group" refers to a cyclic alkenyl group containing one or more unsaturated carbon-carbon double bonds. The bicyclic or tricyclic ring system includes spiro rings, bridged rings, and fused rings, and the alkyl group may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0032] The term "cycloalkynyl group" refers to a cyclic alkyl group containing one or more carbon-carbon triple bonds, and includes monocyclic, bicyclic, and polycyclic ring systems. Bicyclic and polycyclic ring systems include spiro rings, fused rings, and bridging rings. The alkyl group may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0033] The term "heterocycloalkyl" refers to a cyclized heteroalkyl group, including monocyclic, bicyclic, and polycyclic ring systems. Bicyclic and polycyclic ring systems include spiro rings, fused rings, and bridging rings.

[0034] The term "heterocycloalkenyl group" refers to a cyclized heteroalkenyl group, including monocyclic, bicyclic, and polycyclic ring systems. Bicyclic and polycyclic ring systems include spiro rings, fused rings, and bridging rings.

[0035] The term "heterocycloalkynyl group" refers to a cyclized heterocycloalkynyl group, including monocyclic, bicyclic, and polycyclic ring systems. Bicyclic and polycyclic ring systems include spiro rings, fused rings, and bridging rings.

[0036] The terms "aromatic ring" or "aryl group" refer to a polyvalent unsaturated carbon ring system, including monocyclic, bicyclic, and polycyclic ring systems, in which at least one ring is aromatic and may be monovalent, divalent, or polyvalent, and may be monosubstituted or polysubstituted.

[0037] The term "substituted" refers to the substitution of one or more hydrogen atoms in a particular atom with a substituent, and may include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is an oxy group (i.e., =O), it means that two hydrogen atoms are substituted. Oxy group substitution does not occur in aryl compounds. The term "optionally substituted" means that substitution is optional, and unless otherwise specified, the type and number of substituents may be arbitrary as long as they can be realized chemically stably. [Effects of the Invention]

[0038] Compared to the prior art, the present invention has the following beneficial effects.

[0039] This invention discloses the use of a compound represented by general formula (I), its pharmaceutically acceptable salt, solvate, or deuteride in the manufacture of cancer therapeutics. The compound enhances the sensitivity of NK cells to killing tumor cells by inhibiting the interaction process between GSDME and CD97 in tumor cells, thereby inducing an immunoantitumor effect. Furthermore, the compound enhances the sensitivity of tumor cells to pyrotosis by blocking the protective effect of CD97 against GSDME protein cleavage within tumor cells. Therefore, the compound can be used in the treatment of GSDME and CD97-double positive malignancies, as well as in combination with chemotherapy and immunotherapy, to improve the sensitivity of malignancies to chemotherapy and immunotherapy, and further extend the overall survival of malignancy patients. [Brief explanation of the drawing]

[0040] [Figure 1] The project involves the construction of a manufacturing process and screening platform for two types of Raman probes, and the screening of inhibitors from a combinatorial compound library. Here, (a) is a schematic diagram of the manufacturing process and screening platform for two types of Raman probes, and (b) is a schematic diagram of the inhibitor screening process from a combinatorial compound library (20 compounds, 7 μM each). [Figure 2] This figure shows the results of the NK cell killing sensitivity of tumor cells by compounds, and the data are expressed as mean ± SEM. [Figure 3] This is a schematic diagram of the screening system and a diagram showing the results of Elisa detection of IFN-γ release, where a is a schematic diagram of the screening system and b is a diagram showing the results of Elisa detection of IFN-γ release. [Figure 4]This data shows the effect of CD97 molecular knockdown on the immunotoxicity of tumor cells. Here, a is an immunoblot showing the effect of short interfering RNA (siRNA) on ADGRE5 gene (encoding the CD97 protein) expression in NCI-H446, NCI-H1688, and A375 cells, Ctrl is scrambled siRNA, KD1 is CD97-specific siRNA1, and KD2 is CD97-specific siRNA2. b shows cell lysis induced for 4 hours by proliferated human NK cells (effector cell / target cell (E / T) ratio of 5:1) of NCI-H446, NCI-H1688, or A375 tumor cells treated with Ctrl, KD1, or KD2. The data are as follows: 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 a microscopic image of NCI-H446 cells (Ctrl, KD1, or KD2) co-cultured for 16 hours in or without primary human NK cells, with arrows indicating pyrotose vesicles and quantifying the number of viable tumor cells (n=3 donors per group), with a scale bar of 50 μm; and f shows cell lysis of GSDME- / - or GSDME+ / + tumor cells induced by primary human NK cells under CD97 knockdown (KD) or Ctrl conditions (E / T ratio=5:1, 16h, N=5 donors per group). All data are expressed 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. [Figure 5] The figures show the results of in vivo treatment with CD97-targeted siRNA, where a is the in vivo B16 tumor growth curve, b is the in vivo CT26 tumor growth curve, c is an immunoblot showing ectopic expression of low GSDME expression in 4T1 cells of mouse GSDME (mGSDME), and c is a right-hand figure showing the growth of empty vector (EV) or mGSDME 4T1 tumors (n=7 mice per group) treated with Chol-scramble or Chol-siCD97 in BALB / c mice. [Figure 6]The results are from the interaction between CD97 and GSDME. Here, the left figure in a shows the cleavage products of NT and n-terminal GSDME, and full-length GSDME, after 16 hours of GSDME stimulation or destimulation in primary human NK cells treated with scramble (Ctrl), CD97-specific siRNA1 (KD1), or CD97-specific siRNA1 (KD2) in NCI-H446. The right figure in a shows the quantitative measurement of the ratio of NT to FL. The left figure in b shows the cleavage of GSDME by recombinant Caspase-3 (rCASP3). The results of adding P3 (0, 5, 25 × 10⁻² IU) to NCI-H446 cell lysates transfected with CD97 expression plasmid (CD97) or empty vector (EV) are shown. The right figure in b shows the results of quantifying the NT / FL ratio. c shows the results of immunoblotting, where CD97-KD or Ctrl NCI-H446 cells were pretreated with a Caspase-3 inhibitor (zDEVD, 100 μM) for 1 hour, then stimulated with primary human NK cells for 16 hours, and GSDME was detected in the cell lysates. d shows the results of CD97 knockdown (KD) or The results shown are from Ctrl processing, where primary human NK cells induced cytolysis of NCI-H446 tumor cells in the presence of DMSO, zDEVD (100 μM, tumor cells pretreated for 1 hour), or GZMB inhibitor (100 μM, NK cells pretreated for 1 hour). Figures e-f show the results of immunoblotting (IP) experiments, where NCI-H446 tumor cell lysates were pulled down with IgG or anti-GSDME antibody (e), and CD97-his (rCD97-his) and GAPDH (rGAPDH) were mixed and pulled down with IgG or anti-his antibody (f). The diagram shows that g represents different deletion mutants designed around the cleavage site of GSDME based on different domains of GSDME, h represents the evaluation of the interaction ability of GSDME deletion mutants with GSDME by IP detection (left figure of h), and the results of quantifying the binding affinity by measuring the amount of binding to GSDME (right figure of h), and I represents the results of immunoblotting in which rCASP3 or recombinant granzyme B (rGZMB) was cultured in vitro with recombinant GSDME (rGSDME) for 1 hour, with quantitative results also shown, where 1.25 μg of rGSDME was cultured with specified amounts of rCASP3 (0, 3.125, 6.25, 12.5, 25, 50 × 10⁻² IU) (left panel of I) or rGZMB (0, 0.75, 1.5, 3, 6 ng / μl) (right panel of i) and recombinant CD97 (rCD97, 2.5 μg). The data shown are representative from three independent experiments. [Figure 7] This is a characterization diagram of silver nanoparticles in Example 5, where a is the ultraviolet-visible spectrum (3-fold dilution) of silver nanoparticles (AgNPs), and b is a transmission electron microscope (TEM) image of AgNPs. [Figure 8] This is an electron microscope characterization diagram of the silver magnetic nanoparticle composite in Example 5. [Figure 9] The image shows the SERS screening results and a histogram of the intensity corresponding to the feature peak 1587 cm⁻¹ in Example 5, where a is the SERS screening results and b is the histogram of the intensity corresponding to the feature peak 1587 cm⁻¹. [Figure 10] The image shows the SERS detection results processed with gradient concentration WXLL-5471 in Example 5, and a scatter plot of intensity corresponding to the feature peak 1587 cm⁻¹. Here, a is the SERS detection results processed with gradient concentration WXLL-5471, and b is a scatter plot of intensity corresponding to the feature peak 1587 cm⁻¹. [Figure 11] This figure shows the results of inhibiting the interaction between CD97 and GSDME by the compound. Here, a shows the results of treating lysates of 293T cells expressing Flag-GSDME with WXLL-5471 (0.5, 1, 5 μm) for 18 hours and immunoprecipitation with anti-flag antibody. b shows the results of pre-treating cells with DSMO or WXLL-5471 (5 μm) for 18 hours and inducing NCI-H446 cell lysis with primary human NK cells (E / T ratio was 5:1, 16 hours, N=5 donors per group). c shows the results of pre-treating NCI-H446 with DSMO or WXLL-5471 (5 μm) for 18 hours and then stimulating cells with primary human NK cells for 16 hours without stimulation (left figure in c). The ratio of NT to FL was quantitatively measured (right figure in c). Data are expressed as mean ± SEM. [Figure 12] This graph shows the weight changes and blood test results during treatment of mice in the compound and control groups. Here, a is the weight change graph for mice in the compound and control groups during treatment, b is the results of routine blood tests after 7 consecutive days of treatment for mice in the compound and control groups, and c is the biochemical parameter results after 7 consecutive days of treatment for mice in the compound and control groups: white blood cells, hemoglobin, AST (aspartate aminotransferase), ALT (alanine aminotransferase), lactate dehydrogenase, CK (creatine kinase), ALP (alkaline phosphatase), and BUN (blood urea nitrogen). All data are expressed as mean ± SEM. An unpaired t-test was performed. Ns, p>0.05, *p<0.05. [Figure 13]This figure shows the results of the in vivo therapeutic effect of the compound and its impact on the immune microenvironment. Here, a shows the growth status of CT26 and B16 tumors (8 mice in each group) in BALB / c and C57BL / 6 mice after intraperitoneal injection of physiological saline or 100 mg / kg of WXLL-5471, and b shows the results of mGSDMEWT and mGSDMED267A / D after treatment of C57BL / 6 mice with physiological saline or 100 mg / kg of WXLL-5471. The graph shows the growth status of 270A and mGSDME- / -B16 tumors (5 mice per group). ⑧ is a mass cytometry T-sne plot showing the intratumoral immune cell composition (n=5 mice per group) in B16 tumors treated with saline or WXLL-5471 for 7 consecutive days. ⑧ is a volcano plot showing the line changes in immune cells and adjusted p-values. ⑧ is the CD8+ marker in B16 tumors treated with or not treated with WXLL-5471. The radar plots show the expression levels of the T (left) and TIL-NK (right) markers GZMB, CD69, Ki-67, IFN-γ, CD107a, and PD1. 'f' represents the combined immune score of the six markers in 'e' for each group, 'g' shows the expression status of PD1 in TIL-NK and CD8+ T within B16 tumors, and 'h' to 'i' represent tumor volume (h) and survival rate (i) (n=10 mice per group) when B16 tumors were treated with combination therapy (WXLL-5471 and anti-PD-1) or monotherapy (WXLL-5471 or anti-PD-1), respectively. WXLL-5471 is administered once daily, and anti-PD-1 is administered twice weekly. Data are expressed as mean ± SD. The area under the tumor growth curves in a, b, and g were calculated using two-sided Student's t-test and one-way ANOVA, respectively, with Holm-Sidak correction for type I errors. Data analysis was performed using 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. [Modes for carrying out the invention]

[0041] To better understand the technical features, objectives, and beneficial effects of the present invention, the technical means of the present invention will be described in detail below, but this should not be understood as limiting the scope of the invention. The experimental methods described in the following examples are all conventional methods unless otherwise specified, and the reagents and materials are all commercially available unless otherwise specified.

[0042] Example 1: Screening of CD97 molecules using a horizon G-PCR siRNA library As shown in Figure 3a, a screening system was first established. GSDME-positive cell line NCI-H446 was seeded in a 96-well plate, and after 24 hours, the system was transfected with a horizon G-PCR siRNA library (396 candidate genes). After 48 hours of transfection, naive NK cells were added according to a 5:1 effector cell to target cell ratio. The system was co-cultured overnight, the supernatant was taken, and IFN-γ release was detected using Elisa. The degree of NK cell activation indirectly reflects the susceptibility of tumor cells to NK cell killing. Using this screening system, genes FFAR1, FFAR2, FZD3, CXCR4, PTGER2, and ADGRE5 were screened for significantly increased IFN-γ release after knockdown. Based on this, further NK cell killing experiments were conducted, and it was ultimately found that knockdown of ADGRE5, i.e., the CD97 protein, significantly enhanced the susceptibility of SCLC cells to NK cell killing (Figure 3b).

[0043] Example 2: Knockdown of CD97 can enhance the sensitivity of GSDME-positive tumor cells to immunokilling. CD97 (CD97-KD) was knocked out in three tumor cell lines expressing GSDME using siRNA: NCI-H446, NCI-H1688, and A375 (Figure 4a). After transfecting the cells and culturing them with donor-grown NK cells for 4 hours, the tumor cells' susceptibility to NK cell-mediated cytotoxicity was evaluated by the release of calcein. In all cases, knocking out CD97 made the tumor cells more susceptible to NK cell-mediated cytotoxicity (Figure 4b). Conversely, in NCI-H446 tumor cells, ectopic expression of CD97 restored sensitivity to CD97-KD-induced NK cell toxicity (Figure 4c and d). A large number of pyrotose bodies were observed in CD97-KD tumor cells co-cultured with NK cells (Figure 4e). To determine whether NK cell-induced killing is mediated by GSDME-mediated pyrotosis when CD97 expression is reduced, we constructed GSDME-deficient NCI-H446 and A375 cell models and tested how NK cells react to GSDME-deficient NK cells. + / + It exhibits strong cytotoxicity against tumor cells, but GSDME - / - We found that it was not toxic to tumor cells (Figure 4f). As these findings clearly indicate, knocking out CD97 makes tumor cells sensitive to NK cell killing through a mechanism that requires GSDME.

[0044] Example 3: Treatment with cholesterol-modified CD97 siRNA can control the in vivo growth of malignant tumors. C57BL / 6 mouse with 2x10 5 B16F0 cells were subcutaneously injected, and when the subcutaneous tumor grew to 5 × 5 mm, treatment was initiated by injecting cholesterol-modified CD97 siRNA (Chol-siCD97) or unrelated sequence siRNA (Chol-scramble) into the tumor, and the growth of the subcutaneous tumors in mice was recorded (Figure 5a). Subsequently, the growth of mouse tumors after the above treatment was recorded using CT26 and 4T1 mouse tumor models (Figure 5b and c), demonstrating that treatment with cholesterol-modified CD97 siRNA can control the in vivo growth of malignant tumors.

[0045] Example 4: CD97 can bind to the cleavage site of GSDME, protecting it from Caspase-3 and GZMB-mediated cleavage. When human primary NK cells were stimulated and the cleavage status of GSDME was detected by Western blot experiments, it was found that the GSDME protein was more easily cleaved after CD97 knockdown in malignant tumor cells (Figure 6a). 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 cleavage status of GSDME was detected by Western blot. It was found that the GSDME cleavage rate of malignant tumor cells in the CD97 overexpression group was significantly reduced (Figure 6b). This effect on cleavage activity was recovered (rescue) after pretreatment of tumor cells with the Caspase-3 inhibitor zDEVD 30 minutes prior (Figure 6c). It was also recovered (rescue) after pretreatment of NK cells with the GZMB inhibitor zIETD-fmk 30 minutes prior (Figure 6d). Furthermore, experiments using endogenous immunoprecipitation (Figure 6e) and direct interaction of in vitro purified proteins (Figure 6f) revealed that CD97 protein and GSDME protein interact directly. Based on different domains of GSDME, different deletion mutants (Figure 6g) were designed, centering on the cleavage site of GSDME. After introducing each into 293T cells, the pull-down status of the interacting protein CD97 was detected using immunoprecipitation technology. The results showed that a 250-290 amino acid deletion mutant (△250-290) in the cleavage site region of GSDME affected CD97 and its binding, suggesting that the binding site between CD97 and GSDME is located in the cleavage site region of GSDME (Figure 6h). In addition, in in vitro cleavage experiments, adding CD97 protein to recombinant Caspase-3 and GZMB cleavage systems significantly reduced their cleavage efficiency toward GSDME (Figure 6I).

[0046] Example 5 Screening of CD97-GSDME interaction inhibitors using surface-enhanced Raman spectroscopy 1. Preparation of silver nanoparticles (AgNPs): A 250 mL three-necked flask was immersed in an acid tank overnight. The next day, it was removed and thoroughly rinsed, then washed three times with pure water to ensure the inner wall was clean and free of impurities. It was dried in an oven and cooled to room temperature. Meanwhile, 0.018 g of silver nitrate powder was accurately weighed and carefully added to the three-necked flask. 100 mL of ultrapure water was added and shaken uniformly. The solution was connected to a condenser, and the mantle heater was set to 100°C. The solution was heated while stirring until it was slightly boiling, and then 2 mL of 1% sodium citrate solution was rapidly added. The color change of the solution was observed, from pale yellow to dark yellow, and then to grayish-green. When the color stopped changing, the temperature was slightly reduced to 90°C, and stirring was continued for 40 minutes with a magnetic stirrer to obtain a silver nanoparticle dispersion. The absorbance curve of the silver nanoparticles was measured using an ultraviolet spectrophotometer. By diluting the silver nanoparticles threefold and detecting them, it was preliminaryly determined that the synthesized particle size was uniform, as the maximum absorption wavelength range (420-480 nm) was correct and there were no other impurity peaks (Figure 7a). The transmission electron microscope image (Figure 7b) shows that the particle size is approximately 50 nm, indicating uniform size and good dispersion. A=kbc(A=Amax, k=3×10) 11 M -1 cm -1 The concentration of silver nanoparticles was calculated using the formula (b = 1 cm). The samples were stored at 4°C and prepared for use.

[0047] 2. Manufacturing of magnetic nanoparticles: Commercial carboxyl-based magnetic beads (500 nm) manufactured by Shanghai Aladdin Co., Ltd. are prepared to a concentration of 0.5 mg / mL, washed three times with magnetic bead storage solution and magnetically separated, resuspended in the storage solution to prevent aggregation of the magnetic beads, stored at 4°C, and prepared for use.

[0048] 3. Synthesizing an interaction inhibitor of CD97-GSDME and screening two related Raman probes, the specific process is shown in Figure 1.

[0049] (1) Preparation of AgNPs@GSDME silver probe: 1 mL of silver nanoparticle AgNPs (0.25 nM) and 20 μL of Tween 20 were homogeneously mixed in a 1.5 mL EP tube for 20 min to prevent aggregation during the surface modification process 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 and reacted for 1 hour. The mixture was then centrifuged at 4000 g for 7 min to remove excess activator and resuspended. Subsequently, 2 μL of GSDME (1 mg / mL) and 25 μL of 4-MBA (1 mM; if using a 96-well plate, do not add 4-MBA in this step) were added to the solution and reacted at room temperature for 1 hour. Unreacted molecules were removed by centrifugation (4000 g, 7 min) and resuspended. Store the manufactured AgNPs@GSDME at 4°C and prepare for use.

[0050] (2) Preparation of MNs@CD97 magnetic probe: Take 500 μL of carboxyl group magnetic beads (0.5 mg / mL), add 5 μL of Tween 20, mix homogeneously for 30 min, then add 10 μL of EDC / NHS (100 mM), activate for 1 hour, remove excess activator by magnetic separation, wash the magnetic beads again with PBS buffer, and resuspend. Add 2 μL of CD97 (1 mg / mL) to the magnetic bead solution and react at room temperature for 1 hour. Remove the magnetic beads by magnetic separation and wash three times to remove nonspecifically bound proteins, then resuspend in PBS buffer to obtain MNs@CD97, store at 4°C and prepare for use.

[0051] 4. Development of a screening platform for CD97-GSDME interaction inhibitors Using surface-enhanced Raman spectroscopy (SERS) as the sensing principle, we constructed a high-throughput targeted drug screening platform (SERScreen) for screening CD97-GSDME interaction inhibitors.

[0052] Negative control: Evaluation of immunoaffinity structure: Raman probes were prepared immediately before use, and silver probes and magnetic probes were mixed in a 3:1 volume ratio. After incubation for 60 minutes, samples were taken, and the Raman signal was immediately detected and plotted. This signal was used as the negative control. The electron micrograph (Figure 8) is shown below. After incubation and washing three times, a large amount of silver probes (small spheres) adhered to the surface of the magnetic probe (large sphere) in the visible field, demonstrating that the probe preparation was successful. The two formed a silver-magnetic probe complex under protein-protein interaction. After magnetic aggregation under magnetic field drive, Raman detection was performed on the aggregated probe sites in a standard PCR tube (exposure time was 1 s, exposure count was 10, and the average value was taken). The result showed enhancement of the characteristic peak of the 4-MBA signal molecule.

[0053] 5. High-throughput screening of small molecule combinatorial compound libraries for CD97-GSDME interaction targets using SERScreen High-throughput screening was performed using well plates, with a 96-well plate used as an example. 150 μL of AgNPs@GSDME (unlabeled with 4-MBA) was added to each well, followed by the addition of a different small molecule compound (7 μM) corresponding to each well. Then, 50 μL of MNs@CD97 was added to each well, the mixture was reacted for 1 hour, magnetically separated, washed three times, and resuspended in PBS buffer. 4 μL of 4-MBA (1 mM) was added to each well, the mixture was reacted for 1 hour, magnetically separated, washed three times, and magnetic aggregation was performed again. SERS detection was performed at the aggregation sites of the probe (633 nm, exposure time 1 s, 10 exposures, average value taken). During data processing, batch detection was performed three times for each small molecule, and parallel measurements were performed at least three times for each detection. After removing impurity peaks and reducing the baseline using NGSLabSpec software, the final spectral data for each well was processed and plotted (Figure 9).

[0054] The specific process is as follows:

[0055] (1) Negative control: Evaluation of immunoaffinity structure: Raman probes were prepared immediately before use, and silver probes and magnetic probes were mixed in a volume ratio of 150 μL:50 μL in a 96-well plate. After incubation for 60 minutes, samples were taken and Raman signal detection was performed immediately.

[0056] (2) Experimental group: Evaluation of immunoaffinity inhibitory efficacy: 150 μL of silver probe and 50 μL of magnetic probe were accurately aspirated, and the screening compound was added simultaneously. After incubation for 60 minutes, Raman signal detection was performed in a 96-well plate. The exposure time was set to 1 s and the number of exposures to 10, and the average value was taken to determine whether or not the compound had binding inhibitory activity. A weaker Raman signal indicates a potential inhibitory effect. Potentially promising small molecules were selected from the compound library and subjected to subsequent experiments, and finally screened to obtain new small molecule inhibitors. A small molecule compound to be screened was added to each group at a final concentration of 7 μM. The final group and number 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). After culturing with the probe for 60 minutes, Raman detection was performed, and the number of the small molecule that significantly reduced the Raman signal was recorded. As can be seen from the screening results, small molecule number 71 (WXLL-5471) showed good inhibitory activity (Figure 9 a and b) and is a potential small molecule inhibitor of the interaction target of CD97-GSDME.

[0057] (3) Quantitative detection of candidate small molecule WXLL-5471: Quantitative experiments were performed on the screened inhibitor WXLL-5471. The inhibitor was diluted in buffer solution to the corresponding concentration gradient (Figure 10a). Raman measurements were performed in parallel, using the same detection method as above, and a corresponding concentration gradient scatter plot was created (Figure 10b). As a result of evaluating this, it was demonstrated that as the small molecule concentration increased, the SERS signal weakened regularly, and the inhibitory effect on the interaction target of CD97-GSDME increased stepwise, confirming that the small molecule is a potential inhibitor.

[0058] Example 6: Compound WXLL-5471 can enhance the cleavage of tumor cell GSDME protein in the immunokilling process. Flag-GSDME was introduced into 293T cells, and after pretreatment for 18 hours with different concentrations of the compound WXLL-5471, pull-down enrichment was performed using Flag magnetic beads. Finally, the enrichment status of the CD97 protein was detected using a western blot. The results showed that the efficiency of CD97 enrichment decreased as the concentration of the WXLL-7451 compound increased (Figure 11a), indicating that the compound WXLL-5471 has a significant inhibitory effect on the interaction between CD97 and GSDME. GSDME-positive malignant tumor cells were pretreated for 18 hours with 5 μm of the compound WXLL-5471 or DMSO, then the malignant tumor cells were stained with calcein, and human primary NK cells were added in an effector cell to target cell ratio of 5:1. After co-culture for 16 hours, the degree of calcein release was detected (Figure 11b). After processing under the above conditions, malignant tumor cells were selected and further Western blot detection was performed to detect the cleavage status of GSDME. The results showed that after treatment with the compound WXLL-7451, GSDME cleavage was clearly increased in malignant tumor cells under NK cell immunostimulation (Figure 11c).

[0059] Example 7: Demonstration of the safety of compound WXLL-5471 by in vivo experiment. For C57BL / 6 mice, body weight was recorded starting one day before intraperitoneal injection of physiological saline or WXLL-5471, which was designated as day 0. Subsequently, injections were administered continuously for 8 days, and monitoring was performed (10 mice per group). Body weight changes are shown in Figure 12a. For B16 tumor-bearing mice, intraperitoneal injection of physiological saline or WXLL-5471 was administered continuously for 8 days. Orbital blood samples were then taken, and routine blood tests and biochemical parameters were evaluated (routine blood tests were performed on 8 mice per group, and biochemical parameters on 7 mice per group). The results of routine blood tests and biochemical parameters are shown in Figures 12b and c, respectively. As can be seen from the results, no significant weight loss, hematological toxicity, or liver, kidney, or myocardial dysfunction was observed.

[0060] Example 8: Demonstration of the immunotoxicity-enhancing effect of compound WXLL-5471 against malignant tumors by in vitro experiment. Human primary NK cells and mouse spleen NK cells pretreated with NCI-H446 (left) and B16 (right) using DSMO or WXLL-5471 (5.0 μm), respectively, were used to induce 18 hours of cell lysis (human NK E / T ratio was 5:1, mouse NK E / T ratio was 20:1, N=5 donors / mice in each group). In vitro toxication experiments verified that the compound WXLL-5471 significantly enhances immunotoxicity against malignant tumor cells (Figure 2).

[0061] Example 9: In vivo therapeutic effect of compound WXLL-5471 on malignant tumors. To evaluate the in vivo therapeutic effect of WXLL-5471, BALB / c mice carrying CT26 tumors and C57 mice carrying B16 tumors were administered 100 mg / kg intraperitoneally. Four days after subcutaneous injection of tumor cells, WXLL-5471 or saline was administered intraperitoneally daily to monitor subcutaneous tumor growth. The results showed that WXLL-5471 had a significant inhibitory effect on tumor growth (Figure 13a). However, this therapeutic effect is associated with mGSDME. D267A / D270A and mGSDME - / -It was not present in B16 tumors (Figure 13b), indicating that the in vivo antitumor effect of WXLL-5471 is dependent on the fission activity of GSDME in tumors. After continuous administration of WXLL-5471 or saline for 7 days, the difference in the number and activity of individual immune cells in the B16 tumor microenvironment was compared using mass cytometry (n=4 mice). The mass cytometry t-SNE plot shows the composition of intratumoral immune cells in B16 tumors treated with saline or WXLL-5471 for 7 days (n=5 mice in each group) (Figure 13c), and the volcano plot shows the line changes in immune cell types and adjusted p-values ​​(Figure 13d). As can be seen from the results in the figures, WXLL-5471 treatment significantly increased the number of invasive NK cells (Figures 13c and d), and also increased the number of invasive NK and CD8 cells. + T cell activity was significantly increased, and the expression of CD69, GZMB, CD107a, IFN-γ, and Ki-67 was elevated (Figure 13e and f). PD1 / PDL1 immune checkpoint therapy is an important treatment option for various malignant tumors, including malignant melanoma. The exploration of effective combination therapy strategies is expected to improve the clinical efficacy of malignant tumor immunotherapy. Therefore, we preliminaryly investigated the effect of WXLL-5471 treatment on PD1 expression in B16 tumor-infiltrating immune cells. WXLL-5471 treatment increased CD8 + WXLL-5471 significantly increased PD1 expression in T (Figure 13g). Based on this, the combination of WXLL-5471 and an anti-pd1 antibody more effectively controlled the growth of mouse B16 tumors (Figure 13h) and significantly extended the overall survival of mice (Figure 13i).

[0062] Example 10 Synthesis of Novel Pyrimidine Derivatives (A) Synthesis scheme 1 of aromatic amide-substituted pyrimidine biphenyl compound 5:

[0063] [ka]

[0064] Synthesis scheme 2 for aromatic amide-substituted pyrimidine biphenyl compound 5:

[0065] [ka]

[0066] The synthesis process for compound 2 is as follows: Add bis(pinacolato)diborone (7.626 g, 30 mmol) and 1,4-dioxane (40 mL) to a 250 mL three-necked flask. Under the protection of nitrogen gas, add tetrakis(triphenylphosphine)palladium (1.156 g, 1 mmol), potassium acetate (5.888 g, 60 mmol), and p-bromobenzene 1 (20 mmol). Heat while stirring until reflux is achieved, and after 8 hours, monitor the reaction. After the starting materials have completely reacted, cool the reaction mixture to room temperature and, under the protection of nitrogen gas, add 2,4-dichloro-5-methylpyrimidine or 2,4-dichloro Pyrimidine (30 mmol) and saturated sodium carbonate solution (24 mL) were added, and the mixture was heated under stirring until reflux was achieved. The mixture was allowed to react overnight, and after monitoring that the intermediate product had completely reacted, water (100 mL) was added, and the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phase was combined, anhydrous sodium sulfate was added, and the mixture was dried. The solvent was evaporated to dryness, and the mixture was purified by elution using column chromatography [V(petroleum ether):V(ethyl acetate) = 10:1 to 4:1]. Subsequently, the mixture was recrystallized using [V(petroleum ether):V(ethyl acetate) = 3:1] to obtain target product 2.

[0067] [ka]

[0068] The above formula is 2-chloro-5-methyl-4-(4-nitrophenyl)pyrimidine 2.1. mp: 118~120℃; 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 + ].

[0069] [ka]

[0070] The above formula is 2-chloro-4-(4-nitrophenyl)pyrimidine 2.2. mp: 54~55℃; 54%; ES-MS m / z 236.1[M+H + ].

[0071] [ka]

[0072] The above formula is 2-chloro-5-methyl-4-phenylpyrimidine 2.3. mp: 123~125℃; 38%; ES-MS m / z 205.1[M+H + ].

[0073] [ka]

[0074] The above formula is 2-chloro-4-(4-methoxyphenyl)-5-methylpyrimidine 2.4. mp: 93~94℃; 94%; ES-MS m / z 235.1[M+H + ].

[0075] [ka]

[0076] The above formula is 4-(2-chloro-5-methylpyrimidine-4-yl)benzonitrile 2.5. mp: 152~154℃; 83%; ES-MS m / z 230.0[M+H + ].

[0077] [ka]

[0078] The above formula is N-(4-(2-chloro-5-methylpyrimidine-4-yl)phenyl)-3,3-dimethylbutanamide 2.6. mp: 159~161℃; 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 + ].

[0079] The synthesis process for compound 3 is as follows: Compound 2 (11 mmol) was added to a 150 mL thick-walled pressure flask, then 50 mL of a 7 mol / L ammonia-methanol solution was added, and the temperature was raised to 110°C to carry out the reaction. After the starting materials had completely reacted, the reaction mixture was evaporated to dryness, 100 mL of water was added, the aqueous phase was extracted with ethyl acetate (3 × 50 mL), the organic phase was combined, washed twice with saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent was evaporated to dryness, and the product was purified by column chromatography [V(petroleum ether):V(ethyl acetate) = 2:1~1:1] to obtain the target product 3.

[0080] [ka]

[0081] The above formula is 2-amino-5-methyl-4-phenylpyrimidine 3.1. mp: 174~178℃; 65%; ES-MS m / z 186.1[M+H + ].

[0082] [ka]

[0083] The above formula is 4-(4-methoxyphenyl)-5-methylpyrimidine-2-amine 3.2. mp: 136~138℃; 65%; 1H 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 + ].

[0084] The synthesis scheme 1 for compound 4 is as follows:

[0085] [ka]

[0086] The process for the above synthesis scheme 1 is as follows: Compound 3 (14 mmol) was weighed and added to a 250 mL round-bottom flask, dissolved in 70 mL of dichloromethane, and benzoic acid (17.5 mmol), EDCI (4.026 g, 21 mmol), and DMAP (855 mg, 7 mmol) were added in order. The mixture was stirred at room temperature for 96 hours. After the reaction was stopped, 350 mL of water was added, the aqueous phase was extracted with dichloromethane (3 × 175 mL), the organic phase was combined, washed twice with saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent was evaporated to dryness, and the mixture was purified by column chromatography [V (petroleum ether):V (ethyl acetate) = 4:1~1:1] to obtain the target product 4.

[0087] [ka]

[0088] The above formula is N-(5-methyl-4-phenylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.1. mp: 158~159℃; 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); 13C 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 + ].

[0089] [ka]

[0090] The above formula is N-(4-(4-methoxyphenyl)-5-methylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.2. mp: 141~142℃; 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 C NMR(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 + ].

[0091] The synthesis scheme 2 for compound 4 is as follows:

[0092] [ka]

[0093] The process for the above synthesis scheme 2 is as follows: The corresponding benzamide (10 mmol), compound 2 (12 mmol), Pd2(dba)3 (458 mg, 0.5 mmol), Xantphos (868 mg, 1.5 mmol), and Cs2CO3 (4.561 g, 14 mmol) were weighed and placed in a 250 mL three-necked flask. Under positive nitrogen gas pressure, three vacuum evacuation and nitrogen gas replacement cycles were performed under high vacuum. 1,4-dioxane (100 mL) was added under positive nitrogen gas pressure, and the mixture was reacted overnight at reflux temperature. After the reaction was stopped, 350 mL of water was added, the aqueous phase was extracted with ethyl acetate (3 × 175 mL), the organic phase was combined, washed twice with saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent was evaporated to dryness, and the mixture was purified by column chromatography [V(petroleum ether):V(ethyl acetate) = 2:1~1:1] to obtain the target product 4.

[0094] [ka]

[0095] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.3. mp: 191~192℃; 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 + ].

[0096] [ka]

[0097] The above formula is N-(4-(4-nitrophenyl)pyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.4. mp: 181~183℃; 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); 13 C 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 + ].

[0098] [ka]

[0099] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidine-2-yl)-3-(trifluoromethyl)benzamide 4.5. mp: 195~196℃; 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 + ].

[0100] [ka]

[0101] The above formula is N-(5-methyl-4-(4-nitrophenyl)pyrimidine-2-yl)-4-(trifluoromethyl)benzamide 4.6. mp: 187~188℃; 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); 13 C 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 + ].

[0102] [ka]

[0103] The above formula is N-(4-(4-cyanophenyl)-5-methylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 4.7. mp: 214~216℃; 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 + ].

[0104] The synthesis process for compound 5 is as follows: 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 are weighed and added sequentially to a 100 mL round-bottom flask. The mixture is heated under stirring until reflux occurs, and the reaction is monitored after 4 hours. After the starting materials have completely reacted, the reaction solution is filtered through diatomaceous earth, the filter cake is washed with 400 mL of ethanol, and the filtrate is evaporated to dryness. 300 mL of water is added, the aqueous phase is extracted with ethyl acetate (3 × 150 mL), the organic phase is combined, and the mixture is washed twice with saturated sodium chloride solution. Anhydrous sodium sulfate is added and the mixture is dried. The solvent is evaporated to dryness, and the mixture is purified by column chromatography [V(petroleum ether):V(ethyl acetate)=2:1] to obtain the target product 5.

[0105] [ka]

[0106] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 5.1 (also known as WXLL-5471). mp: 108~110℃; 75%; 1 H NMR(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 ES-MS m / z 441.1[M+H + ].

[0107] [ka]

[0108] The above formula is N-(4-(4-aminophenyl)pyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 5.2. mp: 145~146℃; 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 C NMR(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 + ].

[0109] [ka]

[0110] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidine-2-yl)-3-(trifluoromethyl)benzamide 5.3. mp: 114~116℃; 61%; 1 H 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 + ].

[0111] [ka]

[0112] The above formula is N-(4-(4-aminophenyl)-5-methylpyrimidine-2-yl)-4-(trifluoromethyl)benzamide 5.4. mp: 161~163℃; 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 + ].

[0113] (B) The synthesis scheme for bis-aromatic amide-substituted pyrimidine biphenyl compound 6 is as follows:

[0114] [ka]

[0115] The synthesis process for compound 6 is as follows: Weigh compound 5.1 (0.3 mmol) and add it to a 50 mL round-bottom flask, then add 5 mL of dichloromethane to dissolve it, and R 6COOH (0.375 mmol), EDCI (83 mg, 0.45 mmol), and DMAP (18 mg, 0.15 mmol) were added in sequence, and the mixture was stirred at room temperature for 9 hours to allow it to react. After the reaction was stopped, 10 mL of water was added, the aqueous phase was extracted with dichloromethane (3 × 5 mL), and the organic phase was combined. The mixture was washed twice with saturated sodium chloride solution, anhydrous sodium sulfate was added, and the mixture was dried. The solvent was evaporated to dryness, and the mixture was purified by column chromatography [V (petroleum ether):V (ethyl acetate) = 2:1 to 1:1] to obtain the target product 6.

[0116] [ka]

[0117] The above formula is N-(4-(2-(3,5-di(trifluoromethyl)benzamide)-5-methylpyrimidine-4-yl)phenyl)nicotinamide 6.1. mp: 231~232℃; 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-MS m / z 546.1[M+H + ].

[0118] [ka]

[0119] The above formula is N-(4-(4-(3,3-dimethylbutanamide)phenyl)-5-methylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.2. mp: 121~122℃; 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 / z 539.2[M+H + ].

[0120] [ka]

[0121] The above formula is N-(5-methyl-4-(4-(3-(o-tolyl)propionamide)phenyl)pyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.3. mp: 107~109℃; 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 + ].

[0122] [ka]

[0123] The above formula is N-(5-methyl-4-(4-(3-(m-tolyl)propionamide)phenyl)pyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.4. mp: 108~110℃; 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. ES-MS m / z 587.0[M+H + ].

[0124] [ka]

[0125] The above formula is N-(4-(4-formamidophenyl)-5-methylpyrimidine-2-yl)-3,5-bis(trifluoromethyl)benzamide 6.5. mp: 237~239℃; 70%; 1 H NMR(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 + ].

[0126] (C) The synthesis scheme for amine-substituted pyrimidine biphenyl compound 8 is as follows.

[0127] [ka]

[0128] The synthesis process for compound 7 is as follows: Compound 2.1 (249 mg, 1 mmol) was weighed and added to a 50 mL round-bottom flask. 10 mL of 1,4-dioxane was added to dissolve it. TsOH H2O (304 mg, 1.6 mmol) and amine (4 mmol) were added in sequence. The temperature was raised to reflux temperature to start the reaction. After monitoring for the absence of starting materials by TLC, the reaction was stopped, the solvent was evaporated, 30 mL of water was added, and the mixture was extracted with dichloromethane (3 × 20 mL). The organic phase was combined, dried over anhydrous sodium sulfate, and the organic phase was evaporated under reduced pressure. The mixture was purified by column chromatography [V(petroleum ether):V(ethyl acetate):V(acetone) = 5:1:1] to obtain the target product 7.

[0129] [ka]

[0130] The above formula is N-(3,5-di(trifluoromethyl)phenyl)-5-methyl-4-(4-nitrophenyl)pyrimidine-2-amine 7.1. mp: 180~182℃; 68%; 1 H NMR(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 / z 443.1[M+H + ].

[0131] [ka]

[0132] The above formula is N-(3,5-di(trifluoromethyl)benzyl)-5-methyl-4-(4-nitrophenyl)pyrimidine-2-amine 7.2. mp: 158~160℃; 52%; 1ES-MS m / z 457.1[M+H + ].

[0133] [ka]

[0134] The above formula is 5-methyl-N-(3-(morpholine sulfonyl)phenyl)-4-(4-nitrophenyl)pyrimidine-2-amine 7.3. mp: 196~198℃; 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 + ].

[0135] The synthesis process for compound 8 is as follows: Compound 7 (3 mmol), iron powder (672 mg, 12 mmol), ammonium chloride (481 mg, 9 mmol), 30 mL of ethanol, and 7.5 mL of water are weighed and added sequentially to a 100 mL round-bottom flask. The mixture is heated under stirring until reflux occurs, and the reaction is monitored by TLC. After the starting materials have completely reacted, the reaction mixture is filtered through diatomaceous earth, the filter cake is washed with 400 mL of ethanol, and the filtrate is evaporated to dryness. 300 mL of water is added, the aqueous phase is extracted with ethyl acetate (3 × 150 mL), the organic phase is combined, and the mixture is washed twice with saturated sodium chloride solution. Anhydrous sodium sulfate is added and the mixture is dried. The solvent is evaporated to dryness, and the mixture is purified by column chromatography [V(petroleum ether):V(ethyl acetate)=2:1] to obtain the target product 8.

[0136] [ka]

[0137] The above formula is 4-(4-aminophenyl)-N-(3,5-bis(trifluoromethyl)phenyl)-5-methylpyrimidine-2-amine 8.1. mp: 189~190℃; 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 + ].

[0138] [ka]

[0139] The above formula is 4-(4-aminophenyl)-N-(3,5-bis(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 C NMR(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 + .

[0140]

Chemical Structure

[0141] The above formula is 4-(4-aminophenyl)-5-methyl-N-(3-(morpholinosulfonyl)phenyl)pyrimidin-2-amine 8.3. mp: 196~198°C; 81%; 1 H 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),7.32~7.13(m,1H),6.66(d,J=8.6Hz,2H),5.58(s,2H),3.66~3.53(m,4H),2.90~2.82(m,4H),2.31(s,3H);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 + .

[0142] (D) The synthetic scheme of N-(4-(4-aminophenyl)-5-methylpyrimidin-2-yl)-N-methyl-3,5-bis(trifluoromethyl)benzamide 10 is as follows.

[0143]

Chemical formula

[0144] The synthesis process of compound 9 is as follows. Weigh 4.3 (235 mg, 0.5 mmol) and add it to a 50 mL round-bottom flask. Add 5.5 mL of N,N-dimethylformamide and dissolve it. Then add iodomethane (142 mg, 1 mmol) and cesium carbonate (163 mg, 0.5 mmol) in sequence, and stir and react at room temperature. After monitoring the completion of the reaction of the raw materials by TLC, stop the reaction, add 15 mL of water, extract the aqueous phase with ethyl acetate (3 × 7.5 mL), combine the organic phases, wash twice with saturated sodium chloride solution, add anhydrous sodium sulfate to dry, evaporate the solvent to dryness, and purify by column chromatography [V(petroleum ether):V(ethyl acetate)=1:1] to obtain the target product 9.

[0145] 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 (300 MHz, CDCl3) δ 8.48 (s, 1H), 8.25~8.19 (m, 2H), 7.8 (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 + .

[0146] The synthesis process for compound 10 is as follows: 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 are weighed and added sequentially to a 100 mL round-bottom flask. The mixture is heated under stirring until reflux occurs, and the reaction is monitored after 4 hours. After the starting materials have completely reacted, the reaction solution is filtered through diatomaceous earth, the filter cake is washed with 400 mL of ethanol, and the filtrate is evaporated to dryness. 300 mL of water is added, the aqueous phase is extracted with ethyl acetate (3 × 150 mL), the organic phase is combined, and the mixture is washed twice with saturated sodium chloride solution. Anhydrous sodium sulfate is added and the mixture is dried. The solvent is evaporated to dryness, and the mixture is purified by column chromatography [V(petroleum ether):V(ethyl acetate)=2:1] to obtain the target product 10.

[0147] N-(4-(4-aminophenyl)-5-methylpyrimidine-2-yl)-N-methyl-3,5-bis(trifluoromethyl)benzamide 10:mp:106~107℃;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 + ].

[0148] The synthesis scheme for (E)4-(4-aminophenyl)-5-methylpyrimidine-2-amine 11 is as follows:

[0149] [ka]

[0150] The synthesis process for compound 11 is as follows: 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 are weighed and added sequentially to a 100 mL round-bottom flask. The mixture is heated under stirring until reflux occurs, and the reaction is monitored after 4 hours. After the starting materials have completely reacted, the reaction solution is filtered through diatomaceous earth, the filter cake is washed with 400 mL of ethanol, and the filtrate is evaporated to dryness. 300 mL of water is added, the aqueous phase is extracted with ethyl acetate (3 × 150 mL), the organic phase is combined, the mixture is washed twice with saturated sodium chloride solution, anhydrous sodium sulfate is added and dried, the solvent is evaporated to dryness, and the solution is purified by column chromatography [V(petroleum ether):V(ethyl acetate)=2:1] to obtain the target product 11.

[0151] 4-(4-aminophenyl)-5-methylpyrimidine-2-amine 11:mp:188~190℃;72%; 1 H NMR(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 + ].

[0152] The synthesis scheme for (F)N-(4-(2-((3-(N-benzylsulfonamide)phenyl)amino)5-methylpyrimidine-4-yl)phenyl)3,3-dimethylbutanamide 12 is as follows.

[0153] [ka]

[0154] The synthesis process for 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 and placed in a 25 mL three-necked flask. The flask was placed under positive nitrogen gas pressure, and three vacuum evacuation and nitrogen gas replacement cycles were performed under high vacuum. Tert-butanol (9 mL) was added under positive nitrogen gas pressure, and the mixture was reacted overnight at reflux temperature. After stopping the reaction, 50 mL of water was added, the aqueous phase was extracted with ethyl acetate (3 × 25 mL), the organic phase was combined, washed twice with saturated sodium chloride solution, dried with anhydrous sodium sulfate, the solvent was evaporated, and the product was purified by column chromatography [V(dichloromethane):V(methanol) = 100:1] to obtain the target product 12.

[0155] N-(4-(2-((3-(N-benzylsulfonamide)phenyl)amino)-5-methylpyrimidine-4-yl)phenyl)-3,3-dimethylbutanamide 12:mp:225~226℃;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 + ].

[0156] Example 11: Analysis of the effect of a compound on enhancing the NK cell killing sensitivity of tumor cells. Experimental method: 5,000 counted NCI-H446 cells were inoculated into 96-well plates, and each was cultured overnight with a compound of 5 μM or less. Then, human primary NK cells (effector cell to target cell ratio 5:1) were added and cultured for a total of 16 hours. Calcein release experiments were then performed to detect the killing activity of different treatment groups of NK cells against NCI-H446 cells.

[0157] The specific experimental results are as follows:

[0158] The following compounds enhance the sensitivity of tumor cells to NK cell toxicity, resulting in higher toxicity rates against NCI-H446 cells compared to negative controls: 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.

[0159] The following compounds did not enhance the susceptibility of tumor cells to NK cell killing and resulted in lower killing rates against NCI-H446 cells compared to the negative control: Compound 5.2, Compound 6.1, Compound 8.2, Compound 10, Compound 11.

Claims

1. The structure is represented by formula (I), 【Chemistry 41】 During the ceremony, R １ This is selected from a methyl group, a deuterated methyl group, and a fluoromethyl group. R ２ This is selected from hydrogen, deuterium, halogen, C1-C3 alkyl groups and C1-C3 alkoxy groups. R ３ and R ４ R is independently selected from cycloalkyl groups, aryl groups, and heteroaryl groups. ３ and R ４ The cycloalkyl group, aryl group, and heteroaryl group are optionally substituted with 1, 2, 3, 4, or 5 R atoms. R is selected from the group consisting of hydrogen, deuterium, halogen, hydroxyl group, amino group, trifluoromethyl group, cyano group, ester group, carbonyl group, acyl group, aminoacyl group, amide group, sulfonyl group, aminosulfonyl group, sulfonamide group, alkyl group, alkoxy group, aryl group, furyl group, thienyl group, pyridyl group, oxazolyl group and pyrazolyl group. Compounds characterized by the above, and pharmaceutically acceptable salts, solvates, or deuterated compounds thereof.

2. R ２ It is selected from hydrogen and deuterium. The compound according to feature 1, or a pharmaceutically acceptable salt, solvate, or deuterated thereof.

3. R ３ and R ４ are selected from an aryl group and a heteroaryl group, The compound according to feature 1, or a pharmaceutically acceptable salt, solvate, or deuterated thereof.

4. Use of a compound according to any one of claims 1 to 3, a pharmaceutically acceptable salt, solvate, or deuteride thereof, in the manufacture of a cancer therapeutic agent as an antitumor-active CD97-GSDME interaction inhibitor.

5. The aforementioned cancer includes a malignant tumor exhibiting double-positive expression of GSDME and CD97, the amino acid sequence of GSDME being shown in SEQ ID NO: 1, and the amino acid sequence of CD97 being shown in SEQ ID NO:

2. The use described in feature 4.

6. The aforementioned malignant tumor is lung cancer. The use described in feature 5.

7. The aforementioned malignant tumor is breast cancer. The use described in feature 5.

8. The aforementioned malignant tumor is colorectal cancer. The use described in feature 5.

9. The aforementioned malignant tumor is malignant melanoma. The use described in feature 5.

Images