GPR65 small molecule regulator, preparation method therefor, and application

Abstract

Provided are a GPR65 small molecule regulator, a preparation method therefor, and an application, relating to the field of medicinal chemistry. The structure of the pyrimidine derivative is as shown in formula (I). The compound of the present invention has excellent antagonistic activity against human G protein-coupled receptor 65, and has broad application prospects in the preparation of anti-epileptic and anti-tumor drugs.

Description

GPR65 small molecule modulator and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of medicinal chemistry, in particular to a GPR65 small molecule modulator and preparation method and application thereof. BACKGROUND

[0002] GPR65 is a receptor belonging to the G protein-coupled receptor (GPR) family, which is widely used as a drug target or biomarker in biomedical research. GPR65 receptor has important biological functions in many physiological systems such as nervous system, endocrine system, immune system, urinary system, etc., and thus has important significance for disease diagnosis, treatment and drug research.

[0003] Under normal physiological conditions, GPR65 is mainly expressed in T and B lymphocytes, as well as macrophages and other innate immune cells (e.g., neutrophils, eosinophils and mast cells). GPR65 plays an important role in tumor microenvironment, especially in tumor-associated macrophages (TAMs). The acidic characteristics of tumor microenvironment and the abundant presence of TAMs are key features of solid tumors, which promote immunosuppression, support tumor growth, and limit the effectiveness of approved therapies. Studies have shown that GPR65 is a key factor of low pH-induced immunosuppression, especially in TAMs. Studies have shown that activation of GPR65 can promote an immunosuppressive immune cell phenotype, thereby preventing immune-mediated cancer cell killing. Therefore, inhibition of GPR65 can help enhance the anti-tumor capacity of the immune system. Studies have found that overexpression of GPR65 in Lewis lung cancer can promote tumor growth through PKA and extracellular signal-regulated kinase (ERK). Moreover, GPR65 is overexpressed in human colon, ovarian and kidney tumor tissues. Pathios Therapeutics Ltd. has developed a GPR65 small molecule inhibitor named PTT-3213, which is currently in preclinical stage. The drug shows significant anti-tumor effect in MC38 mouse symbiotic cancer model, can increase the number of CD8 + T cells and natural killer T cells in the tumor microenvironment, and synergizes with PD-1 antibodies to produce better drug efficacy. In addition, PTT-3213 can also provide a therapeutic effect comparable to twice-weekly injection of PD-1 antibodies in a mouse colorectal cancer model. Based on the strong expression of GPR65 in lymphoid tissues, its activation may represent a potential anti-tumorigenic approach, especially in hematological malignancies (including leukemia, lymphoma and multiple myeloma).

[0004] Recent studies have shown that abnormal activation or overexpression of GPR65 is closely related to the occurrence of behaviors such as convulsions, fear and anxiety, and these behaviors are one of the most common symptoms of seizures. For example, Lauren et al. found that when mice were exposed to a closed environment with a high concentration of CO2, CO2 could combine with water molecules in the body of the mice to cause systemic carbonic acid poisoning, and GPR65 in microglial cells was activated by high concentrations of H + Continuous activation further promotes the release of pro-inflammatory cytokine interleukin 1β (IL-1β), thereby stimulating abnormal firing of subfornical organ neurons, ultimately inducing seizures such as panic, fear and anxiety in mice. Katherine et al. pointed out that compared with wild-type mice, the symptoms of fear and anxiety in gene knockout mice (GPR65- / -) were significantly alleviated in experiments. In addition, a clinical study showed that the expression level of GPR65 mRNA in peripheral blood mononuclear cells of patients with panic disorder was significantly higher than that of healthy volunteers, and was positively correlated with the severity of CO2-induced panic symptoms. Therefore, abnormal activation or overexpression of GPR65 is closely related to the occurrence of symptoms such as convulsions, fear and anxiety in epilepsy, suggesting that targeting GPR65 may be a potential new anti-epileptic therapy.

[0005] Based on the potential role of GPR65 in various pathophysiological states and diseases, and the effectiveness of strategies targeting specific receptors in drug development, it is crucial to develop specific antagonists targeting GPR65. SUMMARY

[0006] The purpose of the present application is to provide a GPR65 small molecule modulator and a preparation method and application thereof.

[0007] The present application provides a compound represented by formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystal form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterium derivative thereof:

[0008] wherein X 1 is selected from CR1, N, X 2 is selected from CR2, N, X 3 is selected from CR3, N, and X 1 , X 2 , X 3 at least one of A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X, Y and Z is N;

[0009] R1, R2, R3 are each independently selected from hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, amino, nitro;

[0010] R4 is selected from hydrogen, halogen, C1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkoxy, C 1-6 alkylthio, halogen-substituted C 1-6 alkylthio, amino, phenyl, 5-6 membered heteroaromatic ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl;

[0011] or R2, R4 are linked to form a benzene ring;

[0012] L is selected from the group consisting of nothing, O, C 1-4 alkylene; n is selected from the group consisting of 0, 1, 2, 3, 4;

[0013] C ring is selected from the group consisting of benzene ring, 5-6 membered heteroaromatic ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl;

[0014] R c each independently selected from the group consisting of hydrogen, unsubstituted or substituted C c1 alkyl; 1-6 alkyl, unsubstituted or substituted C c1 alkyl; 1-6 alkoxy, unsubstituted or substituted C c2 alkyl; 2-10 alkenyl, unsubstituted or substituted C c2 alkyl; 2-10 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted phenyl, unsubstituted or substituted 5-6 membered heteroaryl, unsubstituted or substituted 3-8 membered saturated cycloalkyl, unsubstituted or substituted 3-8 membered saturated heterocyclyl; c3 alkyl; c3 alkyl; c3 alkyl; c3 alkyl; c3 and ring, L0COOL1R6, L0NR8COL1R6, L0CONR8L1R6, OR7, COR9;

[0015] R c1 each independently selected from the group consisting of hydroxyl, halogen, C 1-6 alkyl;

[0016] R c2 each independently selected from the group consisting of hydroxyl, halogen, C 1-6 alkyl;

[0017] R c3 each independently selected from the group consisting of halogen, C1-6 alkyl, hydroxy or halogen substituted C 1-6 alkyl, C 1-6 alkoxy, hydroxy or halogen substituted C 1-3 alkoxy, 3-8 membered saturated heterocyclyl;

[0018] L0is selected from the group consisting of nothing, C 2-4 alkylene;

[0019] L1is selected from the group consisting of nothing, C 1-4 alkylene;

[0020] R6is selected from the group consisting of C 1-6 alkyl, unsubstituted or substituted by one or more R 6a substituted 5-6 membered heteroaryl, phenyl, 3-8 membered saturated heterocyclyl, 3-8 membered saturated cycloalkyl, fused ring; R 6a each independently selected from the group consisting of halogen, C 1-6 alkyl, halogen substituted C 1-6 alkyl, C 1-6 alkoxy, halogen substituted C 1-6 alkoxy, COOR 6b , hydroxy;

[0021] R 6b is selected from the group consisting of C 1-6 alkyl;

[0022] R8is selected from the group consisting of hydrogen, C 1-6 alkyl, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl;

[0023] R7is selected from the group consisting of phenyl, benzyl;

[0024] R9is selected from the group consisting of fused ring unsubstituted or substituted by one or more R 9a substituted fused ring, R 9a is selected from the group consisting of halogen, C 1-6 alkyl;

[0025] or, m R c wherein two adjacent R c are joined to form a ring;

[0026] said R5is selected from the group consisting of L2NR 13 L3L4R 10 , phenyl unsubstituted or substituted by one or more R 11 substituted phenyl, L5L6R 14 ;

[0027] L2is selected from the group consisting of nothing, 3-6 membered saturated nitrogen heterocycle;

[0028] L3is selected from the group consisting of nothing, C 1-6 alkylene, hydroxy substituted C 1-6alkylene;

[0029] L4 is selected from the group consisting of no,

[0030] R 13 is selected from the group consisting of hydrogen, C 1-6 alkyl;

[0031] R 10 is selected from the group consisting of amino protecting groups, C 1-6 alkyl, NR a1 R a2 , OR a3 , CONHL7R a4 , unsubstituted or substituted by one or more than one R 12 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl, 5-6 membered heteroaryl, phenyl, fused ring, spiro ring; R a1 is a phenyl ring, R a2 is selected from the group consisting of hydrogen, C 1-5 alkyl, R a3 is selected from the group consisting of C 1-6 alkyl, phenyl, benzyl; R a4 is selected from the group consisting of 3-6 membered saturated cycloalkyl; L7 is selected from the group consisting of no, 1-4 alkylene; R 12 are each independently selected from the group consisting of halogen, hydroxyl, C 1-6 alkoxy, C 1-6 alkyl, cyano, COOR b1 , R b1 is selected from the group consisting of C 1-6 alkyl;

[0032] R 11 is selected from the group consisting of halogen, C 1-6 alkoxy, C 1-6 alkyl, hydroxyl;

[0033] L5 is selected from the group consisting of no, 3-6 membered saturated nitrogen heterocycle, unsubstituted or substituted by one or more than one R 5a ; R 5a are each independently selected from the group consisting of hydroxyl, halogen;

[0034] L6 is selected from the group consisting of no, 1-4 alkylene;

[0035] R 14 is selected from the group consisting of unsubstituted or substituted by one or more than one R 14a phenyl ring, 3-6 membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, fused ring; R 14a are each independently selected from the group consisting of halogen, halogen substituted C 1-6 alkyl, C 1-6 alkyl; R 14selected from the group consisting of amino protecting groups;

[0036] the compound is not

[0037] Further, the R3is selected from the group consisting of hydrogen, halogen, C selected from one of the following structures:

[0038] Further, the R3is selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl;

[0039] R4is selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkoxy, C 1-6 alkylthio, halogen-substituted C 1-6 alkylthio, amino, phenyl, 3-6 membered saturated cycloalkyl.

[0040] Further, the L is selected from the group consisting of nothing, O, n is selected from the group consisting of 0, 1, 2, 3, 4;

[0041] C ring is selected from the group consisting of phenyl ring, 5-6 membered nitrogen heteroaromatic ring, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl;

[0042] m is selected from the group consisting of 0, 1, 2, 3, 4;

[0043] R c each independently selected from the group consisting of hydrogen, unsubstituted or substituted with one or two or more R c1 alkyl, unsubstituted or substituted with one or two or more R 1-4 alkyl, unsubstituted or substituted with one or two or more R c1 alkyl, unsubstituted or substituted with one or two or more R 1-4 alkoxy, unsubstituted or substituted with one or two or more R c2 alkyl, unsubstituted or substituted with one or two or more R 2-4 alkenyl, unsubstituted or substituted with one or two or more R c2 alkenyl, unsubstituted or substituted with one or two or more R 2-4 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3substituted 5-membered nitrogen heteroaromatic ring fused to a 5-6 membered saturated heterocyclic ring, unsubstituted or substituted by one or two or more R c3 substituted 5-membered nitrogen heteroaromatic ring fused to a 5-6 membered saturated heterocyclic ring, unsubstituted or substituted by one or two or more R

[0044] R c1 each independently selected from the group consisting of hydroxyl, halogen, C 1-4 alkyl;

[0045] R c2 each independently selected from the group consisting of hydroxyl, halogen, C 1-4 alkyl;

[0046] R c3 each independently selected from the group consisting of halogen, C 1-4 alkyl, hydroxyl or halogen substituted C 1-4 alkyl, C 1-4 alkoxy, hydroxyl or halogen substituted C 1-4 alkoxy, 3-6 membered saturated heterocyclyl;

[0047] L0is selected from the group consisting of nothing, C 2-3 alkylene;

[0048] L1is selected from the group consisting of nothing, C 1-3 alkylene;

[0049] R6is selected from the group consisting of C 1-4 alkyl, unsubstituted or substituted by one or two or more R 6a substituted 5-6 membered saturated nitrogen heterocyclic ring fused to a benzene ring, R 6a each independently selected from the group consisting of halogen, C 1-4 alkyl, halogen substituted C 1-4 alkyl, C 1-4 alkoxy, halogen substituted C 1-4 alkoxy, COOR 6b , hydroxyl;

[0050] R 6b selected from the group consisting of C 1-4 alkyl;

[0051] R8is selected from the group consisting of hydrogen, C 1-4 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl;

[0052] R7is selected from the group consisting of phenyl, benzyl;

[0053] R9is selected from the group consisting of unsubstituted or substituted by one or two or more R 9a substituted 5-6 membered saturated nitrogen heterocyclic ring fused to a benzene ring, R 9a selected from the group consisting of halogen, C1-4 alkyl;

[0054] or, m R c are joined to form a ring, said ring being selected from a 5-6 membered saturated oxacyclic ring, a phenyl ring. c are joined to form a ring, said ring being selected from a 5-6 membered saturated oxacyclic ring, a phenyl ring.

[0055] Further, said R5is selected from L2NR 13 R 10 , NR 13 L3L4R 10 , phenyl unsubstituted or substituted by one or more than one R 11 , L5L6R 14 ;

[0056] L2is selected from nothing, a 4-6 membered saturated azacyclic ring;

[0057] L3is selected from nothing, C 1-5 alkylene, C 1-5 alkylene substituted by hydroxy;

[0058] L4is selected from nothing,

[0059] R 13 is selected from hydrogen, C 1-5 alkyl;

[0060] R 10 is selected from an amino protecting group, C 1-5 alkyl, NR a1 R a2 , OR a3 , CONHL7R a4 , unsubstituted or substituted by one or more than one R 12 : 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, 5-6 membered heteroaryl, phenyl, 5-6 membered saturated carbobenzo-fused ring, R a1 is a phenyl ring, R a2 is selected from hydrogen, C 1-5 alkyl, R a3 is selected from C 1-5 alkyl, phenyl, benzyl; R a4 is selected from 3-6 membered saturated cycloalkyl; L7is selected from nothing, C 1-2 alkylene; R 12 are each independently selected from halogen, hydroxy, C 1-5 alkoxy, C 1-5 alkyl, cyano, COOR b1 , R b1 is selected from C 1-5 alkyl;

[0061] R 11selected from halogen, C 1-5 alkoxy, C 1-5 alkyl, hydroxyl;

[0062] L5 is selected from the absence, unsubstituted or substituted with one or more R 5a substituted 4-6 membered saturated nitrogen heterocycle, R 5a each independently selected from hydroxyl, halogen;

[0063] L6 is selected from the absence, C 1-2 alkylene;

[0064] R 14 selected from unsubstituted or substituted with one or more R 14a substituted phenyl ring, 5-membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, 5-membered heteroarylbenzene ring, R 14a each independently selected from halogen, halogen-substituted C 1-5 alkyl, C 1-5 alkyl; R 14 selected from amino protecting groups.

[0065] Further, the structure of the compound is shown as formula II or formula III:

[0066] wherein, the R3 is selected from hydrogen, halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl;

[0067] R4 is selected from hydrogen, halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl;

[0068] R x1 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl;

[0069] R x2 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl;

[0070] R x3 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl, 5-membered nitrogen heteroaryl ring and 5-6 membered saturated heterocycle, 5-membered nitrogen heteroaryl ring and 5-6 membered heteroaryl ring;

[0071] f is selected from 0, 1, 2;

[0072] R 15 each independently selected from hydrogen, halogen, C 1-3alkyl.

[0073] Further, the compound is selected from one of the following compounds:

[0074] The present application also provides a pharmaceutical composition, which is a preparation prepared from the above-mentioned compound, stereoisomer thereof, pharmaceutically acceptable salt thereof, solvate thereof, crystal form thereof, prodrug thereof, metabolite thereof or deuterated derivative thereof as an active ingredient, and a pharmaceutically acceptable adjuvant.

[0075] The present application also provides the use of compound 9, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a crystal form thereof, a prodrug thereof, a metabolite thereof or a deuterated derivative thereof, or the use of the above-mentioned compound, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a crystal form thereof, a prodrug thereof, a metabolite thereof or a deuterated derivative thereof in the preparation of a G protein-coupled receptor antagonist;

[0076] Further, the G protein-coupled receptor antagonist is a G protein-coupled receptor 65 antagonist.

[0077] Further, the G protein-coupled receptor antagonist is a drug for preventing and / or treating a G protein-coupled receptor related disease.

[0078] Further, the G protein-coupled receptor related disease is epilepsy or cancer.

[0079] Further, the cancer is melanoma, renal cell carcinoma, gastric cancer, liver cancer, acute myeloid leukemia, pancreatic adenocarcinoma, triple-negative breast cancer, colorectal cancer, head and neck cancer, colorectal adenocarcinoma, lung cancer, ovarian cancer, glioblastoma or glioma.

[0080] The present application also provides the use of the above-mentioned compound, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a crystal form thereof, a prodrug thereof, a metabolite thereof or a deuterated derivative thereof in combination with an immune checkpoint inhibitor in the preparation of a drug for treating cancer; the compound is compound 9 or the above-mentioned compound.

[0081] Further, the immune checkpoint inhibition is a PD-1 antibody.

[0082] Definitions of terms used in connection with the present application: Unless otherwise indicated, the initial definition of a group or term provided herein applies throughout the specification; for terms not specifically defined herein, the meaning given such terms by one of ordinary skill in the art in light of the disclosure and context will prevail.

[0083] The minimum and maximum number of carbon atoms in a hydrocarbon group is indicated by a prefix, for example, the prefix C a～b Alkyl refers to a straight or branched chain alkyl group containing 1, 2, 3, 4, 5, or 6 carbon atoms; C 1～6 Alkyl refers to a straight or branched chain alkyl group containing 1, 2, 3, 4, 5, or 6 carbon atoms; C 1-4 Alkylene refers to a straight or branched chain alkylene group containing 1, 2, 3, or 4 carbon atoms; and so on.

[0084] "Heteroaryl" or "heteroaromatic" refers to a heteroaromatic group containing one to several heteroatoms. The heteroatoms referred to herein include, but are not limited to, oxygen, sulfur, and nitrogen.

[0085] 5-6 membered heteroaryl refers to a heteroaromatic group containing 5 or 6 ring atoms, and 5-6 membered heteroaromatic ring refers to a heteroaromatic ring containing 5 or 6 ring atoms.

[0086] "Nitrogen heterocycle" refers to a heterocycle containing nitrogen atoms, and the heteroatoms in the heterocycle can be only nitrogen atoms, or can include nitrogen atoms and other heteroatoms, which include, but are not limited to, oxygen and sulfur.

[0087] "Annulated" refers to a polycycle having two rings sharing two adjacent ring atoms.

[0088] "Spiro" refers to a polycycle having two rings sharing one atom.

[0089] Amino protecting group refers to a chemical group used to protect an amino (-NH2) group, such as tert-butoxycarbonyl, trifluoromethanesulfonyl, benzyloxycarbonyl, dibenzoyl, methanesulfonyl, and the like.

[0090] Halogen is fluorine, chlorine, bromine.

[0091] Experimental results show that the compound of the present application has excellent antagonistic activity on human G protein-coupled receptor 65, and can be used for preparing G protein-coupled receptor 65 antagonists. It is known to those skilled in the art that G protein-coupled receptor 65 antagonists can effectively resist epilepsy and tumors, and therefore the compound of the present application has broad application prospects in the preparation of drugs for resisting epilepsy and tumors.

[0092] Obviously, according to the above content of the present application, other various forms of modification, replacement or change can be made according to the common technical knowledge and usual means in the art without departing from the above basic technical idea of the present application.

[0093] The above content of the present application will be further explained in detail by a specific embodiment in the form of example. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following example. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0094] Fig. 1 is a graph of anti-tumor effect of compound 121: (A) is a graph of subcutaneous tumor effect in mice; (B) is a graph of tumor weight; (C) is a graph of days-tumor volume curve; (D) is a graph of days-body weight curve.

[0095] Fig. 2 is a graph of anti-epilepsy effect of compound 121: (A) is a graph of first seizure latency; (B) is a graph of total seizure time; (C) is a graph of highest seizure grade; (D) is a graph of seizure frequency.

[0096] Fig. 3 is a graph of anti-tumor effect of compound 300: (A) is a graph of subcutaneous tumor effect in mice; (B) is a graph of tumor weight; (C) is a graph of days-tumor volume curve; (D) is a graph of days-body weight curve.

[0097] Fig. 4 is a graph of anti-epilepsy effect of compound 300: (A) is a graph of first seizure latency; (B) is a graph of seizure frequency; (C) is a graph of highest seizure grade. DETAILED DESCRIPTION

[0098] The raw materials and equipment used in the specific embodiment of the present application are known products, which are obtained by purchasing commercially available products.

[0099] Example 1 Preparation of compound 10

[0100] Step a: Preparation of intermediate 1 (4-chloro-5-fluoro-6-(methyloxy)-2-methylpyrimidine.

[0101] The raw material 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) was dissolved in acetonitrile, and 2,4,6-trimethylphenol (136 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added in sequence, and the reaction was carried out at 80°C for 6 hours. TLC monitoring was performed, and after the reaction was completed, the filtrate was extracted and dried by rotary evaporation. The sample was mixed with silica gel and column chromatography was performed, and 224 mg of intermediate 1 was obtained in a yield of 80%. MS (ESI) m / z: 281.1 [M+H] + .

[0102] Step b: Preparation of product 10 5-fluoro-6-(methyloxy)-2-methyl-N-(pent-3- yl)pyrimidin-4-amine.

[0103] Intermediate 1 (112 mg, 0.4 mmol), 3-aminopentane (44 mg, 0.5 mmol) were dissolved in 5 mL of dimethylsulfoxide, N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added and the reaction was left to react at 120 °C overnight. After the end of the reaction, it was diluted with 20 mL of water and extracted twice with 10 mL of ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The product 10 was isolated by column chromatography as a white solid with a yield of 53%. 1 H NMR (400 MHz, CDC13) δ 6.87 (s, 2H), 4.54 (d, J = 8.4 Hz, 1H), 4.11 - 4.01 (m, 1H), 2.29 (s, 3H), 2.24 (s, 3H), 2.11 (s, 6H), 1.70 - 1.62 (m, 2H), 1.56 - 1.47 (m, 2H), 0.94 (t, J = 7.4 Hz, 6H). 13 C NMR (101 MHz, CDC13) δ 160.36, 160.25, 152.61, 152.55, 152.06, 151.98, 146.62, 133.46, 130.01, 129.54, 127.98, 127.54, 51.89, 26.37, 24.59, 19.80, 15.46, 9.04. HRMS (ESI-TOF) m / z calcd. for C 19 H 26 FN3O[M+H] + 332.2133, found 332.2126.

[0104] Example 2 Preparation of compound 21

[0105] Step a: Same as example 1 step a.

[0106] Step b: Preparation of product 21 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6- (methyloxy)-2-methylpyrimidine.

[0107] Intermediate 1 (112 mg, 0.4 mmol), (4-fluoro-2-isopropoxyphenyl)boronic acid (79.2 mg, 0.4 mmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (29.2 mg, 0.04 mmol), potassium carbonate (110.4 mg, 0.8 mmol) were weighed in a vial, dissolved in dioxane / water (10 ml:1 ml), protected by nitrogen, and reacted overnight at 100 °C. After the reaction was completed, it was filtered, the filtrate was concentrated under reduced pressure, and the product 21 was separated by column chromatography to obtain a white solid with a yield of 78%. MS (ESI) m / z: 399.2 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.50-7.38 (m, 1H), 6.83 (s, 2H), 6.71-6.65 (m, 1H), 6.61 (d, J = 11.1 Hz, 1H), 4.54-4.44 (m, 1H), 2.44 (s, 3H), 2.23 (s, 3H), 2.03 (s, 6H), 1.24 (d, J = 5.7 Hz, 6H).

[0108] Example 3 Preparation of compound 62

[0109] Step a: Same as example 1 step a.

[0110] Step b: Preparation of product GP142 5-fluoro-6-(trioxyl)-2-methyl-N-(1- phenylcyclobutyl)pyrimidin-4-amine.

[0111] Intermediate 1 (112 mg, 0.4 mmol), 1-phenylcyclobutylamine hydrochloride (91.8 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N- diisopropylethylamine (209 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, it was diluted with 20 mL of water, extracted with 10 mL of ethyl acetate twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and the product 62 was separated by column chromatography to obtain 156.5 mg of a white solid with a yield of 66%. 1 H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.4 Hz, 1H), 7.33 (t, J = 7.6 Hz, 2H), 7.25-7.19 (m, 1H), 6.84 (s, 2H), 5.36 (s, 1H), 2.76-2.62 (m, 4H), 2.27 (s, 3H), 2.20-2.15 (m, 1H), 2.13 (s, 3H), 2.06 (s, 6H), 1.95-1.87 (m, 1H). 13C NMR (101 MHz, CDC13) δ 160.89, 160.78, 153.78, 153.71, 151.55, 151.48, 147.59, 146.06, 134.56, 131.56, 130.61, 129.03, 127.97, 126.52, 126.23, 60.49, 34.81, 25.45, 20.86, 16.53, 15.59. HRMS (ESI-TOF) m / z calcd for C 24 H 26 FN3O[M+H] + 392.2133, found 392.2126.

[0112] Example 4 Preparation of compound 90

[0113] Step a: Same as example 1 step a.

[0114] Step b: Preparation of product 90 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6- (methyloxy)-2-methylpyrimidine.

[0115] Intermediate 1 (112 mg, 0.4 mmol), o-benzylhydroxylamine (61.2 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 mL of water was added for dilution, and 10 mL of ethyl acetate was added for extraction twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product 90 was separated by column chromatography in the form of off-white solid with a yield of 43%. MS (ESI) m / z: 368.2 [M+H] + .

[0116] Example 5 Preparation of compound 93

[0117] Step a: Preparation of intermediate 1 6-chloro-5-fluoro-2-methyl-N-(l- phenylcyclobutyl)pyrimidin-4-amine.

[0118] Intermediate 1 (146 mg, 0.5 mmol), 2-cyclohexylethanamine (63.5 mg, 0.5 mmol) were dissolved in 5 mL of dimethylsulfoxide, N,N-diisopropylethylamine (260 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 ml of water was added for dilution, and 10 ml of ethyl acetate was added twice for extraction. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 93, a white solid, in a yield of 59%. MS (ESI) m / z: 383.3 [M+H] + .

[0119] Step b: Preparation of product 93 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6- (methyloxy)-2-methylpyrimidine.

[0120] Intermediate 1 (146 mg, 0.5 mmol), 2-cyclohexylethanamine (63.5 mg, 0.5 mmol) were dissolved in 5 mL of dimethylsulfoxide, N,N-diisopropylethylamine (260 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 ml of water was added for dilution, and 10 ml of ethyl acetate was added twice for extraction. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 93, a white solid, in a yield of 59%. MS (ESI) m / z: 383.3 [M+H] + .

[0121] Example 6 Preparation of compound 94

[0122] Step a: Same as Example 1, step a.

[0123] Step b: Preparation of intermediate 2 1-((5-fluoro-6-(methyloxy)-2- methylpyrimidin-4-yl)amino)cyclobutane-1-carboxylic acid methyl ester.

[0124] Intermediate 1 (112 mg, 0.4 mmol), 1-aminocyclobutane-1-carboxylic acid methyl ester (51.6 mg, 0.5 mmol) were dissolved in 5 mL of dimethylsulfoxide, N,N- diisopropylethylamine (209 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 ml of water was added for dilution, and 10 ml of ethyl acetate was added twice for extraction. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 86.6 mg of intermediate 1, a white solid, in a yield of 58%. MS (ESI) m / z: 374.2 [M+H] + .

[0125] Step c: Preparation of intermediate 3, 1-((5-fluoro-6-(methyloxy)-2- methylpyrimidin-4-yl)amino)cyclobutane-1 -carboxylic acid.

[0126] The intermediate 2 obtained in the previous step was dissolved in 5 mL of methanol, 2 mL of lithium hydroxide solution (2 mol / L) was added, and the reaction was carried out at 50 °C for 2 h. After the reaction was completed, the reaction solution was directly rotary evaporated, diluted with dilute hydrochloric acid to pH = 4, extracted with EA, and rotary evaporated to obtain intermediate 3, which was directly used in the next step.

[0127] Step d: Preparation of product 94, N-(1-cyclopropylethyl)-1-((5-fluoro-6- (methyloxy)-2-methylpyrimidin-4-yl)amino)cyclobutane-1-carboxamide.

[0128] Intermediate 3 (71.9 mg, 0.2 mmol), 1-cyclopropylethan-1-amine (17 mg, 0.2 mmol), N,N,N',N'-tetramethyl-O-(7-azabenzotriazol-1-yl)urea hexafluorophosphate (114 mg, 0.3 mmol), N,N-diisopropylethylamine (130 μL, 0.6 mmol) were dissolved in 10 mL of dichloromethane, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was diluted with 20 mL of water, extracted with dichloromethane 10 mL twice, combined organic phase, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 75 mg of product 94, white solid, yield 88%. MS (ESI) m / z: 427.2 [M+H] + .

[0129] Example 7 Preparation of compound 95

[0130] Step a: The same as step a of Example 5.

[0131] Step b: Preparation of product 95, 5-fluoro-4-(4-fluoro-2- isopropoxyphenyl)-6-(methyloxy)-2-methylpyrimidine.

[0132] Intermediate 1 (146 mg, 0.5 mmol), m-amino benzonitrile (59, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (325 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, the reaction solution was diluted with 20 mL of water, extracted with ethyl acetate 10 mL twice, combined organic phase, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain product 95, white solid, yield 40%. MS (ESI) m / z: 374.2 [M+H] + . 1H NMR (400 MHz, CDC13) δ 7.55 - 7.50 (m, 2H), 7.48 - 7.44 (m, 2H), 7.42 - 7.40 (m, 1H), 7.37 - 7.32 (m, 3H), 5.51 (s, 1H), 2.80 - 2.60 (m, 4H), 2.22 (s, 3H), 2.19 - 2.09 (m, 1H), 1.98 - 1.89 (m, 1H).

[0133] Example 8 Preparation of compound 121

[0134] Step a: Preparation of intermediate 1 4-chloro-5-fluoro-6-(4-fluoro-2,6-dimethylphenoxy)- 2-methylpyrimidine.

[0135] The starting material 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) was dissolved in acetonitrile, 4-fluoro-2,6-dimethylphenol (140 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were added successively and the reaction was carried out at 80 °C for 6 hours. TLC monitoring, after the reaction was completed, suction filtration, rotary evaporation of the filtrate, silica gel sample mixing, column chromatography, 218 mg of intermediate 1 was obtained, the yield was 77%. MS (ESI) m / z: 285.0 [M+H] + .

[0136] Step b: Preparation of product 121 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methyloxy)- 2-methylpyrimidine.

[0137] Intermediate 1 (113.6 mg, 0.4 mmol), 1-phenylcyclobutylamine hydrochloride (91.8 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N- diisopropylethylamine (209 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, it was diluted with 20 ml of water, extracted with 10 ml of ethyl acetate twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 115.6 mg of product 121, white solid, the yield was 73%. 1 H NMR (400 MHz, CDC13) δ 7.54 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.7 Hz, 2H), 7.23 (t, J = 7.3 Hz, 1H), 6.74 (d, J = 8.9 Hz, 2H), 5.39 (s, 1H), 2.76 - 2.63 (m, 4H), 2.20 - 2.15 (m, 1H), 2.13 (s, 3H), 2.08 (s, 6H), 1.95 - 1.87 (m, 1H). 13C NMR (101 MHz, CDC13) δ 160.89, 160.78, 158.38, 153.45, 153.39, 151.61, 151.54, 145.96, 145.54, 145.52, 132.93, 132.84, 131.44, 128.95, 127.97, 126.55, 126.24, 114.72, 114.49, 60.50, 34.77, 25.39, 16.77, 15.59. HRMS (ESI-TOF) m / z calcd for C 23 H 23 F2N3O[M+H] + 396.1882, found 396.1877.

[0138] Example 9 Preparation of compound 133

[0139] Step a: Same as example 1 step a.

[0140] Step b: Preparation of product 133 6-fluoro-3-(1-(5-fluoro-6-(methylthioxy)-2- methylpyrimidin-4-yl)piperidin-4-yl)benzo[d]isoxazole.

[0141] Intermediate 1 (112 mg, 0.4 mmol), 6-fluoro-3-piperidin-4-yl-1,2-benzisoxazole hydrochloride (128 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N- diisopropylethylamine (209 μL, 1.2 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 mL of water was added for dilution, and 10 mL of ethyl acetate was added for extraction twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The product 133 was separated by column chromatography and obtained as a white solid in a yield of 70%. MS (ESI) m / z: 465.2 [M+H] + . 1 H NMR (400 MHz, CDC13) δ 7.71 - 7.65 (m, 1H), 7.30 - 7.23 (m, 1H), 7.12 - 7.05 (m, 1H), 6.88 (s, 2H), 4.68 - 4.59 (m, 2H), 3.43 - 3.32 (m, 1H), 3.27 - 3.18 (m, 2H), 2.30 (s, 3H), 2.25 (s, 3H), 2.22 - 2.13 (m, 4H), 2.11 (s, 6H).

[0142] Example 10 Preparation of compound 137

[0143] Step a: Same as example 5 step a.

[0144] Step b: Preparation of product 137 5-fluoro-2-methyl-N-(1-phenylcyclobutyl)-6-(4- propylphenyl)pyrimidin-4-amine.

[0145] Intermediate 1 (112 mg, 0.4 mmol), (4-propylphenyl)boronic acid (65.6 mg, 0.4 mmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (29.2 mg, 0.04 mmol), potassium carbonate (110.4 mg, 0.8 mmol) were weighed in a pear shaped flask, dissolved in dioxane / water (10 ml: 1 ml), protected by nitrogen, 100 degree Celsius overnight. After the reaction was completed, filtered, the filtrate was concentrated under reduced pressure, separated by column chromatography to obtain 129 mg of product 137, white solid, yield 86%. MS (ESI) m / z: 376.2 [M+H] + . 1 H NMR (400 MHz, CDC13) δ 7.80 (d, J = 6.8 Hz, 2H), 7.61 - 7.52 (m, 2H), 7.33 (t, J = 7.7 Hz, 2H), 7.26 - 7.19 (m, 3H), 5.54 (s, 1H), 2.80 - 2.66 (m, 4H), 2.65 - 2.58 (m, 2H), 2.21 - 2.13 (m, 1H), 1.99 - 1.89 (m, 1H), 1.71 - 1.59 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H).

[0146] Example 11 Preparation of compound 149

[0147] Step a: Preparation of intermediate 1 4-chloro-6-(2,6-dimethyl-4-nitrophenoxy)-5- fluoro-2-methylpyrimidine.

[0148] The starting material 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) was dissolved in acetonitrile, 2,6-dimethyl-4-nitrophenol (167 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were added in turn, and reacted at 80°C for 6 hours. TLC monitoring, after the reaction was completed, suction filtration, the filtrate was dried, and silica gel was mixed, column chromatography, to obtain 245 mg of intermediate 1, yield 79%. MS (ESI) m / z: 312.0 [M+H] + .

[0149] Step b: Preparation of intermediate 2 6-(2,6-dimethyl-4-nitrophenoxy)-5-fluoro-2- methyl-N-(1-phenylcyclobutyl)pyrimidin-4-amine.

[0150] Intermediate 1 (180 mg, 0.5 mmol), 1-phenylcyclobutylamine hydrochloride (91.9 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 mL of water was added for dilution, and 10 mL of ethyl acetate was added for extraction twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Column chromatography was performed to obtain 116 mg of intermediate 2 at a yield of 55%. MS (ESI) m / z: 423.2 [M+H] + .

[0151] Step c: Preparation of intermediate 3 6-(4-amino-2,6-dimethylphenoxy)-5-fluoro-2-methyl-N-(1- phenylcyclobutyl)pyrimidin-4-amine.

[0152] Intermediate 2 (116 mg, 0.275 mmol) was dissolved in 10 mL of an ethanol / water (v / v = 10:1) solution, iron powder (61 mg, 1.1 mmol), ammonium chloride (29.4 mg, 055 mmol) were added, and the reaction was carried out at 80 °C for 2 h. TLC was used for monitoring the reaction. After the reaction was completed, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. Column chromatography was performed to obtain 94 mg of intermediate 3 at a yield of 88%. MS (ESI) m / z: 393.2 [M+H] + .

[0153] Step d: Preparation of product 149 2-fluoro-N-(4-((5-fluoro-2-methyl-6-((1- phenylcyclobutyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylphenyl)benzamide.

[0154] Intermediate 3 (94 mg, 0.242 mmol), o-fluorobenzoic acid (34 mg, 0.242 mmol), HATU (137.9 mg, 0.363 mmol), DIPEA (126 μL, 0.726 mmol) were weighed in an eggplant flask, dissolved in dichloromethane, and the reaction was carried out at room temperature for 2 h. TLC was used for monitoring the reaction. After the reaction was completed, it was concentrated under reduced pressure, and column chromatography was performed to obtain 93 mg of product 149 at a yield of 75%. MS (ESI) m / z: 515.2 [M+H] + . 1H NMR (400 MHz, CDC13) δ 8.36 (d, J = 15.3 Hz, 1H), 8.15 (t, J = 7.5 Hz, 1H), 7.59 - 7.51 (m, 2H), 7.51 - 7.45 (m, 1H), 7.36 (s, 2H), 7.34 - 7.29 (m, 2H), 7.28 - 7.23 (m, 1H), 7.23 - 7.06 (m, 2H), 5.42 (s, 1H), 2.77 - 2.60 (m, 4H), 2.21 - 2.15 (m, 1H), 2.13 (s, 3H), 2.08 (s, 6H), 1.96 - 1.86 (m, 1H).

[0155] Example 12 Preparation of compound 151

[0156] Step a: Preparation of intermediate 1 4-(4-bromo-2,6-dimethylphenoxy)-6-chloro-5-fluoro-2-methylpyrimidine.

[0157] The starting material 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) was dissolved in acetonitrile, 4-bromo-2,6-dimethylphenol (167 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were added successively and the reaction was carried out at 80 °C for 6 hours. TLC monitoring was performed, and after the reaction was completed, the filtrate was extracted with 20 ml of water and 10 ml of ethyl acetate twice, combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by column chromatography to obtain 300 mg of intermediate 1 at a yield of 87%. MS (ESI) m / z: 347.0 [M+H] + .

[0158] Step b: Preparation of intermediate 2 6-(4-bromo-2,6-dimethylphenoxy)-5-fluoro-2-methyl-N-(1-phenylcyclobutyl)pyrimidin-4-amine.

[0159] Intermediate 1 (227 mg, 0.5 mmol) and 1-phenylcyclobutylamine hydrochloride (91.9 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, the reaction mixture was diluted with 20 ml of water and extracted with 10 ml of ethyl acetate twice, combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by column chromatography to obtain 102 mg of intermediate 2 at a yield of 45%. MS (ESI) m / z: 456.1 [M+H] + .

[0160] Step c: Preparation of product GP403 5-fluoro-6-((3'-fluoro-3,5-dimethyl-[l,l'-biphenyl]-4- yl)oxy)-2-methyl-N-(l-phenylcyclobutyl)pyrimidin-4-amine.

[0161] Intermediate 2 (102 mg, 0.225 mmol), m-fluorobenzeneboronic acid (32 mg, 0.225 mmol), 1,1-bis(diphenylphosphino)ferrocene palladium dichloride (22 mg, 0.03 mmol), potassium carbonate (93 mg, 0.675 mmol) were weighed in a pear shape flask, dissolved in dioxane / water (10 ml: 1 ml), protected by nitrogen, and reacted at 100 degrees Celsius overnight. After the reaction was completed, it was filtered, the filtrate was concentrated under reduced pressure, and 80 mg of product 151 was separated by column chromatography as a white solid with a yield of 75%. MS (ESI) m / z: 472.2 [M+H] + . 1 H NMR (400 MHz, CDC13) δ 7.57 - 7.55 (m, 1H), 7.55 - 7.53 (m, 1H), 7.38 - 7.32 (m, 4H), 7.30 - 7.27 (m, 1H), 7.25 (s, 2H), 7.24 - 7.22 (m, 1H), 7.04 - 6.97 (m, 1H), 5.40 (s, 1H), 2.78 - 2.64 (m, 4H), 2.27 - 2.19 (m, 1H), 2.16 (s, 6H), 2.15 (s, 3H), 1.97 - 1.88 (m, 1H).

[0162] Example 13 Preparation of compound 184

[0163] Step a: Preparation of intermediate 1 methyl 4-((6-chloro-5-fluoro-2-methylpyrimidin-4- yl)oxy)-3,5-dimethylbenzoate.

[0164] The starting material 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) was dissolved in acetonitrile, and methyl 4-hydroxy-3,5-dimethylbenzoate (180 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were sequentially added and reacted at 80 degrees Celsius for 6 hours. TLC monitoring was performed, and after the reaction was completed, it was suction filtered, the filtrate was rotary evaporated, and the sample was mixed with silica gel and column chromatography to obtain 259 mg of intermediate 1 with a yield of 80%. MS (ESI) m / z: 325.1 [M+H] + .

[0165] Step b: Preparation of intermediate 2 methyl 4-((5-fluoro-2-methyl-6-((l- phenylcyclobutyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzoate.

[0166] Intermediate 1 (162 mg, 0.5 mmol), 1-phenylcyclobutylamine hydrochloride (91.9 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 mL of water was added for dilution, and 10 mL of ethyl acetate was added for extraction twice. The organic phase was combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column chromatography to obtain 131 mg of intermediate 2 at a yield of 60%. MS (ESI) m / z: 436.2 [M+H] + .

[0167] Step c: Preparation of intermediate 3 6-(4-amino-2,6-dimethylphenoxy)-5-fluoro-2-methyl-N-(1- phenylcyclobutyl)pyrimidin-4-amine.

[0168] Intermediate 2 (131 mg, 0.3 mmol) was dissolved in 10 mL of methanol solution, 1 mL of 1 N lithium hydroxide solution was added, and the reaction was carried out at room temperature overnight. TLC was used for monitoring, after the reaction was completed, concentrated under reduced pressure, added with appropriate amount of water, diluted with hydrochloric acid to adjust the pH to 3-4, extracted with EA for 3 times, combined the organic phase, dried over anhydrous sodium sulfate, filtered, and concentrated the filtrate to obtain intermediate 3, which was directly used in the next step.

[0169] Step d: Preparation of product GP516 (S)-4-((5-fluoro-2-methyl-6-((1-phenylcyclobutyl)amino)pyrimidin-4- yl)oxy)-N,3,5-trimethyl-N-(1-phenylethyl)benzamide.

[0170] Intermediate 3 (84 mg, 0.2 mmol), (S)-N-methyl-1-phenylethan-1-amine (32 mg, 0.24 mmol), HATU (114 mg, 0.3 mmol), DIPEA (104 μL, 0.6 mmol) were weighed in an eggplant flask, dissolved in dichloromethane, and the reaction was carried out at room temperature for 2 h. TLC was used for monitoring the reaction, after the reaction was completed, concentrated under reduced pressure, and separated by column chromatography to obtain 86 mg of product 184 at a yield of 82%. MS (ESI) m / z: 539.3 [M+H] + . 1H NMR (400 MHz, CDC13) δ 7.50 - 7.43 (m, 2H), 7.32 - 7.26 (m, 4H), 7.25 - 7.20 (m, 2H), 7.18 - 7.11 (m, 2H), 7.05 (s, 2H), 5.32 (s, 1H), 2.72 - 2.54 (m, 4H), 2.12 - 2.05 (m, 1H), 2.04 (s, 3H), 2.03 (s, 6H), 1.87 - 1.81 (m, 1H), 1.54 (s, 3H), 1.51 (s, 3H).

[0171] Example 14 Preparation of compound 212

[0172] Step a: Same as example 12 step a.

[0173] Step b: Same as example 12 step b.

[0174] Step c: Preparation of product 212 6-(2,6-dimethyl-4-(pyrazolo[l,5-a]pyrimidin-3- yl)phenoxy)-5-fluoro-2-methyl-N-(l-phenylcyclobutyl)pyrimidin-4-amine.

[0175] Intermediate 2 (102 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)pyrazolo[l,5-a]pyrimidine (55 mg, 0.225 mmol), l,l-bis(diphenylphosphino) ferrocenedichloropalladium (22 mg, 0.03 mmol), potassium carbonate (93 mg, 0.675 mmol) were weighed in a pear shaped flask, dissolved in dioxane / water (10 ml: 1 ml), protected by nitrogen, 100 degree Celsius overnight. After the reaction was completed, filtered, the filtrate was concentrated under reduced pressure, separated by column chromatography to obtain 74 mg of product 212, white solid, yield 66%. MS (ESI) m / z: 495.2 [M+H] + . 1 H NMR (400 MHz, DMSO) δ 9.16 (d, J = 7.1 Hz, 1H), 8.75 - 8.70 (m, 1H), 8.69 - 8.64 (m, 1H), 8.15 (s, 1H), 7.90 - 7.81 (m, 2H), 7.54 (d, J = 8.0 Hz, 2H), 7.32 (t, J = 6.8 Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 7.14 - 7.08 (m, 1H), 2.71 - 2.55 (m, 4H), 2.10 (s, 6H), 2.04 - 2.01 (m, 1H), 2.00 (s, 3H), 1.85 - 1.77 (m, 1H). 13C NMR (101 MHz, DMSO) δ 160.49, 160.39, 156.03, 155.95, 151.07, 151.03, 149.49, 147.92, 144.09, 143.29, 132.72, 131.63, 131.14, 129.13, 128.87, 128.10, 127.33, 126.73, 126.39, 124.07, 117.72, 116.41, 114.18, 93.57, 91.84, 61.22, 61.20, 56.92, 56.71, 42.70, 42.56, 25.48, 16.66.

[0176] Example 15 Preparation of compound 220

[0177] Step a: Preparation of intermediate 1 4-(4-bromo-2,6-dimethylphenoxy)-2,6- chloro-5-fluoropyrimidine.

[0178] The starting material 2,4,6-trichloro-5-fluoropyrimidine (201 mg, 1 mmol) was dissolved in acetonitrile, 4-bromo-2,6-dimethylphenol (201 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were added successively and the reaction was carried out at 80 °C for 6 hours. TLC monitoring, after the reaction was completed, suction filtration, rotary evaporation of the filtrate, silica gel sample mixing, column chromatography, 310 mg of intermediate 1 was obtained, the yield was 85%. MS (ESI) m / z: 366.9 [M+H] + .

[0179] Step b: Preparation of intermediate 2 6-(4-bromo-2,6-dimethylphenoxy)-2-chloro-5- fluor-N-(1-phenylcyclobutyl)pyrimidin-4-amine.

[0180] Intermediate 1 (183 mg, 0.5 mmol), 1-phenylcyclobutylamine hydrochloride (91.9 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N- diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, it was diluted with 20 ml of water, extracted with 10 ml of ethyl acetate twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and then separated by column chromatography to obtain 253 mg of intermediate 2, the yield was 53%. MS (ESI) m / z: 478.0 [M+H] + .

[0181] Step c: Preparation of product 220 2-chloro-6-(2,6-dimethyl-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)phenoxy)-5-fluoro-N-(1-phenylcyclobutyl)pyrimidine-4-amine.

[0182] Intermediate 2 (107 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)

[0183] -4,5,6,7-Tetrahydropyrazole[1,5-a]pyridine (56 mg, 0.225 mmol), 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (22 mg, 0.03 mmol), and potassium carbonate (93 mg, 0.675 mmol) were weighed into a round-bottom flask, dissolved in dioxane / water (10 ml: 1 ml), and incubated overnight at 100°C under nitrogen protection. After the reaction was complete, the mixture was filtered, the filtrate was concentrated under reduced pressure, and separated by column chromatography to obtain 91 mg of product 220, a white solid, with a yield of 78%. MS (ESI) m / z: 518.2 [M+H] + . 1 H NMR (400MHz, CDCl3) δ7.56 (s, 1H), 7.51–7.43 (m, 2H), 7.30 (t, J = 7.7Hz, 2H), 7.18–7.15 (m, 1H), 6.99 (s, 2H), 5.54 (s, 1H), 4.12 (t,J=6.1Hz,2H),2.91–2.85(m,2H),2.73–2.57(m,4H),2.12–2.08(m,1H),2.06(s,6H),2.04–1.93(m,3H),1.88–1.79(m,3H).

[0184] Example 16 Preparation of compound 262

[0185] Step a: Same as step a in Example 12.

[0186] Step b: Preparation of intermediate 2,4-(4-bromo-2,6-dimethylphenoxy)-5-fluoro-2-methyl-6-(2-(pyridin-3-yl)pyrrolidine-1-yl)pyrimidine.

[0187] Intermediate 1 (183 mg, 0.5 mmol), 3-(pyrrolidin-2-yl)pyridine (74 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, 20 mL of water was added, and the mixture was extracted with 10 mL of ethyl acetate twice. The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was separated by column chromatography to obtain 135 mg of intermediate 2 at a yield of 59%. MS (ESI) m / z: 457.1 [M+H] + .

[0188] Step c: Preparation of product GP626 3-(4-((5-fluoro-2-methyl-6-(2-(pyridin-3-yl)pyrrolidin-1- yl)pyrimidin-4-yl)oxy)-3,5,6,7-tetrahydropyrazolo[1,5-a]pyridine.

[0189] Intermediate 2 (103 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4,5,6,7- tetrahydropyrazolo[1,5-a]pyridine (56 mg, 0.225 mmol), 1,1'-bis(diphenylphosphino)ferrocene palladium dichloride (22 mg, 0.03 mmol), potassium carbonate (93 mg, 0.675 mmol) were weighed in a flask, dissolved in dioxane / water (10 mL:1 mL), and the reaction was carried out overnight at 100 °C under nitrogen atmosphere. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography to obtain 92 mg of product 262 as a white solid at a yield of 82%. MS (ESI) m / z: 499.3 [M+H] + . 1 H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H), 7.21-7.15 (m, 2H), 7.05 (s, 2H), 6.99 (t, J = 8.7 Hz, 2H), 5.44-5.37 (m, 1H), 4.19 (t, J = 6.1 Hz, 2H), 4.09-4.02 (m, 1H), 3.86-3.79 (m, 1H), 2.95 (t, J = 6.3 Hz, 2H), 2.40-2.31 (m, 1H), 2.17 (s, 3H), 2.10 (s, 6H), 2.08-2.03 (m, 2H), 2.02-1.94 (m, 3H), 1.92-1.88 (m, 2H).

[0190] Example 17 Preparation of compound 265

[0191] Step a: Preparation of intermediate 1 4-(4-bromo-2-chloro-6-methylphenoxy)-6-chloro-5-fluoro-2-methylpyrimidine.

[0192] The starting material 2,4,6-trichloro-5-fluoropyrimidine (201 mg, 1 mmol) was dissolved in acetonitrile, 4-bromo-2-chloro-6-methylphenol (221 mg, 1 mmol), potassium carbonate (276 mg, 2 mmol) were added successively and the reaction was carried out at 80 °C for 6 hours. TLC monitoring was performed and after the reaction was completed, the filtrate was suction filtered, dried in vacuo, and the sample was mixed with silica gel and column chromatography was performed to obtain 274 mg of intermediate 1 at a yield of 75%. MS (ESI) m / z: 366.9 [M+H] + .

[0193] Step b: Preparation of intermediate 2 6-(4-bromo-2-chloro-6-methylphenoxy)-5-fluoro-N-(1-(3-fluorophenyl)cyclopropyl)-2-methylpyrimidin-4-amine.

[0194] Intermediate 1 (183 mg, 0.5 mmol), 1-(3-fluorophenyl)-cyclopropylamine hydrochloride (93 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, N,N-diisopropylethylamine (262 μL, 1.5 mmol) was added, and the reaction was carried out at 120 °C overnight. After the reaction was completed, the mixture was diluted with 20 mL of water, extracted with 10 mL of ethyl acetate twice, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and column chromatography was performed to isolate 152 mg of intermediate 2 at a yield of 63%. MS (ESI) m / z: 482.0 [M+H] + .

[0195] Step c: Preparation of product GP629 6-(2-chloro-6-methyl-4-(4,5,6,7-tetrahydropyrazolo[1,5-a]pyridin-3-yl)phenoxy)-5-fluoro-N-(1-(3-fluorophenyl)cyclopropyl)-2-methylpyrimidin-4-amine.

[0196] Intermediate 2 (109 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)- 4,5,6,7-tetrahydropyrazolo[l,5-a]pyridine (56 mg, 0.225 mmol), 1,1-bis(diphenylphosphino) ferrocene palladium dichloride (22 mg, 0.03 mmol), potassium carbonate (93 mg, 0.675 mmol) were weighed in a pear shape flask, dissolved in dioxane / water (10 ml: 1 ml), protected by nitrogen, 100 Celsius overnight. After the reaction was completed, filtered, the filtrate was concentrated under reduced pressure, separated by column chromatography to obtain 89 mg of product 265, white solid, the yield was 76%. MS (ESI) m / z: 522.2 [M+H] + . 1 H NMR (400 MHz, CDC13) δ 7.68 (s, 1H), 7.32 - 7.29 (m, 1H), 7.28 - 7.25 (m, 1H), 7.20 - 7.16 (m, 1H), 7.14 - 7.06 (m, 2H), 6.91 (t, J = 9.0 Hz, 1H), 5.65 (s, 1H), 4.23 (t, J = 6.1 Hz, 2H), 2.98 (t, J = 6.4 Hz, 2H), 2.26 (s, 3H), 2.24 (s, 3H), 2.14 - 2.07 (m, 2H), 1.98 - 1.90 (m, 2H), 1.43 - 1.37 (m, 4H).

[0197] Example 18 Preparation of compound 300

[0198] Step a: Preparation of intermediate 1 tert-butyl (S)-4-[(tert-butyldimethylsilyl)oxy]- 2-oxopyrrolidine-l-carboxylate

[0199] To a solution of (S)-4-hydroxy-2-pyrrolidinone (5.0 g, 49.5 mmol) in super-dry dimethylformamide (25 mL) at 0 °C was added tert-butyldimethylsilyl chloride (7.8 g, 52 mmol) and imidazole (5.1 g, 25.74 mmol). The reaction was allowed to warm to room temperature and stirred for 3 h. The mixture was poured into water and the resulting precipitate was extracted with ethyl acetate. The product must be dried to ensure no residual moisture. This gave 2(S)-4-[(tert-butyldimethylsilyl)oxy]pyrrolidin-2-one. A solution of 2 (10.9 g, 50.7 mmol) in super-dry acetonitrile (100 mL) was charged with triethylamine (TEA, 8.5 mL, 61 mmol), 4-dimethylaminopyridine (DMAP, 3.1 g, 25.45 mmol) and di-tert-butyl dicarbonate (14.4 g, 66.2 mmol) at 0 °C under a nitrogen atmosphere. The mixture was allowed to warm to room temperature and stirred overnight. The mixture was poured into water and extracted with ethyl acetate. The organic layer was separated, washed with 1 N hydrochloric acid, 1 N sodium hydroxide and saturated aqueous brine, dried over sodium sulfate, filtered and concentrated to give tert-butyl (S)-4-[(tert-butyldimethylsilyl)oxy]-2-oxopyrrolidin-1- carboxylate. [M+H] + = 316.2.

[0200] Step b: Preparation of Intermediate 2 tert-butyl N-[(2S)-2-[(tert-butyldimethylsilyl)oxy]-4-hydroxy-4-phenylbutyl]carbamate

[0201] To a solution of Intermediate 1 (13.6 g, 43.2 mmol) in super-dry tetrahydrofuran (100 mL) at 0 °C under a nitrogen atmosphere was added phenyl magnesium bromide (52 mL, 1 M in tetrahydrofuran, 51.84 mmol) slowly over 1 h. The reaction mixture was stirred at 0 °C for 1 h. To this mixture was added super-dry methanol (80 mL) followed by sodium borohydride (2.45 g, 64.8 mmol) at 0 °C. The mixture was stirred at 0 °C for 1 h and then poured into 10% aqueous ammonium chloride solution. The mixture was extracted with ethyl acetate, washed with saturated aqueous brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography on silica gel using a gradient of ethyl acetate in hexane to give tert-butyl N-[(2S)-2-[(tert-butyldimethylsilyl)oxy]-4-hydroxy-4-phenylbutyl]carbamate. [M+H] + = 396.3.

[0202] Step c: Preparation of Intermediate 3 tert-butyl (4S)-4-[(tert-butyldimethylsilyl)oxy]-2-phenylpyrrolidine-1-carboxylate

[0203] To a solution of intermediate 2 (15.8 g, 38.2 mmol) in super-dry dichloromethane (120 mL) was added triethylamine (TEA, 16 mL, 114.6 mmol) and methanesulfonyl chloride (MsCl, 3.3 mL, 42.0 mmol) at -60 °C under nitrogen atmosphere. The resulting mixture was stirred at -60 °C for 1 h. The reaction was poured into water, washed with saturated brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by silica gel column chromatography using ethyl acetate / hexane gradient as eluent to give the target product (4S)-4-[(tert-butyldimethylsilyl)oxy]-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester. [M+H] + = 378.2.

[0204] Step d: Preparation of intermediate 4 (4S)-4-hydroxy-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester

[0205] To a solution of intermediate 3 (18.1 g, 38.2 mmol) in super-dry tetrahydrofuran (76 mL) was added tetra-n-butylammonium fluoride (TBAF, 50 mL, 1.0 M in tetrahydrofuran, 49.7 mmol) at room temperature. The mixture was stirred at room temperature for 2 h, then poured into water. The mixture was extracted with ethyl acetate, washed with water and saturated brine, dried over sodium sulfate, filtered and concentrated. The mixture was purified by silica gel column chromatography using ethyl acetate / hexane gradient as eluent to give the product (4S)-4-hydroxy-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester. [M+H] + = 264.2.

[0206] Step e: Preparation of intermediate 5 (2S,4R)-4-fluoro-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester

[0207] To a solution of intermediate 4 (2.7 g, 9.6 mmol) in super-dry dichloromethane (25 mL) was added diethylaminosulfur trifluoride (DAST, 2.5 mL, 19.2 mmol) at -78 °C. The mixture was stirred at -78 °C for 2 h, then slowly warmed to room temperature and stirred overnight. To the mixture was added aqueous sodium bicarbonate solution dropwise at 0 °C, then extracted with dichloromethane. The organic layers were combined, washed with saturated brine, dried over sodium sulfate, filtered and concentrated. The two diastereomers (2S,4R)-4-fluoro-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester and (2R,4R)-4-fluoro-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester were separated by silica gel column chromatography using ethyl acetate / hexane gradient as eluent, the less polar product (2S,4R)-4-fluoro-2-phenylpyrrolidine-1 -carboxylic acid tert-butyl ester was the target product. [M+H] + = 266.2.

[0208] Step f: Preparation of intermediate 6 (2S,4R)-4-fluoro-2-phenylpyrrolidine

[0209] Intermediate 5 was dissolved in dichloromethane, 5 equivalents of hydrochloric acid in dioxane was added and the reaction was stirred for 4 hours. After spinning down, the solution was dissolved in sodium bicarbonate and extracted with ethyl acetate. The organic layers were combined, filtered and concentrated to yield the product (2S,4R)-4-fluoro-2-phenylpyrrolidine. [M+H] + = 166.1.

[0210] Step g: Preparation of intermediate 7 4-(4-bromo-2,6-dimethylphenoxy)-5-fluoro-6-[(2S,4R)-4-fluoro-2-phenylpyrrolidin-l-yl]-2-methylpyrimidine

[0211] Intermediate 6 was dissolved in dimethyl sulfoxide, 1 equivalent of 4-(4-bromo-2,6- dimethylphenoxy)-6-chloro-5-fluoro-2-methylpyrimidine, 4 equivalents of DIPEA were added and the reaction was stirred at 120 °C overnight. The solution was poured into water and extracted with ethyl acetate. The organic layers were combined, filtered and concentrated. The mixture was purified by silica gel column chromatography using a gradient of ethyl acetate / hexanes as eluent to yield the product 4-(4-bromo-2,6-dimethylphenoxy)-5-fluoro-6-[(2S,4R)-4-fluoro-2-phenylpyrrolidin-l-yl]-2-methylpyrimidine. [M+H] + = 476.1.

[0212] Step h: Preparation of intermediate 8 tert-butyl 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2- phenylpyrrolidin-l-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-lH-pyrrolo[2,3- b]pyridine- 1 -carboxylate

[0213] Intermediate 9 was dissolved in dioxane, 3 mL of water, 1 equivalent of l-tert-butoxycarbonyl-7-azaindole-3-boronic acid pinacol ester, 4 equivalents of potassium carbonate and 0.15 equivalents of 1,1'-bis(di-cyclohexylphosphino)ferrocene palladium dichloride were added and the reaction was stirred at 100 °C overnight under a nitrogen atmosphere. The mixture was purified by silica gel column chromatography using a gradient of ethyl acetate / hexanes as eluent to yield the product tert-butyl 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2-phenylpyrrolidin-l-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-lH-pyrrolo[2,3-b]pyridine-l-carboxylate. [M+H] + = 612.3.

[0214] Step i: Preparation of final product 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2- phenylpyrrolidin-1-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H- pyrrolo[2,3-b]pyridine

[0215] Intermediate 8 was dissolved in dichloromethane, 5 equivalents of hydrochloric acid in dioxane was added, and the reaction was allowed to proceed for 4 hours. After spinning down, the reaction was dissolved in sodium bicarbonate and extracted with ethyl acetate. The organic layers were combined, filtered, and concentrated. The mixture was purified using silica gel column chromatography with a gradient of methanol / dichloromethane as eluent to yield compound 300 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2-phenylpyrrolidin-1-yl)-2- methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridine. 1 H NMR (400 MHz, DMSO) δ 11.86 (s, 1H), 8.31 (d, J = 8.1 Hz, 1H), 8.28 - 8.23 (m, 1H), 7.83 (s, 1H), 7.43 (s, 2H), 7.37 - 7.29 (m, 4H), 7.26 - 7.19 (m, 1H), 7.16 - 7.11 (m, 1H), 5.56 - 5.35 (m, 2H), 4.28 - 4.12 (m, 2H), 2.85 - 2.67 (m, 1H), 2.22 - 2.11 (m, 1H), 2.07 (s, 3H), 2.05 (s, 6H). 13 C NMR (101 MHz, DMSO) δ 160.59, 160.48, 157.12, 157.02, 151.57, 149.51, 148.01, 143.30, 139.65, 132.82, 132.23, 131.16, 129.72, 128.96, 128.09, 127.70, 127.46, 126.80, 124.08, 117.76, 116.41, 114.22, 69.49, 66.78, 55.31, 55.25, 42.41, 42.35, 25.66, 16.71. [M+H] + = 512.2

[0216] The remaining compounds in Table 1 of the present application were prepared according to the synthetic methods described above for Examples 1-16, in combination with methods conventional in the art, and have structure- corresponding characterization data as shown in Table 2.

[0217] Table 2 Numbering and characterization data for GPR65 antagonists

[0218] The beneficial effects of the present application are demonstrated by the following specific experimental examples.

[0219] Experimental Example 1, Study on the efficacy of the compound of the present application

[0220] 1. Experimental method

[0221] 1.1 Luminescence-based activity test system

[0222] GloSensor is a modified luciferase, which has low catalytic activity when not combined with cAMP, and weak luminescence after adding substrate; when combined with cAMP, the sensor catalytic activity is significantly improved, and the luminescence is enhanced after adding substrate.

[0223] When the density of HEK293T cells reached 60%-70%, pcDNA3.1-Flag-GPR65 and pGloSensor TM 22F plasmid (mass ratio 1:3), 24 h later, the cells were resuspended in a white sterile 96-well plate coated with poly-L-lysine (100 ng / mL) at a concentration of 2.5 x 10 4 After the cells adhered, the supernatant was discarded, 40 μL of HBSS solution containing 30 μM D-luciferin potassium salt (containing 20 mM TAPS solution, pH = 8.40) was added to each well, and the plate was balanced in a 5% CO2, 37°C cell incubator for 90 minutes. The test drug was diluted with HBSS solution containing 100 μM IBMX and 0.1% BSA (containing 20 mM HEPES solution, pH = 7.20), and the final concentration of the drug screening was 10 μM. The balanced plate was taken out, the supernatant was discarded, and 45 μL of the test drug solution was added to each well. The pH 7.20 buffer was used as the positive well, and the pH 8.40 buffer was used as the negative well. After 1 hour of action at room temperature, the multifunctional enzyme labeler was used to detect the bioluminescence signal. The antagonistic rate (%) = (test drug signal value - pH 8.40 signal value) / (pH 7.20 signal value - pH 8.40 signal value) x 100%. Compounds with an antagonistic rate < 50% were further retested at a concentration of 30 μM, three-fold dilution, and at least 3 replicates per concentration. The concentration-antagonistic rate was plotted, and the activity was fitted using Graphpad 8.3.

[0224] 1.2 Evaluation of the anti-MC38 tumor pharmacodynamics of compound 121

[0225] C57BL / 6 mice, male, 20-22 g, were raised in a SPF level animal room. MC38 was cultured using DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin double antibody), and no contamination was ensured during the culture. The log phase MC38 cells were collected, washed with sterile PBS three times, and then resuspended with DMEM medium to adjust the cell concentration to 1 x 10 7 Compound 121 was dissolved in 10% DMSO, and a drug solution of 3 mg / mL was prepared by adding 30% PEG400, 5% HS-15, and 55% physiological saline, and the solution was clear. PD-1 monoclonal antibody was diluted to 0.6 mg / mL with a special buffer and used on ice. The tumor volume was 100 cm 3The mice were randomly divided into four groups, namely: solvent group, PD-1 monoclonal antibody group (dose 6 mg / kg), compound 121 group (dose 30 mg / kg), and compound 121 + PD-1 monoclonal antibody combination group (dose 6 mg / kg of PD-1 monoclonal antibody and 30 mg / kg of compound 121). The mice were orally administered compound 121 twice a day, and the tumor volume of the mice was measured every other day. The tumor volume (cm 3 ) = (length x width 2 ) / 2. When the PD-1 monoclonal antibody group reached 200 cm 3 , the mice in the PD-1 monoclonal antibody group and the combination group were injected intraperitoneally with PD-1 monoclonal antibody solution every other day. When the tumor volume of the mice in the solvent group reached 1500 cm 3 , the experiment was terminated, and the subcutaneous tumors of the mice were fixed in 4% paraformaldehyde.

[0226] 1.3 Anti-epileptic efficacy evaluation of compound 121

[0227] SD rats, male, 220-250 g, were raised in a SPF level animal room. Compound 121 and 284 were dissolved in 10% DMSO, and then added to 30% PEG400, 5% HS-15 and 55% physiological saline to prepare a drug solution of 1 and 3 mg / mL, and the solution was clear. Pentylenetetrazole (PTZ) is a central nervous system stimulant that can induce acute epilepsy. PTZ was prepared as a 18 mg / mL solution with physiological saline and kept on ice. The rats were randomly divided into four groups, namely: solvent + PTZ group, 121 (5 mg / kg) + PTZ group, 121 (10 mg / kg) + PTZ group, and 284 (10 mg / kg) + PTZ group, wherein 284 is a negative control. The rats were given intraperitoneal injection of the corresponding drug solution, and 1 hour later, 65 mg / kg of PTZ solution was injected intraperitoneally. The rats were continuously observed for 1 hour and the corresponding indicators were recorded. The seizure condition of each rat was graded according to the Racine scoring system, which is as follows: 1st grade, slurred speech, facial twitching; 2nd grade, head twitching; 3rd grade, limb lifting, twitching; 4th grade, falling, limb twitching; and 5th grade, turning over, strong whole body twitching with limb rigidity.

[0228] 1.4 Anti-CT26 tumor efficacy evaluation of compound 300

[0229] Since the human and mouse activity of compound 300 is quite different (human IC 50 = 0.009 μM, mouse IC 50 > 30 μM), a Gpr65 humanized mouse was used for efficacy evaluation.

[0230] Humanized Gpr65-Balbc mice, female, 4-5 weeks old, were raised in SPF level animal house. CT26 cells were cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin double antibody) without contamination. The logarithmic growth phase of CT26 cells was collected, washed with sterile PBS for three times, and then resuspended in DMEM medium. The cell concentration was adjusted to 5x10 6 6 / mL, and 100 μL of cell suspension was inoculated in the right axillary subcutaneous position of mice. Compound 300 was dissolved in 10% DMSO, and then prepared into a 1 mg / mL drug solution by adding 30% PEG400, 5% HS-15 and 55% normal saline. The solution was clear. PD-1 monoclonal antibody was diluted to 0.6 mg / mL with a special buffer and used on ice. Mice with a tumor volume of about 100 cm 3 3> were randomly divided into four groups: solvent group, PD-1 monoclonal antibody group (dose 6 mg / kg), compound 300 group (dose 10 mg / kg), and compound 300+PD-1 monoclonal antibody combination group (PD-1 monoclonal antibody dose 6 mg / kg, compound 300 dose 10 mg / kg). Mice were orally administered with compound 300 once a day, and the tumor volume of mice was measured every other day. Tumor volume (cm 3 3> = (length x width 2 ) / 2. When the tumor volume of the PD-1 monoclonal antibody group reached 200 cm 3 3>, the mice in the PD-1 monoclonal antibody group and the combination group were injected with PD-1 monoclonal antibody solution intraperitoneally every other day. When the tumor volume of the solvent group reached 1500 cm 3 3>, the experiment was terminated, and the subcutaneous tumors of mice were fixed in 4% paraformaldehyde.

[0231] 1.5 Anti-epileptic pharmacodynamic evaluation of compound 300

[0232] Since the human and mouse activity of compound 300 has a large difference (human IC 50 = 0.009 μM, mouse IC 50 > 30 μM), the humanized Gpr65 mouse was used for pharmacodynamic evaluation.

[0233] Humanized Gpr65-Balb / c mice and common wild-type Balb / c mice, both male, 8-9 weeks old, were raised in the SPF level animal room. Compound 300 was dissolved with 10% DMSO, added with 30% PEG400, 5% HS-15 and 55% physiological saline to prepare a drug solution of 1 mg / mL, and the solution was clear. Pentylenetetrazole (PTZ) is a central nervous system stimulant that can induce acute epilepsy. Pentylenetetrazole was prepared into an 8 mg / mL solution with physiological saline and used on ice. The humanized and wild-type mice were randomly divided into two groups, respectively, namely: solvent + PTZ group, 300 (1 mg / kg) + PTZ group. The mice were given intraperitoneal injection of the corresponding drug solution, and 30 minutes later, 80 mg / kg of PTZ solution was subcutaneously injected, and the mice were continuously observed for 1 hour and the corresponding indicators were recorded. According to the Racine scoring system, the seizure condition of each mouse was graded, specifically: 1st grade, slurred speech, facial twitching; 2nd grade, head twitching; 3rd grade, limb lifting, twitching; 4th grade, falling, limb twitching; 5th grade, turning over, strong whole body twitching with limb rigidity.

[0234] 2. Experimental results

[0235] 2.1 GPR65 antagonistic activity of the compound of the present application

[0236] All the compounds of the present application were detected by the cAMP-Glosensor method, and the concentration-antagonism rate was plotted, and the activity was fitted by Graphpad8.3. The activity is shown in Table 3.

[0237] Table 3 Antagonistic effect of the compound of the present application on hGPR65

[0238] 2.2 Compound 121 has an anti-tumor effect

[0239] Compared with the solvent group, compound 121 single drug and combination with PD-1 monoclonal antibody showed obvious anti-tumor effect, which was manifested as the reduction of tumor volume (Fig. 1A, Fig. 1C) and tumor dry weight (Fig. 1B). And the 15-day administration did not affect the body weight of mice, and no adverse effects were shown (Fig. 1D).

[0240] 2.3 Compound 121 has an anti-epileptic effect

[0241] Compared with the solvent group, compound 121 (10 mg / kg, i.p.) can alleviate acute epilepsy induced by PTZ in rats, which is manifested by prolonging the first seizure latency (Figure 2A), reducing the seizure grade (Figure 2C), reducing the total seizure time (Figure 2B) and the number of seizures (Figure 2D).

[0242] 2.4 Compound 300 has an antitumor effect

[0243] In Gpr65 humanized mice, compared with the solvent group, PD-1 monoclonal antibody does not show obvious antitumor effect, and compound 300 single drug and combination with PD-1 monoclonal antibody both show obvious antitumor effect, which is manifested by reducing the tumor volume (Figure 3A) and the tumor dry weight (Figure 3B). However, in wild-type mice, compound 300 loses efficacy (Figure 3C, Figure 3D), indicating that the compound depends on antagonizing GPR65 to exert the antitumor effect. This is consistent with the cell activity experiment results, and compound 300 shows good targeting.

[0244] 2.5 Compound 300 has an antiepileptic effect

[0245] In Gpr65 humanized mice, compared with the solvent group, compound 300 (10 mg / kg, i.p.) can alleviate acute epilepsy induced by PTZ in mice, which is manifested by prolonging the first seizure latency (Figure 4A), reducing the number of seizures (Figure 4B), and reducing the seizure grade (Figure 4C). However, in wild-type mice, compound 300 loses efficacy, indicating that the compound depends on antagonizing GPR65 to exert the antiepileptic effect. This is consistent with the cell activity experiment results, and compound 300 shows good targeting.

[0246] The experimental results show that the compound of the present application has excellent antagonistic activity on human G protein-coupled receptor 65, and the compound of the present application has a broad application prospect in the preparation of drugs for antiepileptic and antitumor.

Claims

1. A compound represented by Formula I, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystal form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof: Formula I wherein, X 1 is selected from CR1, N, X 2 is selected from CR2, N, X 3 is selected from CR3, N, and X 1 , X 2 , X 3 at least one of X R1, R2, R3are each independently selected from the group consisting of hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, amino, nitro; R4is selected from hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkoxy, C 1-6 alkylthio, halogen-substituted C 1-6 alkylthio, amino, phenyl, 5-6 membered heteroaromatic ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl; or R2, R4 are linked to form a phenyl ring; L is selected from the group consisting of none, O, C 1-4 alkylene; n is selected from 0, 1, 2, 3, 4; C ring is selected from phenyl ring, 5-6 membered heteroaromatic ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl; R c each independently selected from hydrogen, unsubstituted or substituted C c1 substituted C 1-6 alkyl, unsubstituted or substituted by one or two or more R c1 substituted C 1-6 alkoxy, unsubstituted or substituted by one or two or more R c2 substituted C 2-10 alkenyl, unsubstituted or substituted by one or two or more R c2 substituted C 2-10 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted by one or two or more R c3 substituted phenyl, unsubstituted or substituted by one or two or more R c3 substituted 5-6 membered heteroaryl, unsubstituted or substituted by one or two or more R c3 substituted 3-8 membered saturated cycloalkyl, unsubstituted or substituted by one or two or more R c3 substituted 3-8 membered saturated heterocyclyl, unsubstituted or substituted by one or two or more R c3 substituted fused ring, L0COOL1R6, L0NR8COL1R6, L0CONR8L1R6, OR7, COR9; R c1 each independently selected from the group consisting of hydroxyl, halogen, C 1-6 alkyl; R c2 each independently selected from the group consisting of hydroxyl, halogen, C 1-6 alkyl; R c3 each independently selected from halogen, C 1-6 alkyl, hydroxyl or halogen substituted C 1-6 alkyl, C 1-6 alkoxy, hydroxyl or halogen substituted C 1-3 alkoxy, 3-8 membered saturated heterocyclyl; L0is selected from the absence, C 2-4 alkylene; L1is selected from the group consisting of none, C 1-4 alkylene; R6is selected from C 1-6 alkyl, unsubstituted or substituted by one or more R 6a substituted by one or more R 6a each independently selected from halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkoxy, COOR 6b hydroxy; R 6b selected from C 1-6 alkyl; R8is selected from hydrogen, C 1-6 alkyl, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl; R7 is selected from phenyl, benzyl; R9is selected from the group consisting of unsubstituted or substituted phenyl, R 9a substituted cycloalkyl, R 9a selected from the group consisting of halogen, C 1-6 alkyl; or m R c two adjacent R c are linked to form a ring; R5is selected from L2NR 13 L3L4R 10 , phenyl which is unsubstituted or substituted by one or two or more R 11 L5L6R 14 ; L2 is selected from nothing, 3-6 membered saturated nitrogen heterocycle; L3 is selected from the group consisting of none, C 1-6 alkylene, hydroxy-substituted C 1-6 alkylene; L4 is selected from the group consisting of no, R 13 selected from hydrogen, C 1-6 alkyl; R 10 is selected from amino protecting groups, C 1-6 alkyl, NR a1 R a2 , OR a3 , CONHL7R a4 , is unsubstituted or substituted by one or more than one R 12 is selected from the group consisting of 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclyl, 5-6 membered heteroaryl, phenyl, fused ring, spirocyclic; R a1 is phenyl ring, R a2 is selected from hydrogen, C 1-5 alkyl, R a3 is selected from C 1-6 alkyl, phenyl, benzyl; R a4 is selected from 3-6 membered saturated cycloalkyl; L7 is selected from null, C 1-4 alkylene; R 12 is each independently selected from halogen, hydroxyl, C 1-6 alkoxy, C 1-6 alkyl, cyano, COOR b1 , R b1 is selected from C 1-6 alkyl; R 11 selected from halogen, C 1-6 alkoxy, C 1-6 alkyl, hydroxy; L5 is selected from none, unreplaced, or replaced by one or more Rs. 5a Substituted 3-6 membered saturated nitrogen heterocycles, R 5a Each is independently selected from hydroxyl and halogen; L6is selected from the group consisting of none, C 1-4 alkylene; R 14 selected from unsubstituted or substituted by one or more than one R 14a substituted phenyl ring, 3-6 membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, fused ring, R 14a each independently selected from halogen, halogen-substituted C 1-6 alkyl, C 1-6 alkyl; R 14 selected from amino protecting groups; The compound is not 2. The compound of claim 1, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystalline form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof, wherein, The selected from one of the following structures:

3. The compound of claim 2, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystalline form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof, wherein, R3is hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl; R4is selected from hydrogen, halogen, C 1-6 alkyl, halogen-substituted C 1-6 alkyl, C 1-6 alkoxy, halogen-substituted C 1-6 alkoxy, C 1-6 alkylthio, halogen-substituted C 1-6 alkylthio, amino, phenyl, 3- to 6-membered saturated cycloalkyl.

4. The compound of any one of claims 1-3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystalline form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof, wherein, said L is selected from the group consisting of none, O, n is selected from 0, 1, 2, 3, 4; C ring is selected from phenyl ring, 5-6 membered nitrogen heteroaromatic ring, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl; m is selected from 0, 1, 2, 3, 4; R c each independently selected from hydrogen, unsubstituted or substituted C c1 substituted C 1-4 alkyl, unsubstituted or substituted with one or two or more R c1 substituted C 1-4 alkoxy, unsubstituted or substituted with one or two or more R c2 substituted C 2-4 alkenyl, unsubstituted or substituted with one or two or more R c2 substituted C 2-4 alkynyl, cyano, halogen, nitro, amino, unsubstituted or substituted with one or two or more R c3 substituted phenyl, unsubstituted or substituted with one or two or more R c3 substituted 5-6 membered heteroaryl, unsubstituted or substituted with one or two or more R c3 substituted 3-6 membered saturated cycloalkyl, unsubstituted or substituted with one or two or more R c3 substituted 3-6 membered saturated heterocyclyl, unsubstituted or substituted with one or two or more R c3 substituted 5 membered azacyclic ring fused to a 5-6 membered saturated heterocyclic ring, unsubstituted or substituted with one or two or more R c3 substituted 5 membered azacyclic ring fused to a 5-6 membered heteroaryl ring, L0COOL1R6, L0NR8COL1R6, L0CONR8L1R6, OR7, COR9; R c1 each independently selected from the group consisting of hydroxyl, halogen, C 1-4 alkyl; R c2 each independently selected from the group consisting of hydroxyl, halogen, C 1-4 alkyl; R c3 each independently selected from halogen, C 1-4 alkyl, hydroxyl, or halogen substituted C 1-4 alkyl, C 1-4 alkoxy, hydroxyl, or halogen substituted C 1-4 alkoxy, 3-6 membered saturated heterocyclyl; L0is selected from the absence, C 2-3 alkylene; L1is selected from the group consisting of none, C 1-3 alkylene; R6is selected from C 1-4 alkyl, unsubstituted or substituted by one or more R 6a substituted by one or more R 6a each independently selected from halogen, C 1-4 alkyl, halogen-substituted C 1-4 alkyl, C 1-4 alkyl, halogen-substituted C 1-4 alkoxy, COOR 6b , hydroxy; R 6b selected from C 1-4 alkyl; R8is selected from hydrogen, C 1-4 alkyl, 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl; R7 is selected from phenyl, benzyl; R9is selected from the group consisting of unsubstituted or substituted 5-6 membered saturated nitrogen heterocycle, R 9a substituted 5-6 membered saturated nitrogen heterocycle, R 9a selected from the group consisting of halogen, C 1-4 alkyl; or m R c wherein two adjacent R c are linked to form a ring selected from a 5-6 membered saturated oxacyclic ring, a phenyl ring.

5. The compound of any one of claims 1-3, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystalline form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof, wherein, R5is selected from L2NR 13 R 10 , NR 13 L3L4R 10 , phenyl which is unsubstituted or substituted by one or two or more R 11 L5L6R 14 ; L2 is selected from nothing, 4-6 membered saturated nitrogen heterocycle; L3 is selected from the group consisting of none, C 1-5 alkylene, hydroxy-substituted C 1-5 alkylene; L4 is selected from the group consisting of no, R 13 selected from hydrogen, C 1-5 alkyl; R 10 selected from amino protecting groups, C 1-5 alkyl, NR a1 R a2 , OR a3 , CONHL7R a4 , unsubstituted or substituted by one or more than one R 12 3-6 membered saturated cycloalkyl, 3-6 membered saturated heterocyclyl, 5-6 membered heteroaryl, phenyl, 5-6 membered saturated carbocyclophenyl ring, R a1 is a phenyl ring, R a2 is selected from hydrogen, C 1-5 alkyl, R a3 is selected from C 1-5 alkyl, phenyl, benzyl; R a4 is selected from 3-6 membered saturated cycloalkyl; L7 is selected from none, C 1-2 alkylene; R 12 are each independently selected from halogen, hydroxy, C 1-5 alkoxy, C 1-5 alkyl, cyano, COOR b1 , R b1 is selected from C 1-5 alkyl; R 11 selected from halogen, C 1-5 alkoxy, C 1-5 alkyl, hydroxy; L5 is selected from none, unreplaced, or replaced by one or more Rs. 5a Substituted 4-6 membered saturated nitrogen heterocycles, R 5a Each is independently selected from hydroxyl and halogen; L6is selected from the group consisting of none, C 1-2 alkylene; R 14 selected from unsubstituted or substituted by one or more than one R 14a substituted phenyl ring, 5-membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, 5-membered heteroarylbenzene ring, R 14a each independently selected from halogen, halogen-substituted C 1-5 alkyl, C 1-5 alkyl; R 14 selected from amino protecting groups.

6. The compound of any one of claims 1-5, or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, or a solvate thereof, or a crystalline form thereof, or a prodrug thereof, or a metabolite thereof, or a deuterated derivative thereof, wherein, The structure of the compound is shown as formula II or formula III: wherein R3is selected from hydrogen, halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl; R4is selected from hydrogen, halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl; R x1 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl; R x2 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl; R x3 selected from halogen, C 1-3 alkyl, halogen-substituted C 1-3 alkyl, 5-membered nitrogen heteroaromatic ring fused to a 5-6 membered saturated heterocyclic ring, 5-membered nitrogen heteroaromatic ring fused to a 5-6 membered heteroaromatic ring; f is selected from 0, 1, 2; R 15 each independently selected from hydrogen, halogen, C 1-3 alkyl.

7. The compound of claim 1, stereoisomer thereof, pharmaceutically acceptable salt thereof, solvate thereof, crystal form thereof, prodrug thereof, metabolite thereof or deuterated derivative thereof, characterized in that: The compound is selected from one of the following compounds:

8. A pharmaceutical composition, characterized by, The pharmaceutical composition is prepared by taking the compound, stereoisomer thereof, pharmaceutically acceptable salt thereof, solvate thereof, crystal form thereof, prodrug thereof, metabolite thereof or deuterated derivative thereof of any one of claims 1-7 as an active ingredient, plus pharmaceutically acceptable adjuvants.

9. Use of compound 9, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a crystal form thereof, a prodrug thereof, a metabolite thereof, or a deuterated derivative thereof, or a compound of any one of claims 1-6, a stereoisomer thereof, a pharmaceutically acceptable salt thereof, a solvate thereof, a crystal form thereof, a prodrug thereof, a metabolite thereof, or a deuterated derivative thereof, in the manufacture of a G protein-coupled receptor antagonist; 10. Use according to claim 9, characterized in that: The G protein-coupled receptor antagonist is a G protein-coupled receptor 65 antagonist.

11. Use according to claim 9 or 10, characterized in that: The G protein-coupled receptor antagonist is a drug for preventing and / or treating G protein-coupled receptor related diseases.

12. Use according to claim 11, characterized in that: The G protein-coupled receptor related disease is epilepsy or cancer.

13. Use according to claim 12, characterized in that: The cancer is melanoma, renal cell carcinoma, gastric cancer, liver cancer, acute myeloid leukemia, pancreatic adenocarcinoma, triple-negative breast cancer, colorectal cancer, head and neck cancer, colorectal adenocarcinoma, lung cancer, ovarian cancer, glioblastoma or glioma.

14. Use of a compound, stereoisomer thereof, pharmaceutically acceptable salt thereof, solvate thereof, crystal form thereof, prodrug thereof, metabolite thereof or deuterated derivative thereof in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating cancer; the compound is compound 9 or the compound of any one of claims 1-6.

15. Use according to claim 14, characterized in that: The immune checkpoint inhibition is a PD-1 antibody.

Images