A CD4-enhancing + Compounds that differentiate T cells into Th9 cells

Abstract

The invention discloses a method for enhancing CD4 + The invention relates to a compound for differentiating T cells into Th9 cells, and belongs to the technical field of medicine; the compound for enhancing CD4 + Compounds that differentiate T cells into Th9 cells have an inverse agonist effect on the GPR34 receptor, which is different from the effect of agonists or antagonists of the GPR34 receptor. The GPR34 inverse agonist provided by the present invention can effectively enhance the differentiation of T cells into Th9 cells, solve the technical problem of low in vitro differentiation efficiency of Th9 cells in the prior art, and can significantly enhance the effect of Th9 cells in adoptive transfer treatment of solid tumors, achieving excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology and particularly relates to a method for enhancing CD4 + Compounds that differentiate T cells into Th9 cells. Background Art

[0002] Adoptive T cell therapy is an immunotherapy that selectively expands or modifies the body's own T cells in vitro and then returns them to cancer patients to specifically attack tumor cells. Common adoptive T cell therapy methods include "tumor infiltrating lymphocytes" (TIL) and chimeric antigen receptor T cell therapy (CAR-T). It takes 2 to 8 weeks to culture T cells in the laboratory. During this period, patients receive chemotherapy and radiotherapy to reduce the number of other immune cells and make the transplanted T cells more effective. After the treatment, the cultivated T cells are returned to the patient through intravenous infusion. TIL therapy uses tumor infiltrating lymphocytes (TILs) in the patient's body to fight tumors. The steps include collecting TILs samples from the patient's tumor tissue, expanding and activating them in the laboratory, and then re-injecting them into the patient. TILs can recognize and attack tumor cells, trigger an immune response, and inhibit tumor growth. Although TIL therapy shows potential efficacy, it also faces challenges such as the complexity of preparing cells, low anti-tumor efficacy, and easy exhaustion. CAR-T cell therapy is an immunotherapy that uses genetic engineering technology to transform the patient's own T cells to have stronger tumor recognition and attack capabilities. The basic principle is to collect the patient's T cells, introduce the CAR gene into these cells, and then re-inject them into the patient after in vitro amplification. CAR-T cells can recognize and attack tumor cells, and have achieved remarkable therapeutic effects in the treatment of certain types of hematological malignancies, such as B-cell lymphoma and acute lymphoblastic leukemia. However, the therapeutic effect on solid tumors is poor. Although CAR-T cell therapy has made significant progress as a revolutionary immunotherapy, it also faces some challenges and difficulties, including treatment-related side effects, treatment sustainability, treatment adaptability, treatment costs, and antigen escape. Although adoptive T cell therapy shows potential advantages in cancer treatment, there are limitations to the current in vitro expansion methods, especially IL-2-cultured Th1 cells are prone to exhaustion and apoptosis, and are difficult to expand in vivo or have low persistence; therefore, there is an urgent need to develop new T cell subsets for in vitro culture and expansion. Since 2008, a new CD4 +A subpopulation of T cells was discovered and named Th9 cells, which are characterized by the ability to secrete high levels of IL-9. Studies have shown that Th9, as an immune regulatory cell with multiple advantages, enhances the recognition and killing ability of immune cells by producing IL-9, and has low exhaustion and apoptosis characteristics. However, the content of Th9 cells in the body is extremely limited and cannot be directly isolated and amplified from the body; the current standard method for culturing Th9 cells in vitro is to use TGF-β and IL-4, but the content of IL-9 secreting cells produced is low, which limits its better efficacy in anti-tumor treatment.

[0003] G protein-coupled receptors (GPCRs) are the largest seven-segment transmembrane protein receptor superfamily discovered by scientists so far. They are involved in regulating many physiological processes such as cardiovascular, nervous, immune, metabolic and endocrine systems, and are the most common drug targets. The latest studies have shown that the abnormal function and dysregulated expression of GPCRs are directly related to the tumor itself. They may participate in the occurrence and development of tumors as proto-oncogenes or tumor suppressor genes, including proliferation, survival, angiogenesis, invasion, metastasis, treatment resistance and immune escape. GPCRs also play an important role in tumor immunotherapy. Their importance is reflected in the multiple roles played by this receptor family in inflammation, coordinating immune cell transport and regulating the tumor microenvironment. GPR34 belongs to G protein-coupled receptors. GPR34 is closely related to the human immune system and is highly expressed in immune cells such as mast cells and macrophages, immune system-derived cell lines and lymphoma cells. GPR34 is also preferentially expressed in microglia, and its mRNA level is upregulated in activated microglia after treatment with demyelinating toxins, suggesting that this receptor plays a role in neuroinflammation. GPR34 is highly expressed in multiple solid tumors, such as metastatic melanoma, MALT lymphoma, gastric cancer, and colon cancer, and promotes the proliferation, invasion, and metastasis of these malignancies. Studies have shown that high expression of GPR34 in lymphoma and Hela cells causes phosphorylation of ERK, PKC, and CREB, further inducing CRE, AP1, and NF-κB-mediated gene transcription and promoting cell proliferation. These reports suggest that our small molecule ligand compounds of GPR34 may be involved in blocking the occurrence and development of tumors. However, there have been no reports on inverse agonist compounds for the GPR34 receptor, and their research in tumor immunity has not yet been reported.

[0004] An inverse agonist is a substance that binds to the same receptor as an agonist but causes the opposite pharmacological reaction. Inverse agonists are a new type of drug that acts on receptors. In fact, antagonist compounds include two major categories: neutral antagonists (or silent antagonists) and inverse agonists. After binding to receptors, antagonists and inverse agonists can block the effects of agonists; the difference is that inverse agonists can also bind to receptors in the absence of agonists, thereby blocking the constitutive activity of receptors and making the receptors ineffective. The different receptor protein conformations caused by the two will largely cause differences in downstream protein transcription and gene regulation, resulting in different physiological activities and functions. For example, benzodiazepine receptors will produce sedation after excitement, no biological effects after antagonism, and excitement after inverse excitement. Almost all atypical antipsychotic drugs that were previously considered antagonists have been shown to have inverse agonist activity on type II serotonin receptors, suggesting that the inverse agonist effect of drugs is of great importance in the therapeutic effect. However, there is no feasible strategy for the discovery of inverse agonist drug molecules, which are currently only obtained through random virtual screening in compound libraries. Summary of the invention

[0005] The purpose of the present invention is to provide a CD4+ T cell receptor agonist that has an inverse agonist effect on the GPR34 receptor, can significantly enhance the differentiation of Th9 cells and thus improve their anti-tumor function, especially in the adoptive transfer treatment of solid tumors. + Compounds that differentiate T cells into Th9 cells.

[0006] To achieve the above object, in a first aspect of the present invention, the present invention provides a method for enhancing CD4 + A compound for differentiating T cells into Th9 cells, wherein the structural formula of the compound is as shown in Formula I,

[0007]

[0008] Wherein, R1 is selected from any one of hydrogen, halogen, cyano, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted aromatic, substituted or unsubstituted heteroaromatic;

[0009] R2 is selected from any one of hydrogen, halogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted aromatic group;

[0010] R3 is selected from any one of hydrogen, substituted or unsubstituted C1-C8 alkyl;

[0011] Y is selected from any one of a substituted or unsubstituted C2-C6 alkenyl group, a substituted or unsubstituted aromatic group, and a substituted or unsubstituted heteroaromatic group;

[0012] L is selected from any one of an amide group, an ester group, a thioamide group, and an ether group;

[0013] The substituted group is any one of a halogen, a C1-C4 alkyl group, and an aromatic group.

[0014] The compound of the structure shown in Formula I provided by the present invention has an inverse agonist effect on the GPR34 receptor, which is different from the effect of the agonist or antagonist of the GPR34 receptor. The GPR34 inverse agonist provided by the present invention can effectively enhance the differentiation of T cells into Th9 cells and significantly enhance the anti-tumor function of Th9 cells, especially in the adoptive transfer treatment of solid tumors.

[0015] As the CD4 + A preferred embodiment of the compound for differentiating T cells into Th9 cells, wherein R1 is selected from any one of hydrogen, halogen, substituted or unsubstituted C1-C5 alkyl, substituted or unsubstituted aromatic group;

[0016] The R2 is selected from any one of hydrogen and halogen;

[0017] Said R3 is selected from hydrogen or methyl;

[0018] The Y is selected from any one of C2-C4 alkenyl, substituted or unsubstituted heteroaromatic groups;

[0019] The L is selected from any one of an amide group and an ester group.

[0020] As the CD4 + A preferred embodiment of the compound for differentiating T cells into Th9 cells, wherein R1 is selected from any one of hydrogen, chlorine, cyano, isobutyl, n-butyl, tert-butyl, substituted isobutyl, substituted or unsubstituted methyl, substituted or unsubstituted phenyl, thienyl, and furanyl;

[0021] Said R2 is selected from hydrogen or chlorine;

[0022] Said R3 is selected from hydrogen;

[0023] The Y is selected from vinyl or imidazo[1,2-a]pyridinyl;

[0024] The L is selected from an amide group or an ester group;

[0025] The substituent group is selected from any one of chlorine, fluorine, methyl, ethyl and propyl.

[0026] Preferably, the enhanced CD4 + The chemical structural formula of the compound for T cell differentiation into Th9 cell is any one of Formulas 1-24:

[0027]

[0028]

[0029] The present invention has found that when further selection is made to enhance CD4 + When the compound that differentiates T cells into Th9 cells is the above-mentioned substance, it is possible to more effectively enhance the differentiation of T cells into Th9 cells and the anti-tumor effect of Th9 cells, especially in the adoptive transfer treatment of solid tumors.

[0030] More preferably, the enhanced CD4 + The compounds that can differentiate T cells into Th9 cells are compound 8, compound 15, and compound 17.

[0031] Most preferably, the enhanced CD4 + The compound that differentiates T cells into Th9 cells is compound 8.

[0032] The present invention has found that when further selection is made to enhance CD4 + When the compounds that promote T cell differentiation into Th9 cells are the above three substances, the results show that they can more effectively promote Th9 cell differentiation.

[0033] In a second aspect of the present invention, the present invention provides the enhanced CD4 + A method for preparing a compound for differentiating T cells into Th9 cells, the method comprising the following steps:

[0034] The compound represented by formula C is mixed with carboxylic acid to carry out amide condensation reaction, ester condensation reaction and de-tert-butylation reaction in sequence, and after the reaction, post-treatment is carried out to obtain an enhanced CD4 + Compounds that differentiate T cells into Th9 cells;

[0035] And / or, any one of the compounds represented by formula G, formula G1, formula I, and formula M is mixed with carboxylic acid to sequentially carry out amide condensation reaction and methyl ester hydrolysis reaction, and after the reaction, post-treatment is carried out to obtain an enhanced CD4 + Compounds that differentiate T cells into Th9 cells;

[0036]

[0037] The preparation method provided by the present invention can effectively synthesize the substances corresponding to compounds 1-24 in the present invention.

[0038] As a preferred embodiment of the preparation method of the present invention, the raw materials for synthesizing the substance corresponding to the compound 1-24 are:

[0039] The synthesis of compound 1-4 is carried out by using the compound represented by formula C and the carboxylic acid represented by formula D as raw materials, sequentially carrying out amide condensation reaction, ester condensation reaction, and de-tert-butylation reaction, and performing post-treatment after the reaction to obtain compound 1-4;

[0040] The synthesis of compound 5-20 is carried out by using the compound represented by formula G and the carboxylic acid represented by formula D as raw materials, sequentially carrying out amide condensation reaction and methyl ester hydrolysis reaction, and performing post-treatment after the reaction to obtain compound 5-20;

[0041] The synthesis of compound 21 is carried out by using the compound represented by formula G1 and the carboxylic acid represented by formula D as raw materials, sequentially carrying out amide condensation reaction and methyl ester hydrolysis reaction, and performing post-treatment after the reaction to obtain compound 21;

[0042] The synthesis of compound 22 is carried out by using the compound represented by formula I and the carboxylic acid represented by formula D as raw materials, sequentially carrying out amide condensation reaction and methyl ester hydrolysis reaction, and performing post-treatment after the reaction to obtain compound 22;

[0043] The synthesis of compound 23-24 is carried out by using the compound represented by formula M and the carboxylic acid represented by formula D as raw materials, sequentially carrying out amide condensation reaction and methyl ester hydrolysis reaction, and performing post-treatment after the reaction to obtain compound 23-24;

[0044] Wherein, the carboxylic acid represented by formula D includes the compound represented by formula D1-18, and the structural formula of the formula D1-18 is as follows;

[0045]

[0046]

[0047] As a preferred embodiment of the preparation method of the present invention, at least one of the following (a)-(d):

[0048] (a) The amide condensation reaction is carried out under an inert gas environment at a temperature of 20-30° C. and a reaction time of 6-12 h;

[0049] (b) the ester condensation reaction is carried out under an inert gas environment, at a reaction temperature of 20-30° C., and for a reaction time of 4-8 h;

[0050] (c) the temperature of the de-tert-butylation reaction is 20-30° C. and the reaction time is 20-28 h;

[0051] (d) The temperature of the methyl ester hydrolysis reaction is 40-60° C. and the reaction time is 20-50 min.

[0052] In the third aspect of the present invention, the present invention provides the enhanced CD4 +Use of a compound or a pharmaceutically acceptable salt thereof that causes T cells to differentiate into Th9 cells as a GPR34 inverse agonist.

[0053] The present invention has found that the enhanced CD4 + The compound that induces T cells to differentiate into Th9 cells has a significant inverse agonist effect on GPR34 and can therefore serve as a GPR34 inverse agonist.

[0054] In a fourth aspect of the present invention, the present invention provides the enhanced CD4 + Use of a compound that causes T cells to differentiate into Th9 cells or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting Th9 cell differentiation.

[0055] The present invention has found that the enhanced CD4 + Compounds that induce T cells to differentiate into Th9 cells can effectively enhance the differentiation of Th9 cells, and can be used in the preparation of drugs that promote Th9 cell differentiation.

[0056] In a fifth aspect of the present invention, the present invention provides the enhanced CD4 + Use of a compound or a pharmaceutically acceptable salt thereof that can differentiate T cells into Th9 cells in the preparation of a drug for promoting adoptive T cell therapy for tumors.

[0057] The present invention has found that the GPR34 inverse agonist provided by the present invention can significantly enhance the ability of Th9 adoptive immunity to kill tumor cells, and can be applied to drugs that promote adoptive T cell therapy for tumors.

[0058] Preferably, the tumor is a solid tumor, such as melanoma.

[0059] In a sixth aspect of the present invention, the present invention provides the enhanced CD4 + Use of a compound or a pharmaceutically acceptable salt thereof for differentiating T cells into Th9 cells in the preparation of tumor immunotherapy drugs.

[0060] The present invention has found that the enhanced CD4 + Compounds that differentiate T cells into Th9 cells can promote Th9 cell differentiation, thereby enhancing the efficacy of adoptive T cell therapy, and can then be effectively used in the preparation of tumor immunotherapy drugs.

[0061] As a preferred embodiment of the application of the present invention, the tumor is a solid tumor.

[0062] Exemplarily, the solid tumor is melanoma.

[0063] In a seventh aspect of the present invention, the present invention provides a tumor immunity drug, wherein the tumor immunity drug comprises the CD4 enhancer of the present invention. + A compound or a pharmaceutically acceptable salt thereof that induces T cells to differentiate into Th9 cells.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] The enhanced CD4 + The compound that differentiates T cells into Th9 cells is an inverse agonist of the GPR34 receptor, that is, the present invention proposes for the first time the chemical structure of the inverse agonist of the GPR34 receptor. The inverse agonist of the GPR34 receptor provided by the present invention has an inverse agonist effect on the GPR34 receptor, which is different from the effect of the agonist or antagonist of the GPR34 receptor. The GPR34 inverse agonist provided by the present invention can effectively enhance the differentiation of T cells into Th9 cells, solve the technical problem of low in vitro differentiation efficiency of Th9 cells in the prior art, and can significantly enhance the anti-tumor effect of Th9 cells, especially the effect in adoptive transfer treatment of solid tumors, thereby achieving excellent anti-tumor effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is the test result diagram of compound 8 in Example 2:

[0067] (a) is the result of the excitement mode test;

[0068] (b) is the result diagram of antagonistic mode test;

[0069] Figure 2 This is a graph showing the results of the antagonist compounds X01 and X02 promoting Th9 differentiation in vitro in Example 3;

[0070] Figure 3 This is the result of compound 8 promoting Th9 differentiation in vitro in Example 3;

[0071] Figure 4 This is the result of compound 15 promoting Th9 differentiation in vitro in Example 3;

[0072] Figure 5 This is a graph showing the results of compound 17 promoting Th9 differentiation in vitro in Example 3;

[0073] Figure 6 The results of compound 8 in Example 4 enhancing the ability of Th9 adoptive immunity to kill tumors are shown in the figure:

[0074] (a) is a graph showing the changes in tumor volume over time;

[0075] (b) Comparison of tumor photos. DETAILED DESCRIPTION

[0076] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0077] Unless otherwise specified, the reagents, methods and equipment used in the present invention are all conventional reagents, methods and equipment in the art; and unless otherwise specified, the raw materials used in parallel experiments are from the same batch of raw materials.

[0078] Example 1

[0079] The present invention provides a synthesis of the compound of the present invention, which specifically includes the following contents:

[0080] 1. Synthesis of the compound represented by formula C, the synthetic route is shown below:

[0081]

[0082] The specific steps include:

[0083] (1) N,N'-diisopropylcarbodiimide (7.6 mL, 48.5 mmol) and cuprous chloride (62.3 mg, 0.629 mmol) were dissolved in anhydrous tert-butyl alcohol solvent, protected by argon, and stirred at room temperature for 72 hours. Then, 2 g of cross-linked poly(4-vinylpyridine-ethylvinylbenzene) was added, stirred for 20 minutes, and then filtered through diatomaceous earth. The filtrate was taken and the solvent was dried to obtain (Z)-N,N'-diisopropylcarbamide tert-butyl ester compound A;

[0084] (2) ((Benzyloxy)carbonyl)-L-serine (1 g, 4.18 mmol) and compound A (1.6 g, 8.36 mmol) were dissolved in anhydrous dichloromethane. After stirring at room temperature for 12 hours, the reaction solution was filtered through diatomaceous earth, and the filtrate was taken and the solvent was evaporated. The residue was purified twice by silica gel column chromatography (n-hexane / ethyl acetate = 5:1 to 3:1) to obtain ((benzyloxy)carbonyl)-L-serine tert-butyl ester as white needle-shaped crystals (compound B, 821 mg, yield 66.7%), characterized by: HRMS (ESI-Tof [M+Na] + ):Calcd for C 15 H 21NNaO5:318.1312,Found:318.1300.1H NMR(400MHz, CDCl3):7.34-7.26(5H,m),5.96(1H,d,J=7.32Hz),5.08(2H,s),4.30(1H,t,J=3.72Hz),3.98-3.80(2H,m),1.45(9H,s).13C NMR(100MHz, CDCl3):172.98,169.67,156.42,136.19,128.11,128.05,82.59,67.00,63.30,56.65,27.92;

[0085] (3) Compound B (200 mg, 0.67 mmol) and Pd / C (32 mg) were dissolved in tetrahydrofuran and stirred at room temperature for 1.5 hours under hydrogen protection. The mixture was then filtered through diatomaceous earth and the filtrate was evaporated to dryness to obtain L-serine tert-butyl ester as a white solid (Compound C, 93.4 mg, yield 90.6%); the results were as follows: HRMS (ESI-Tof [2M+Na] + ):Calcd for C 14 H 30 N2NaO6:345.1996,Found:345.2000.1H NMR(400MHz,CDCl3):3.73-3.69(1H,m),3.62-3.58(1H,m),3.38(1H,s),2.84(3H,brs),1.39(9H,s).13C NMR (100MHz, CDCl3): 172.98, 81.62, 63.97, 56.46, 28.01.

[0086] 2. Synthesis of the compound represented by formula G, the synthetic route is shown below:

[0087]

[0088] The specific steps include:

[0089] (1) Boc-Dap-OH (88 mg, 0.43 mmol) and TMS-diazomethane (10% hexane solution, 0.7 mL, 0.47 mmol) were dissolved in a mixed solvent of dichloromethane (10 mL) and methanol (1 mL) and stirred at room temperature overnight. The solvent was evaporated on a rotary evaporator to obtain a colorless oil E (90 mg) without further purification. Then 4-(benzyloxy)benzoic acid (D17, 95 mg, 0.42 mmol), HATU (245 mg, 0.65 mmol) and N, N-diisopropylethylamine (DIPEA, 220 μL, 1.3 mmol) were added, anhydrous dichloromethane was used as solvent, and the mixture was stirred at room temperature overnight under argon protection. Water was added to the reaction solution, and the mixture was extracted with dichloromethane three times (80 mL in total). The combined organic layer was washed once with brine, dried over anhydrous sodium sulfate, filtered and the solvent was dried. The residue was purified by column chromatography (n-hexane / ethyl acetate = 5:1 to 1:1) to obtain compound F as a colorless oil (154 mg, two-step yield 87.5%), characterized by: HRMS (ESI-Tof [M+Na] + ):Calcd for C 23 H 28 N2NaO6:451.1840,Found:451.1820.1H NMR (400MHz, CDCl3): 7.73 (2H, d, J = 8.84Hz), 7.42-7.29 (5H, m), 7.13 (1H, t, J = 5.54Hz), 6.94 (2H, d,J=8.8Hz),5.07(2H,s),4.48-4.46(1H,m),3.78(2H,t,J=5.4Hz),3.74(3H,s),1.40(9H,s).13C NMR(100MHz, CDCl3):171.21,167.30,161.65,136.48,129.27,129.09,128.81,12 8.33,128.29,127.62,126.51,114.79,80.69,70.24,53.96,52.97,43.06,28.41;

[0090] (2) Compound F (154 mg, 0.36 mmol) was dissolved in a dichloromethane (10 mL) solution containing 1.0 mL of TFA and stirred at room temperature overnight. Saturated sodium bicarbonate solution (20 mL) was added to adjust the pH to 10, and the mixture was extracted with dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated. The residue was dried overnight with an oil pump to obtain a white solid compound G (88.5 mg, 77.6%); the compound G was characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C18 H 21 N2O4:329.1496,Found:329.1471.1H NMR (400MHz, CDCl3): 7.74 (2H, d, J = 8.84Hz), 7.44-7.34 (6H, m), 6.99 (2H, d, J = 8.84Hz), 6.66-6 .64(1H,m),5.11(2H,s),3.89-3.83(1H,m),3.77(3H,s),3.72-3.69(1H,m),3.53-3.46(1H,m).

[0091] 3. Synthesis of the compound represented by formula G1, the synthetic route is shown below:

[0092]

[0093] The synthesis route of compound G1 refers to the synthesis of compound G, and the obtained compound F1 is characterized by: RMS (ESI-Tof [M + Na] + ):Calcd for C 23 H 27 ClN2NaO6:485.1450,Found:485.1474. 1 H NMR(400MHz,MeOD):7.77(2H,d,J=8.76Hz),7.45-7.43(2H,m),7.40-7.37(2H,m),7.05(2H, d,J=8.92Hz),5.14(2H,d),4.41-4.37(1H,m),3.73(3H,s),3.72-3.70(2H,m),1.44(9H,s);

[0094] Compound G1 was characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C 18 H 20 ClN2O4:363.1106,Found:363.1110.

[0095] 4. Synthesis of the compound shown in Formula I, the synthetic route is shown below:

[0096]

[0097] The synthetic route of compound I was based on the synthesis of compound G by amide condensation reaction, and the obtained compound H was characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C 29 H 31N4O5:515.2289,Found:515.2298. 1 H NMR (400MHz, CDCl3): 8.34(1H,dd,J=1.0Hz,J=1.36Hz),8.20(1H,s),7.81(1H,t,J=5.8Hz),7.68(2H,d,J=8.36Hz),7.62-7.59(5H,m),7.55(1H,dd,J= 1.72Hz,J=9.44Hz),7.47-7.43(2H,m),7.38-7.34(1H,m),5.86(1H,d,J=7. 6Hz), 4.52 (1H, d, J = 4.88Hz), 3.95-3.83 (2H, m), 3.78 (3H, s), 1.42 (9H, s). 13 C NMR(100MHz, CDCl3):171.22,163.41,155.51,143.65,141.05,140.05,139.43,135.23,128.84,12 7.78,127.62,127.50,127.16,126.93,123.42,117.78,114.88,80.01,54.22,52.60,40.93,28.21;

[0098] Compound I was characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C 24 H 23 N4O3:415.1765,Found:415.1774.

[0099] 5. The synthesis of the compound represented by formula M, the synthesis route is shown below:

[0100]

[0101] The specific steps include:

[0102] (1) Boc-Asp-OH (300 mg, 1.29 mmol) and TMS-diazomethane (10% hexane solution, 6 mL, 3.87 mmol) were dissolved in a mixed solution of dichloromethane (10 mL) and methanol (1 mL) and stirred at room temperature. The reaction was terminated after 10 minutes, and the solvent was dried by spin drying to obtain a colorless oil;

[0103] (2) Then add 0.1M KOH aqueous solution (13mL) and methanol (5mL) and hydrolyze overnight. The entire solution is spin-dried and dried with an oil pump to obtain a white solid K without purification. Then add 4-(benzyloxy)aniline (200mg, 1.00mmol), HATU (458mg, 1.21mmol) and N,N-diisopropylethylamine (DIPEA, 340μL, 2.00mmol), anhydrous dichloromethane as solvent, and stir at room temperature overnight under argon protection. Add water to the solution, extract with dichloromethane 3 times (a total of 120mL), wash the combined organic layer once with saturated brine, dry over anhydrous sodium sulfate, filter and evaporate the solvent. The residue was separated by column chromatography (n-hexane / ethyl acetate = 3:1 to 1:1) to obtain compound L as a white solid (227.2 mg, total yield of three steps 53.08%), characterized by: HRMS (ESI-Tof [M+Na] + ):Calcd for C 23 H 28 N2NaO6:451.1840,Found:451.1815. 1 H NMR (400MHz, CDCl3): 8.43 (1H, s), 7.43-7.26 (7H, m), 6.92 (2H, d, J = 8.9Hz), 5.04 (2H, s), 4.63 (1H ,s),3.72(3H,s),3.04(1H,dd,J=4.3Hz,J=4.3Hz),2.74(1H,dd,J=6.4Hz,J=6.1Hz),1.48(9H,s);

[0104] (3) Compound L (227.2 mg, 0.53 mmol) was dissolved in a dichloromethane solution containing 1.5 mL of TFA and stirred at room temperature for 8 hours. Saturated sodium bicarbonate solution (20 mL) was added to adjust the pH to 10, and the mixture was extracted with a dichloromethane solution. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was dried by rotary evaporation. The mixture was dried overnight with an oil pump to obtain a white solid compound M (180.2 mg, yield 99%); the results were as follows: HRMS (ESI-Tof [M+H] + ):Calcd for C 18 H 21 N2O4:329.1496,Found:329.1509. 1H NMR (400MHz, CDCl3): 10.03 (1H, s), 7.49 (2H, d, J = 8.72Hz), 7.46-7.26 (7H, m), 6.8 0(2H,d,J=8.8Hz),4.88(2H,s),4.67-4.60(1H,m),3.54(3H,s),3.16-3.09(2H,m).

[0105] 6. Synthesis of the compound of formula D, wherein among the compounds of formula D provided, D1-16 is homemade, and D17 and D18 are commercially available. Therefore, the synthesis of the compound of formula D1-16 is provided;

[0106] 1) Synthesis of formula D1, the synthetic route is shown below:

[0107]

[0108] The specific steps include:

[0109] Commercially available reagents trans-4-bromocinnamic acid (113.5 mg, 0.5 mmol) and (4-chlorophenyl)boric acid (117.3 mg, 0.75 mmol) were dissolved in 1,2-dimethoxyethane (20 mL) solvent, and catalyst tetrakis(triphenylphosphine)palladium Pd(PPh3)4 (28.5 mg, 0.025 mmol) and sodium carbonate aqueous solution (219 mg, 2.0 mmol) were added. The reaction was refluxed at 90°C for 4 hours under argon protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was separated by column chromatography (n-hexane / ethyl acetate = 2:1 to 0:1) to obtain (E)-3-(4'-chloro-[1,1'-biphenyl]-4-yl)acrylic acid as a white solid (compound D1, 93.1 mg, yield 70.3%); characterized by: HRMS (ESI-Tof [MH] - ):Calcd for C 15 H 10 ClO2:257.0374,Found:257.0393. 1 H NMR (400MHz, DMSO-d6): 7.80-7.72 (6H, m), 7.62 (1H, d, J = 16.0Hz), 7.53 (2H, d, J = 8.6Hz), 6.59 (1H, d, J = 16.0Hz).

[0110] 2) Synthesis of Formula D2 Referring to the synthesis of D1, the compound of Formula D2 is characterized by: HRMS (ESI-Tof [MH] - ):Calcdfor C 15 H 10ClO2:257.0375,Found:257.0393.

[0111] 3) Synthesis of the compound of formula D3-16, the synthetic route is shown below:

[0112]

[0113] Specifically comprising the following steps: (taking the synthesis of D3 as an example, the synthesis of D4-16 refers to that of D3)

[0114] (1) Dissolve the commercially available reagents 5-bromo-2-aminopyridine (1 g, 5.8 mmol) and ethyl bromopyruvate (1.23 g, 6.35 mmol) in ethanol (30 mL), and reflux the solution for 6 hours. Add 50 mL of water to the reaction solution, and extract it three times with dichloromethane (200 mL in total). Combine the organic layers, wash twice with saturated brine, dry over anhydrous sodium sulfate, filter and spin dry the solvent to obtain ethyl 6-bromoimidazo[1,2-a]pyridine-2-carboxylate as a yellow solid without further purification (1.34 g, yield 86.1%); characterized by: HRMS (ESI-Tof[M+H] + ):Calcd for C 10 H 10 BrN2O2:268.9920,Found:268.9937. 1 H NMR(400MHz, CDCl3):8.30-8.29(1H,m),8.14(1H,d,J=0.72Hz),7.60-7.57(1H,m), 7.32(1H,dd,J=1.84Hz,J=1.84Hz), 4.46(2H,q,J=7.13Hz), 1.44(3H,t,J=7.18Hz);

[0115] (2) The ester (180 mg, 0.67 mmol) and (4-chlorophenyl)boronic acid (198.1 mg, 1.01 mmol) obtained in the previous step were dissolved in 1,2-dimethoxyethane (20 mL) solvent, and a catalyst tetrakis(triphenylphosphine)palladium Pd(PPh3)4 (38.8 mg, 0.034 mmol) and an aqueous sodium carbonate solution (285 mg, 2.69 mmol) were added. The reaction was refluxed at 90°C for 4 hours under argon protection. The reaction solution was filtered through diatomaceous earth, and the filtrate was separated by column chromatography (n-hexane / ethyl acetate = 2:1 to 0:1) to obtain 6-(4-chlorophenyl)imidazo[1,2-a]pyridine-2-carboxylic acid ethyl ester as a white solid (121.1 mg, yield 60.2%); characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C 16 H14 ClN2O2:301.0738,Found:301.0871. 1 H NMR(400MHz, CDCl3):8.27-8.291(1H,t,J=1.26Hz),8.23(1H,s),7.75(2H,d, J=9.44Hz),7.50-7.43(5H,m),4.47(2H,q,J=7.13Hz),1.46(3H,t,J=7.12Hz);

[0116] (3) The ester obtained in the previous step (100 mg, 0.33 mmol) was added to a mixed solution of ethanol (10 mL) and water (2 mL) with an aqueous solution of LiOH (1 mol / L, 2 equivalents of LiOH). The reaction solution was stirred at 50°C for 1 hour, and the reaction progress was monitored by TLC. After the reaction was completed, a 2M HCl solution was added dropwise to adjust the pH to 2. The white solid was filtered out to obtain 6-(4-chlorophenyl)imidazo[1,2-a]pyridine-2-carboxylic acid (compound D3, 77.4 mg, yield 86.2%); characterized by: HRMS (ESI-Tof[MH] - ):Calcd for C 14 H8ClN2O2:271.0280,Found:271.0299. 1 H NMR (400MHz, DMSO-d6): 8.97 (1H, t, J = 1.28Hz), 8.47 (1H, s), 7.76-7.58 (6H, m).

[0117] According to the above method, the only difference between the preparation method of the compound represented by formula D4-15 and the compound represented by formula D3 is that different boric acids are used in step (2). The boric acids used in the synthesis process of the compound represented by formula D4-15 are 4-biphenylboric acid, 4-tert-butylphenylboric acid, 4-phenylboric acid, 4-trifluoromethylphenylboric acid, 3-biphenylboric acid, 4-n-butylphenylboric acid, 4-n-butylbiphenylboric acid, 4-methylbiphenylboric acid, 4-chlorobiphenylboric acid, 4-(2-thiophene)phenylboric acid, 4-(2-furan)phenylboric acid, and 4-cyanophenylboric acid; the difference between the compound represented by D16 is that commercially available reagents 4-bromo-2-aminopyridine and 4-biphenylboric acid are used, and the characterization of the obtained compound of formula D4-16 is as follows:

[0118] D4: HRMS (ESI-Tof[MH] - ):Calcd for C 20 H 13 N2O2:313.0983,Found:313.0999. 1HNMR(400MHz,DMSO-d6):9.14(1H,s),8.62(1H,s),8.03(1H,dd,J=1.44Hz,J=9.4Hz),7.85-7.81(5H,m),7.76(2H,d,J=7.12Hz),7.51(2H,t),7.41(1H,d,J=7.36Hz);

[0119] D5:HRMS(ESI-Tof[M-H] - ):Calcd for C 18 H 17 N2O2:293.1296,Found:293.1308. 1 HNMR(400MHz,DMSO-d6):8.98(1H,s),8.57(1H,s),7.88-7.48(6H,m),1.32(9H,s);

[0120] D6:HRMS(ESI-Tof[M-H]-):Calcd for C 14 H9N2O2:237.0670,Found:237.0695. 1 HNMR(400MHz,DMSO-d6):8.95(1H,dd,J=1.32Hz,J=1.32Hz),8.49(1H,d,J=0.6Hz),7.76-7.70(4H,m),7.55-7.51(2H,m),7.45-7.41(1H,m).

[0121] D7:HRMS(ESI-Tof[M-H] - ):Calcd for C 15 H8F3N2O2:305.0543,Found:305.0555. 1 HNMR(400MHz,DMSO-d6):9.06(1H,dd,J=1.44Hz,J=1.44Hz),8.50(1H,s),7.95(2H,J=8.28Hz),7.88(2H,d,J=8.44Hz),7.75(2H,d,J=1.76Hz);

[0122] D8:HRMS(ESI-Tof[M-H] - ):Calcd for C 20 H 13 N2O2:313.0983,Found:313.0985;

[0123] D9:HRMS(ESI-Tof[M-H] - ):Calcd for C 18 H 17 N2O2:293.1296,Found:293.1317. 1 HNMR(400MHz,CDCl3):8.92(1H,s),8.49(1H,s),7.77-7.69(2H,m),7.53(2H,d,J=8.2Hz),7.34(2H,d,J=8.24Hz),2.65-2.58(2H,m),1.63-1.55(2H,m),1.36-1.30(2H,m),0.91(3H,t,J=7.28Hz);

[0124] D10:HRMS(ESI-Tof[M-H] - ):Calcd for C 24 H 21 N2O2:369.1609,Found:369.1619. 1 HNMR(400MHz,DMSO-d6):9.04-9.06(1H,m),8.53(1H,s),7.89-7.74(6H,m),7.65(2H,d,J=8.2Hz),2.63(2H,t,J=7.52Hz),1.59(2H,m),1.34(2H,m),0.92(3H,t,J=7.32Hz);

[0125] D11:HRMS(ESI-Tof[M-H] - ):Calcd for C 21 H 25 N2O2:327.1139,Found:327.1146. 1 HNMR(400MHz,CDCl3):9.22(1H,s),8.71(1H,s),8.20(1H,dd,J=1.56Hz,J=9.52Hz),7.88(1H,d,J=9.52Hz),7.84(4H,s),7.66(2H,d,J=8.12Hz),7.31(2H,d,J=8.12Hz),4.49(2H,q,J=7.12Hz),2.36(3H,s);

[0126] D12:HRMS(ESI-Tof[M-H] - ):Calcd for C 20 H 12ClN2O2:347.0593,Found:347.0628. 1 H NMR(400MHz,DMSO-d6):9.02(1H,dd,J=1.04Hz,J=1.72Hz),8.49(1H,d,J=0.56Hz),7.83(4H,s),7.80-7.77(3H,m),7.73-7.71(1H,m),7.55(2H,d,J=8.6Hz);

[0127] D13:HRMS(ESI-Tof[M-H] - ):Calcd for C 18 H 11 N2O2S:319.0547,Found:319.0548. 1 HNMR(400MHz,DMSO-d6):9.09-9.06(1H,m),8.56(1H,d,J=5.0Hz),7.91-7.76(7H,m),7.63-7.58(2H,m),7.19-7.17(1H,m);

[0128] D14:HRMS(ESI-Tof[M-H] - ):Calcd for C 18 H 11 N2O3:303.0775,Found:303.0787;

[0129] D15:HRMS(ESI-Tof[M-H] - ):Calcd for C 15 H8N3O2:262.0622,Found:262.0661;

[0130] D16:HRMS(ESI-Tof[M-H] - ):Calcd for C 20 H 13 N2O2:313.0983,Found:313.0999. 1 HNMR(400MHz,DMSO-d6):8.75(1H,d,J=7.28Hz),8.62(1H,s),7.99-7.95(3H,m),7.85(2H,d,J=8.6Hz),7.77-7.74(2H,m),7.64(1H,dd,J=1.76Hz,J=7.24Hz),7.53-7.46(2H,m),7.43-7.37(1H,m)。

[0131] 7. Synthesis of compound 1-4. The synthesis of compound 1-4 is shown in the following formula:

[0132]

[0133] Specifically, it includes amide condensation reaction, ester condensation reaction and TFA de-tert-butylation reaction. The specific operation steps are taken as an example of compound 4 (the synthesis process of compounds 1-3 refers to compound 4):

[0134] (1) 6-(4-Biphenyl)imidazo[1,2-a]pyridine-2-carboxylic acid (D4, 185 mg, 0.59 mmol) and L-serine tert-butyl ester (intermediate C, 95 mg, 0.59 mmol) were added to an anhydrous dichloromethane solution containing a condensing agent HATU (269 mg, 0.71 mmol) and N,N-diisopropylethylamine (DIPEA, 204 μL, 1.18 mmol), and the reaction solution was stirred at room temperature for 8 hours under argon protection. After the reaction was completed, water (40 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane three times (a total of 100 mL). The organic layers were combined, washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was dried. The residue was purified by column chromatography (n-hexane / ethyl acetate = 3:1 to 0:1) to obtain (6-(4-biphenyl)imidazo[1,2-a]pyridine-2-carbonyl)-L-serine tert-butyl ester as a white solid (198.9 mg, yield 71.4%), characterized by: HRMS (ESI-Tof [M+H] + ):Calcd for C 27 H 28 N3O4:458.2074,Found:458.2069. 1 H NMR(400MHz, CDCl3):8.14(2H,d,J=8.92Hz),8.09(1H,s),7.51-7.46(4H,m),7.43-7.39(3 H,m),7.36-7.26(4H,m),5.03(1H,brs),4.77-4.72(1H,m),4.09-4.08(2H,m),1.46(9H,s). 13 C NMR (100MHz, CDCl3):169.27,162.73,143.49,140.55,139.79,139.26,134.89,128.72 ,127.47,126.83,126.70,126.49,123.21,117.49,114.95,82.25,63.12,55.36,27.70;

[0135] (2) The amide obtained above (79.6 mg, 0.174 mmol) was mixed with 4-(benzyloxy)benzoic acid (40 mg, 0.18 mmol) in anhydrous dichloromethane solution, and EDCI·HCl (50 mg, 0.26 mmol) and DMAP (4.4 mg, 0.036 mmol) were added at the same time. The mixture was stirred at room temperature for 6 hours under argon protection. Water was added to the solution, and the mixture was extracted with dichloromethane three times (a total of 100 mL). The organic layers were combined, washed once with saturated brine (20 mL), and then dried over anhydrous sodium sulfate, filtered, and the solvent was dried. The residue was purified by column chromatography (n-hexane / EtOAc=3:1~0:1) to obtain the ester as a colorless oil (63.4 mg, yield 40.2%), characterized by: HRMS (ESI-Tof[M+H] + ):Calcd for C 41 H 38 N3O6:668.2755,Found:668.2682. 1 H NMR (400MHz, DMSO-d6): 9.04 (1H, t, J = 1.6Hz), 8.63 (1H, s, J = 7.92Hz), 8.44 (1H, s), 7.90 (2H, d, J = 8.92Hz), 7.83-7.72 (8H, m), 7.52-7.45(4H,m),7.41-7.34(4H,m),7.14(2H,d,J=8.96Hz),5.18(2H,s),4.92-4.87(1H,m),4.69-4.60(2H,m),1.41(9H,s). 13 CNMR(100MHz,DMSO-d6):168.34,164.97,162.41,162.01,143.22,139.67,139.33,139.20,136.35,135.17,131.30,129.00,128.46,128. 02,127.85,127.68,127.36,127.06,126.60,126.46,125.59,124.77 ,121.64,117.40,115.44,114.86,81.70,69.53,63.71,51.85,27.55;

[0136] (3) The ester obtained in the previous step (46.2 mg, 0.069 mmol) was dissolved in TFA (1.0 mL) and the tert-butyl group was deprotected. The reaction solution was stirred at room temperature for 24 hours, the solvent was evaporated and dried overnight by an oil pump to obtain a light yellow solid (compound 4, 43 mg, yield 95.1%);

[0137] The compound 4 was characterized by: mp 215.8-217.2°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 28 N3O6:610.1984,Found:610.1954. 1 H NMR (400MHz, DMSO-d6): 9.06 (1H, s), 8.69 (1H, d, J = 7.64Hz), 8.46 (1H, s), 7.89 (2H, d, J = 8.92Hz), 7.84-7.82 (5H, m ),7.76-7.73(3H,m),7.52-7.33(8H,m),7.13(2H,d,J=8.96Hz),5.18(2H,s),4.98-4.93(1H,m),4.74-4.59(2H,m). 13 C NMR(100MHz,DMSO-d6):170.64,165.02,162.35,161.53,142.85,139.74,139.29,138.38,136.33,134.99,131.28,128.99,12 8.45,127.99,127.80,127.69,127.36,127.08,126.59,125.90,124.94,121.70,116.94,115.44,114.82,69.50,63.66,51.21.

[0138] Compound 1-3 was obtained by a similar method. The only difference between the synthesis of compound 1-3 and the synthesis of compound 1 is that the carboxylic acid used in step (1) is different. The carboxylic acids used in compound 1-3 are compounds represented by formula D2, formula D1, and formula D3, respectively. The characterizations of the obtained compounds 1-3 are as follows:

[0139] Compound 1: mp 214.2-215.0℃. HRMS (ESI-Tof [MH] - ):Calcd for C 32 H 25 ClNO6:554.1376,Found:554.1445. 11H NMR (400 MHz, DMSO-d6): 13.12 (1H, brs), 8.66 (1H, d, J = 8.0 Hz), 8.86 (1H, brs), 7.93 (2H, d, J = 9.72 Hz), 7.77 - 7.74 (4H, m), 7.69 - 7.67 (2H, m), 7.55 - 7.50 (3H, m), 7.47 - 7.45 (2H, m), 7.42 - 7.32 (3H, m), 7.14 (2H, d, J = 9.0 Hz), 6.89 (2H, d, J = 15.84), 5.20 (2H, s), 4.88 - 4.83 (1H, m), 4.59 - 4.47 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): 170.80, 164.99, 162.37, 139.69, 138.87, 138.08, 136.37, 134.24, 132.67, 131.46, 128.93, 128.46, 128.37, 128.28, 128.01, 127.81, 127.10, 121.75, 121.70, 114.78, 69.51, 64.01, 51.38. Anal. Calcd for C 32 H 26 ClNO6·0.4H2O; C, 68.24; H, 4.80; N, 2.49. Found C, 68.60; H, 4.82; N, 2.51;

[0140] Compound 2: m.p. 187 - 188 °C. HRMS (ESI-Tof [M - H] - ): Calcd for C 32 H 25 ClNO6: 554.1376, Found: 554.1373. 1 1H NMR (400 MHz, DMSO-d6): 8.60 (1H, d, J = 7.88 Hz), 7.94 (2H, d, J = 9.0 Hz), 7.88 (1H, s), 7.75 (2H, d, J = 8.72 Hz), 7.70 - 7.67 (1H, m), 7.61 - 7.50 (5H, m), 7.47 - 7.34 (5H, m), 7.14 (2H, d, J = 9.04 Hz), 6.96 (1H, d, J = 15.84 Hz), 5.19 (2H, s), 4.86 - 4.82 (1H, m), 4.58 - 4.54 (1H, m), 4.51 - 4.48 (1H, m). 13C NMR(100MHz,DMSO-d6):170.76,165.00,164.94,162.36,139.41,139.17,138.40,136.37,135.57,132.61,131.45,12 9.64,128.89,128.52,128.46,128.00,127.81,127.76,126.80,125.96,122.27,121.72,114.77,69.50,64.12,51.47;

[0141] Compound 3: mp 198.7-199.5°C. HRMS (ESI-Tof [MH] - ):Calcd for C 31 H 23 ClN3O6:568.1281,Found:568.1323. 1 H NMR(400MHz,DMSO-d6):9.14-9.01(1H,m),8.61-8.57(1H,m),7.93-7.87(3H,m),7.85-7.77(3H,m),7.61(2H ,d,J=8.6Hz),7.47-7.31(6H,m),7.12(2H,d,J=8.84Hz),5.18(2H,s),4.99-4.94(1H,m),4.73-4.59(2H,m). 13 C NMR(100MHz,DMSO-d6):170.60,170.48,166.93,165.05,162.38,160.84,136.36,134.68,134.54,133.24,132.76,131.35,131.32,12 9.22,129.07,128.90,128.53,128.48,128.03,127.99,127.83,127.80,125.80,121.70,115.71,114.84,114.60,69.52,63.57,51.41.

[0142] 8. Synthesis of compound 5-24. The synthesis of compound 5-24 is shown in the following formula:

[0143]

[0144] Specifically including amide condensation reaction and methyl ester hydrolysis reaction, the specific operation steps are taken as an example of compound 8 (the synthesis process of compounds 5-7 and 9-24 refers to compound 8):

[0145] (1) 6-([1,1'-biphenyl]-4-yl)imidazo[1,2-a]pyridine-2-carboxylic acid (D4, 50 mg, 0.159 mmol) and intermediate G (62.6 mg, 0.19 mmol) were mixed in anhydrous dichloromethane, and HATU (72.5 mg, 0.19 mmol) and DIPEA (55 μL, 0.32 mmol) were added. The mixture was stirred at room temperature for 8 hours under argon protection. Water (50 mL) was added to the solution, and the mixture was extracted with dichloromethane three times (a total of 100 mL). The organic layers were combined, washed once with saturated brine (20 mL), dried over anhydrous sodium sulfate, filtered, and the solvent was dried. The residue was purified by column chromatography (first eluted with n-hexane / ethyl acetate = 1:1 for 4 column volumes, then eluted with acetate / methanol = 10:1) to give a white solid (41.8 mg, yield 42.2%), characterized by HRMS (ESI-Tof [M+H] + ):Calcd for C 38 H 33 N4O5:625.2445,Found:625.2399.1H NMR (400MHz, CDCl3): 8.66 (1H, d, J = 6.64Hz), 8.39 (1H, s), 8.32 (1H, s), 7.83 (2H, d, J = 8.88Hz), 7.74-7.70 (3H, m), 7.67-7.61 ( 6H,m),7.54-7.46(3H,m),7.42-7.31(7H,m),6.96(2H,d,J=8.88Hz),5.07(2H,s),4.99-4.92(1H,m),4.09-3.95(2H,m),3.81( 3H,s).13CNMR(100MHz,CDCl3):170.58,167.48,161.48,141.66,140.13,136.55,134.84,129.25,129.16,129.08,128.82,12 8.75,128.23,128.11,127.94,127.58,127.45,127.16,126.65,123.80,117.32,115.74,114.66,70.14,53.18,53.06,38.73;

[0146] (2) 15 mg (0.024 mmol) of the product obtained in the previous step was taken for ester hydrolysis, methanol (5 mL) and water (1 mL) were used as solvents, and LiOH aqueous solution (1 mol / L, 2 eq) was added. The reaction solution was heated and stirred at 50°C for 30 minutes, and the reaction was completed. 2M HCl solution was added dropwise to adjust the pH to 2, and the white solid was filtered, washed with ether, and dried with an oil pump to obtain a white solid (Compound 8, 11.1 mg, yield 75.8%), characterized by: mp 244.0-245.3°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 29 N4O5:609.2143,Found:609.1972. 1 H NMR (400MHz, DMSO-d6): 9.25 (1H, s), 9.16 (1H, brs), 8.70-8.66 (2H, m), 8.06 (1H, d, J = 9.52Hz), 7.89-7.82 (6H, m), 7.77-7. 75(2H,m),7.53-7.49(2H,m),7.46-7.31(5H,m),7.07(2H,d,J=9.0Hz),5.16(2H,s),4.72-4.67(1H,m),3.83-3.71(2H,m). 13 C NMR(100MHz,DMSO-d6):171.39,166.24,160.67,140.06,139.20,136.63,129.07,129.01,128 .42,127.90,127.78,127.70,127.42,127.26,126.63,126.54,115.63,114.29,69.30,52.68.

[0147] Compounds 5-7 and 9-24 were obtained by similar methods. The only difference between the synthesis of compounds 5-7 and 9-24 and the synthesis of compound 8 is that the carboxylic acids used in step (1) are different. The carboxylic acids used in compound 5-7 are compounds represented by formula D2, D1, and D3, respectively. The carboxylic acids used in compound 9-24 are compounds represented by formula D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D4, D17, D3, and D4, respectively. The characterizations of the obtained compounds 5-7 and 9-24 are as follows:

[0148] Compound 5: mp 166.1-167.3°C. HRMS (ESI-Tof [MH] - ):Calcd for C32 H 26 ClN2O5: 553.1536, Found: 553.1580. 1 1H NMR (400 MHz, DMSO-d6): δ 8.71 (1H, s), 8.11 (1H, d, J = 6.32 Hz), 7.92 (1H, s), 7.78 - 7.75 (4H, m), 7.54 - 7.44 (7H, m), 7.41 - 7.33 (5H, m), 7.07 (2H, d, J = 8.88 Hz), 7.01 (1H, d, J = 15.96 Hz), 5.15 (2H, s), 4.29 - 4.26 (1H, m), 3.63 - 3.33 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): δ 171.50, 165.49, 164.92, 160.55, 139.31, 138.43, 136.68, 135.78, 132.57, 129.57, 128.87, 128.76, 128.52, 128.43, 127.90, 127.76, 126.99, 125.88, 123.34, 114.37, 69.33, 53.13;

[0149] Compound 6: m.p. 253.3 - 254.4 °C. HRMS (ESI-Tof [M-H] - ): Calcd for C 32 H 26 ClN2O5: 553.1536, Found: 553.1531. 1 1H NMR (400 MHz, DMSO-d6): δ 8.65 (1H, t, J = 5.18 Hz), 8.33 - 8.31 (1H, m), 7.78 (2H, d, J = 9.8 Hz), 7.74 - 7.67 (6H, m), 7.52 (2H, d, J = 8.76 Hz), 7.48 - 7.31 (7H, m), 7.07 (2H, d, J = 8.96 Hz), 6.88 (1H, d, J = 15.8 Hz), 5.15 (2H, s), 4.44 - 4.38 (1H, m), 3.64 - 3.52 (2H, m). 13C NMR(100MHz,DMSO-d6):171.80,165.85,165.02,160.73,139.60,138.33,138.16,136.70,134.40,132.67,128.97,128.95,1 28.90,128.48,128.40,128.33,127.95,127.79,127.09,126.88,126.05,122.47,114.38,106.75,69.37,52.88.Anal.Calcd forC 32 H 27 ClN2O5·H2O; C, 67.07; H, 4.89; N, 5.10. Found C, 67.13; H, 5.07; N, 4.78;

[0150] Compound 7: mp231.2-232.9°C. MHz, DMSO-d6): 9.11 (1H, s), 8.91 (1H, brs), 8.64 (1H, t, J = 5.92 Hz), 8.52 (1H, s), 7.89-7.76 (6H, m), 7.61 (2H, d, J = 8.68 Hz), 7.46-7.27 (5H, m), 7.07 (2H, d, J = 8.96 Hz), 5.16 (2H, s), 4.71-4.66 (1H, m), 3.77-3.72 (2H, m). 13 C NMR(100MHz,DMSO-d6):171.47,166.28,160.65,136.61,134,68,133.07,129.14,129.03,128. 44,128.40,127.88,127.69,126.56,125.54,115.48,114.28,69.29,52.55,28.47.Anal.Calcd for C 31 H 25 ClN4O5·H2O·HCl; C, 59.72; H, 4.53; N, 8.99. Found C, 60.34; H, 4.70; N, 8.72;

[0151] Compound 9: mp 180.8-182.3°C. HRMS (ESI-Tof [MH] - ):Calcd for C 35 H 33 N4O5:589.2456,Found:589.2467. 11H NMR (400 MHz, DMSO-d6): δ 8.92 (1H, d, J = 1.12 Hz), 8.63 - 8.59 (2H, m), 8.38 (1H, s), 7.79 (2H, d, J = 8.92 Hz), 7.70 (1H, s), 7.64 (2H, d, J = 8.44 Hz), 7.53 (2H, d, J = 8.52 Hz), 7.46 - 7.32 (5H, m), 7.07 (2H, d, J = 8.92 Hz), 5.15 (2H, s), 4.70 - 4.65 (1H, m), 3.77 - 3.73 (2H, m), 1.32 (9H, s). 13 13C NMR (100 MHz, DMSO-d6): δ 171.69, 166.33, 162.20, 160.70, 150.57, 143.14, 139.47, 136.68, 133.36, 129.07, 128.46, 127.93, 127.75, 126.66, 126.54, 126.29, 125.96, 124.41, 117.30, 115.26, 114.33, 69.36, 52.51, 34.34, 31.07;

[0152] Compound 10: m.p. 160.5 - 161.8 °C. HRMS (ESI-Tof [M-H] - ): Calcd for C 31 H 25 N4O5: 533.1830, Found: 533.1906. 1 1H NMR (400 MHz, DMSO-d6): δ 9.09 (1H, s), 8.91 (1H, brs), 8.65 (1H, t, J = 5.90 Hz), 8.53 (1H, s), 7.90 - 7.74 (5H, m), 7.56 - 7.52 (2H, m), 7.47 - 7.44 (3H, m), 7.41 - 7.32 (3H, m), 7.07 (2H, d, J = 8.88 Hz), 5.16 (2H, s), 4.71 - 4.66 (1H, m), 3.78 - 3.74 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): δ 171.52, 166.29, 160.69, 136.65, 135.77, 129.23, 129.07, 128.45, 128.28, 127.92, 127.73, 126.71, 126.60, 125.38, 115.50, 114.32, 69.33, 52.60;

[0153] Compound 11: mp 252.0-253.4°C. HRMS (ESI-Tof [MH] - ):Calcd for C 32 H 24 F3N4O5:601.1704,Found:601.1695. 1 H NMR (400MHz, DMSO-d6): 9.21 (1H, s), 8.94 (1H, d, J = 6.76Hz), 8.65 (1H, t, J = 5.96Hz), 8.56 (1H, s), 8.00-7 .80(8H,m),7.45-7.30(5H,m),7.07(2H,d,J=9.0Hz),5.15(2H,s),4.70-4.65(1H,m),3.78-3.74(2H,m). 13 C NMR(100MHz,DMSO-d6):171.45,166.26,160.66,142.84,139.99,136.62,129.04,128.97,128.69,128.41 ,128.25,128.05,127.97,127.88,127.69,127.49,126.56,126.04,126.01,115.66,114.28,69.30,52.61;

[0154] Compound 12: mp 158.1-159.3°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 29 N4O5:609.2143,Found:609.2111. 1 H NMR(400MHz,DMSO-d6):9.29(1H,s),9.16(1H,brs),8.69(1H,t,J=5.92Hz),8.64(1H,s),8.11(1H,d,J=8.84Hz),8.03(1H,s),7.84-7.75(7H,m ),7.64(1H,t,J=7.72Hz),7.52(2H,t,J=7.4Hz),7.45-7.32(6H,m),7.0 7(2H,d,J=8.84Hz),5.16(2H,s),4.71-4.66(1H,m),3.80-3.75(2H,m). 13C NMR(100MHz,DMSO-d6):171.41,166.25,160.69,141.22,139.75,136.65,136.18,129.88,129.09,128 .97,128.44,127.91,127.80,127.72,126.97,126.56,125.86,125.22,115.58,114.31,69.32,52.73;

[0155] Compound 13: m, p. 215.7-217.0℃. HRMS (ESI-Tof [MH] - ):Calcd for C 35 H 33 N4O5:589.2456,Found:589.2505. 1 H NMR(400MHz,DMSO-d6):12.84(1H,brs),8.93(1H,t,J=1.36Hz),8.62-8.60(2H,m),8 .37(1H,s),7.79(2H,d,J=8.88Hz),7.70(2H,s),7.63(2H,d,J=8.2Hz),7.45-7.32(7H ,m),7.07(2H,d,J=8.96Hz),5.15(2H,s),4.70-4.65(1H,m),3.75(2H,t,J=6.32Hz), 2.63(2H,t,J=7.52Hz),1.62-1.55(2H,m),1.37-1.28(2H,m),0.91(3H,t,J=7.32Hz). 13 C NMR(100MHz,DMSO-d6):171.67,166.31,162.15,160.68,143.08,142.32,136.66,133.54,129.09,129.06,128.44,1 27.92,127.74,126.64,126.43,126.04,124.41,117.22,115.24,114.32,69.34,52.49,34.40,33.03,21.72,13.77;

[0156] Compound 14: mp 242.6-244.1°C. HRMS (ESI-Tof [MH] - ):Calcd for C 41 H 37 N4O5:665.2769,Found:665.2807. 11H NMR (400 MHz, DMSO-d6): δ 9.07 (1H, s), 8.74 (1H, d, J = 7.56 Hz), 8.63 (1H, t, J = 5.84 Hz), 8.44 (1H, s), 7.89 - 7.79 (8H, m), 7.75 (1H, d, J = 9.48 Hz), 7.66 (2H, d, J = 9.48 Hz), 7.46 - 7.30 (7H, m), 7.07 (2H, d, J = 8.92 Hz), 5.16 (2H, s), 4.71 - 4.66 (1H, m), 3.79 - 3.75 (2H, m), 2.63 (2H, t, J = 7.64 Hz), 1.63 - 1.56 (2H, m), 1.38 - 1.29 (2H, m), 0.92 (3H, t, J = 7.32 Hz). 13 13C NMR (100 MHz, DMSO-d6): δ 171.59, 166.30, 160.67, 141.95, 139.69, 137.79, 136.64, 134.69, 129.05, 128.94, 128.42, 127.90, 127.72, 127.11, 127.04, 126.62, 126.45, 124.07, 117.15, 115.34, 114.30, 69.32, 52.52, 34.41, 33.04, 21.73, 13.76;

[0157] Compound 15: m.p. 226.1 - 227.4 °C. HRMS (ESI-Tof [M - H] - ): Calcd for C 38 H 31 ClN4O5: 623.2300, Found: 623.2345. 1 1H NMR (400 MHz, DMSO-d6): δ 9.12 (1H, s), 8.86 (1H, brs), 8.63 (1H, t, J = 5.76 Hz), 8.50 (1H, s), 7.93 - 7.76 (8H, m), 7.65 (2H, d, J = 8.16 Hz), 7.45 - 7.29 (7H, m), 7.07 (2H, d, J = 8.88 Hz), 5.15 (2H, s), 4.71 - 4.66 (1H, m), 3.77 - 3.74 (2H, m), 2.36 (3H, s). 13C NMR(100MHz,DMSO-d6):171.54,166.32,160.69,139.78,137.11,136.65,136.36,129.61,129.07 ,128.44,127.91,127.72,127.11,126.62,126.44,125.13,115.44,114.32,69.33,52.57,20.68;

[0158] Compound 16: mp 253.9-255.6°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 28 ClN4O5:643.1754,Found:643.1777. 1 H NMR(400MHz, DMSO-d6):9.23(1H,s),9.10(1H,s),8.69-8.64(2H,m),8.04(1H,d,J=9.32Hz),7.87(4H,s),7.83-7.79(5H, s),7.56(2H,d,J=8.68Hz),7.46-7.32(5H,m),7.07(2H,d,J=8.96Hz),5.16(2H,s),4.71-4.66(1H,m),3.83-3.71(2H,m). 13 C NMR(100MHz,DMSO-d6):171.42,166.27,160.69,138.66,138.03,136.65,134.87,132.66,129.08,128.97 ,128.44,128.42,127.91,127.72,127.40,127.32,126.58,125.70,115.64,114.32,69.33,52.70,37.51;

[0159] Compound 17: mp 225.1-226.5℃HRMS (ESI-Tof [MH] - ):Calcd for C 35 H 27 N4O5S - :615.1708,Found:615.1736. 1H NMR(400MHz,DMSO-d6):9.11(1H,s),8.81(1H,brs),8.63(1H,t,J=5.84Hz),8.47(1H,d,J=5.44Hz),7.88-7.75(9H,m),7.63 -7.58(2H,m),7.46-7.31(5H,m),7.19-7.17(1H,m),7.07(2H,d,J=8.92Hz),5.16(2H,s),4.71-4.66(1H,m),3.76(2H,brs). 13 C NMR(100MHz,DMSO-d6):171.54,166.29,160.66,142.54,136.62,133.49,129.04,128.63,128.41 ,127.89,127.70,127.22,127.12,126.59,126.03,125.93,124.15,115.41,114.29,69.30,52.53;

[0160] Compound 18: mp 219.7-221.1°C. HRMS (ESI-Tof [MH] - ):Calcd for C 35 H 27 N4O6:599.1936,Found:599.1967. 1 H NMR (400MHz, DMSO-d6):9.18-9.11(1H,m),8.97-8.92(1H,m),8.67-8.65(1H,m),8.55-8.52(1H,m),7. 89-7.67(9H,m),7.45-7.31(5H,m),7.08-7.06(2H,m),5.15(2H,s),4.69-4.68(1H,m),3.76(2H,brs). 13 C NMR(100MHz,DMSO-d6):171.45,166.25,160.65,152.38,145.45,143.30,136.61,132.06,131.85,129.04,128 .74,128.70,128.40,127.87,127.69,127.30,127.25,127.11,126.55,124.08,115.52,114.28,69.29,52.61;

[0161] Compound 19: mp 265.0-266.3°C. HRMS (ESI-Tof [MH]- ): Calculated for C 32 H 24 N5O5: 558.1783, Found: 558.1806. 1 1H NMR (400 MHz, DMSO-d6): 9.12 (1H, s), 8.69 - 8.60 (1H, m), 8.40 (1H, s), 8.01 - 7.94 (3H, m), 7.81 - 7.77 (4H, m), 7.45 - 7.32 (5H, m), 7.07 (2H, d, J = 8.88 Hz), 5.15 (2H, s), 4.70 - 4.65 (1H, m), 3.77 - 3.74 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): 171.63, 166.35, 161.87, 160.70, 143.14, 140.87, 136.67, 133.07, 129.98, 129.07, 128.68, 128.46, 127.93, 127.77, 127.75, 127.43, 126.85, 126.65, 126.29, 124.43, 118.71, 117.41, 115.58, 114.33, 110.56, 69.35, 52.55;

[0162] Compound 20: m.p. 243.5 - 244.9 °C. HRMS (ESI-Tof [M - H] - ): Calculated for C 37 H 29 N4O5: 609.2143, Found: 609.2111. 1 1H NMR (400 MHz, DMSO-d6): 9.16 (1H, brs), 8.89 (1H, d, J = 7.08 Hz), 8.70 - 8.67 (2H, m), 8.00 - 7.98 (3H, m), 7.89 - 7.71 (7H, m), 7.53 - 7.49 (2H, m), 7.45 - 7.30 (6H, m), 7.07 (2H, d, J = 8.84 Hz), 5.16 (2H, s), 4.72 - 4.67 (1H, m), 3.82 - 3.73 (2H, m). 13CNMR(100MHz,DMSO-d6):171.33,166.22,160.66,140.96,139.08,136.61,129.06,129.03 ,128.40,127.87,127.68,127.55,127.43,126.66,126.52,115.20,114.28,69.29,52.71;

[0163] Compound 21: mp 264.6-265.7°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 28 ClN4O5:643.1754,Found:643.1731. 1 H NMR (400MHz, DMSO-d6): 9.08 (1H, s), 8.72 (1H, d, J = 7.28Hz), 8.63 (1H, t, J = 5.68Hz), 8.44 (1H, s), 7.86-7. 74(10H,m),7.52-7.38(7H,m),7.07(2H,d,J=8.92Hz),5.16(2H,s),4.71-4.66(1H,m),3.78-3.75(2H,m). 13 C NMR(100MHz,DMSO-d6):171.58,166.26,160.46,139.72,139.28,135.70,135.00,132.45,129.51,129.05 ,128.99,128.42,127.68,127.35,127.08,126.74,126.59,124.93,115.36,114.31,68.44,52.49,32.49;

[0164] Compound 22: mp 265.5-266.9°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 29 N4O5:609.2143,Found:609.2232. 11H NMR (400 MHz, DMSO-d6): δ 9.14 (1H, s), 8.91 (1H, br s), 8.67 (1H, d, J = 7.16 Hz), 8.50 (1H, s), 7.94 - 7.84 (7H, m), 7.75 (3H, d, J = 8.16 Hz), 7.52 - 7.45 (4H, m), 7.42 - 7.33 (4H, m), 7.11 (2H, d, J = 8.84 Hz), 5.18 (2H, s), 4.61 - 4.56 (1H, m), 3.88 - 3.71 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): δ 171.80, 165.90, 160.82, 141.89, 139.89, 139.27, 136.64, 134.76, 129.20, 129.02, 128.45, 127.93, 127.76, 127.40, 127.17, 126.63, 126.23, 115.34, 114.37, 69.35, 53.16;

[0165] Compound 23: m.p. 273.7 - 275.6 °C. HRMS (ESI-Tof [M - H] - ): Calcd for C 31 H 24 ClN4O5: 567.1441, Found: 567.1510. 1 1H NMR (400 MHz, DMSO-d6): δ 10.00 - 9.97 (1H, m), 9.00 (1H, d, J = 5.72 Hz), 8.55 (1H, d, J = 8.44 Hz), 8.39 (1H, d, J = 5.96 Hz), 7.77 - 7.72 (4H, m), 7.58 (2H, d, J = 8.48 Hz), 7.48 (2H, d, J = 8.92 Hz), 7.44 - 7.31 (5H, m), 6.94 (2H, d, J = 9.0 Hz), 5.05 (2H, s), 4.95 - 4.83 (1H, m), 3.02 - 2.85 (2H, m). 13 13C NMR (100 MHz, DMSO-d6): δ 172.20, 167.74, 161.14, 153.81, 142.76, 139.17, 136.76, 134.74, 132.44, 131.99, 128.73, 127.98, 127.36, 127.26, 125.88, 124.66, 124.43, 120.57, 120.13, 117.04, 114.88, 114.41, 68.93, 48.02, 34.56;

[0166] Compound 24: mp 255.3-256.6°C. HRMS (ESI-Tof [MH] - ):Calcd for C 37 H 29 N4O5:609.2143,Found:609.2244. 1 H NMR(400MHz,DMSO-d6):9.98 -10.01(1H,m),9.09-9.14(1H,m),8.70(1H,d,J=8.8Hz),8.58-8.46(1H,m),7.87-7.74(9H,m),7.53-7. 47(4H,m),7.44-7.31(7H,m),6.95(2H,d,J=9.0Hz),5.05(2H,s),4.97-4.86(1H,m),3.02-2.86(2H,m). 13 CNMR(100MHz,DMSO-d6):172.52,171.59,168.10,154.30,139.75,139.35,137.14,135.06,132.38,128.9 4,128.29,127.56,127.30,127.04,126.54,124.94,121.05,120.46,115.42,114.73,69.25,48.39,37.67.

[0167] Example 2

[0168] The present invention embodiment performs a GPR34 receptor activity test on compound 1-24, specifically comprising the following steps:

[0169] HEK293A cells were inoculated in a culture dish and tested by using the TGFα shedding test method (see Inoue, Nature Methods, 2012(9), 1021). The cell suspension volume was 10 mL and the density was 2×10 5Cells were cultured in a CO2 incubator in a 10% (v / v) DMEM medium supplemented with fetal bovine serum (FBS). After 24 hours, plasmids encoding APTGFα (2.5 μg per dish) and LysoPS receptor (2.5 μg per dish) were transfected into the cells using Lipofectamine 2000 reagent (Life Technologies; 12.5 μL per dish). Empty vectors were used to measure receptor-independent responses (i.e., no receptor plasmids were added), and cells were transfected with a mixture of plasmids encoding AP-TGFα (2.5 μg per dish) and empty vectors (2.5 μg per dish). For GPR34 and its controls, a chimeric Gα q / i1 Plasmids of the subunits of GPR34 were added (0.5 μg per dish). After 24 h, cells were harvested with trypsin-EDTA solution, washed with PBS, and suspended in Hank's balanced salt solution (HBSS; 35 mL per dish) containing 5 mM HEPES (pH 7.4). Cells overexpressing GPR34 and their corresponding control cells were seeded into 96-well plates (80 μL per well) and mixed with the LPA receptor antagonist Ki16425 at a final concentration of 10 μM (10 μL per well). After incubation for 30 min, cells were treated with the test compounds diluted in 0.01% (w / v) bovine serum albumin (BSA; fatty acid-free grade; Sigma-Aldrich) containing HBSS (10 μL per well; total volume 100 μL) and incubated at 37°C for 1 h. The cell plate was centrifuged for 2 min, and the supernatant (CM) was transferred to a blank 96-well plate (80 μL per well). p-NPP solution (10 mM p-nitrophenyl phosphate, 40 mM Tris-HCl (pH 9.5), 40 mM NaCl, and 10 mM MgCl2) was added to the CM plate and the cell plate (80 μL per well) and incubated at 37°C. The absorbance (OD) of both plates was read at 405 nm before and after incubation for 1 hour using a microplate reader. 405 ). To evaluate the GPR34 antagonist activity, each test compound should be added with a GPR34 agonist (final concentration: 300nM). We performed activity assays for both mouse GPR34 (Uniprot: Q9R1K6) and human GPR34 (Uniprot: Q9UPC5). The structural formula of the GPR34 agonist used here is shown below:

[0170]

[0171] The results are shown in Table 1.

[0172] Table 1

[0173]

[0174] As can be seen from Table 1, the compounds synthesized in the present invention exhibited good inverse agonist activity and antagonist activity against mouse GPR34 and human GPR34, among which compounds 8, 15, and 17 had the best inverse agonist ability against human GPR34. 50 All <30nm, IC for human GPR34 antagonism 50 <100nm;

[0175] Further provide the test diagram of compound 8, such as Figure 1 As shown, Figure 1 The middle a is the agonist mode, and the EC of compound 8 for inverse agonism of GPR34 is tested. 50 , Figure 1 b is the antagonistic mode, testing the inhibitory activity IC of compound 8 on GPR34 50 .

[0176] Example 3

[0177] The present invention embodiment conducted an in vitro Th9 differentiation promotion study on compound 8, compound 15, and compound 17, specifically comprising the following steps:

[0178] Grind the spleen and lymph nodes of C57 BL / 6 mice (from Southern Medical University, production license number: SCXK (Guangdong) 2021-0041) and select mouse CD4 + T negative selection kit (Biolegend) was used to purify and enrich the undifferentiated CD4 + T cells. TC-treated 48-well plates were pretreated with anti-CD3 antibody (4 μg / mL; 17A2; BioXCell). Undifferentiated CD4 T cells were cultured with anti-CD-28 antibody (1 μg / ml; 37.51; BioXCell), anti-IL-12 antibody (5 μg / ml; C17.8; BioXCell), anti-IFN-γ antibody (20 μg / ml; XMG1.2; BioXCell), mIL-4 (10 ng / ml; peprotech) and hTGF-β1 (1 ng / ml; EPOTO BIOTECH). + T cells were induced to differentiate toward Th9 and GPR34 antagonist compounds (40 μM, final concentration) and inverse agonist compounds (40 μM, final concentration) were added to the culture medium. After three days, the culture conditions were changed to mIL-4 (10 ng / ml; peprotech) and hTGF-β (1 ng / ml; EPOTOBIOTECH) to continue to maintain Th9 differentiation. During this period, the expression of IL-9 was detected to measure the Th9 differentiation.

[0179] The structures of the antagonist compounds X01 and X02 used therein are shown below;

[0180] X01:

[0181] X02:

[0182] The test results are as follows Figure 2-5 shown; from Figure 2-5 It can be seen that the antagonist compounds X01 and X02 cannot effectively promote Th9 cell differentiation, while the compounds 8, 15 and 17 provided by the present invention can effectively promote Th9 cell differentiation.

[0183] Example 4

[0184] The present invention explores the ability of compound 8 to enhance Th9 adoptive immunity to kill tumors, specifically including the following steps:

[0185] B16-OVA (B16 cell line from ATCC, infected with lentivirus expressing OVA) was used to sort GFP + Cell construction) mouse melanoma model was constructed, and each mouse was subcutaneously injected with 5*10^ 5 The subcutaneous solid tumor model was induced by B16-OVA cells. After the model was successfully constructed, the patients were divided into different groups and injected with DC cells (dendritic cells) and different Th9 cells for treatment. The patients were divided into PBS blank control group (PBS group), DMSO treated Th9 control group (Th9-DMSO group) and inverse agonist compound 8 treated Th9 group (Th9-compound 8 group, undifferentiated CD4 + T cells were induced to Th9 under the conditions of hTGF-β1 and mIL-4, and the inverse agonist compound 8 and its solvent DMSO were added during the culture process as a control, with three mice in each group. The differentiated Th9 cells were collected and injected into the tail vein of each mouse with 2*10^ 6 The treated Th9 cells were used to measure the size of the tumor on the back of the living mice with a vernier caliper every three days, and the tumor curve was drawn. When the tumor reached the humane endpoint, the mice were killed, and the solid melanoma of the mice was bluntly separated and recorded. The results were as follows Figure 6 As shown, Figure 6 (a) is a curve diagram of the change of tumor volume over time, and (b) is a comparison diagram of tumor photographs obtained by removing tumor tissue after killing mice;

[0186] from Figure 6 As can be seen, the use of the present invention to enhance CD4 +The tumor volume of Th9 cells treated with compounds that differentiate T cells into Th9 cells increased more slowly and was smaller than that of the control group, even when CD4 + Th9 cells treated with compounds that differentiate T cells into Th9 cells showed stronger tumor suppression effects and could effectively prolong the survival of mice.

[0187] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A CD4 Enhancer + A compound for differentiating T cells into Th9 cells, characterized in that The structural formula of the compound is shown in Formula I, Wherein, R1 is selected from any one of hydrogen, chlorine, cyano, isobutyl, n-butyl, tert-butyl, substituted isobutyl, substituted or unsubstituted methyl, substituted or unsubstituted phenyl, thienyl, and furyl; Said R2 is selected from hydrogen or chlorine; Said R3 is selected from hydrogen; The Y is selected from vinyl or imidazo[1,2-a]pyridinyl; The L is selected from an amide group or an ester group; The substituent group is selected from any one of chlorine, fluorine, methyl, ethyl and propyl.

2. The CD4 enhancement according to claim 1 + Use of a compound that causes T cells to differentiate into Th9 cells or a pharmaceutically acceptable salt thereof in the preparation of a drug for promoting Th9 cell differentiation.

3. The CD4 enhancement according to claim 1 + Use of a compound or a pharmaceutically acceptable salt thereof that can differentiate T cells into Th9 cells in the preparation of a drug for promoting adoptive T cell therapy for tumors.

4. The CD4 enhancement according to claim 1 + Use of a compound or a pharmaceutically acceptable salt thereof for differentiating T cells into Th9 cells in the preparation of tumor immunotherapy drugs.

5. A tumor immunotherapy drug, characterized in that: The tumor immunotherapy drug comprises the CD4 enhancer as claimed in claim 1 + A compound or a pharmaceutically acceptable salt thereof that induces T cells to differentiate into Th9 cells.

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