WO2026051972A1 - Gpr65 small molecule regulator, preparation method therefor, and application - Google Patents

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

A small molecule regulator of GPR65, its preparation method and application Technical Field

This invention relates to the field of medicinal chemistry, specifically to a GPR65 small molecule modulator, its preparation method, and its application.

Background Technology

GPR65 is a receptor belonging to the G protein-coupled receptor (GPR) family and is widely used as a drug target or biomarker in biomedical research. GPR65 receptors play important biological functions in many physiological systems, including the nervous, endocrine, immune, and urinary systems, making them significant for disease diagnosis, treatment, and drug research.

Under normal physiological conditions, GPR65 is primarily expressed in T and B lymphocytes, as well as macrophages and other innate immune cells (e.g., neutrophils, eosinophils, and mast cells). GPR65 plays a crucial role in the tumor microenvironment, particularly in tumor-associated macrophages (TAMs). The acidic nature of the tumor microenvironment and the abundant presence of TAMs are key characteristics of solid tumors, driving immunosuppression, supporting tumor growth, and limiting the effectiveness of approved therapies. Studies have shown that GPR65 is a key factor in low-pH-induced immunosuppression, especially in TAMs. Research indicates that GPR65 activation can promote an immunosuppressive immune cell phenotype, thereby preventing immune-mediated killing of cancer cells. Therefore, inhibiting GPR65 may help enhance the immune system's anti-tumor capabilities. Studies have found that GPR65 overexpression in Lewis lung cancer can promote tumor growth through PKA and extracellular signal-regulated kinases (ERKs). Moreover, GPR65 is overexpressed in human colon, ovarian, and kidney tumor tissues. Pathios Therapeutics Ltd. has developed a small molecule GPR65 inhibitor called PTT-3213, currently in the preclinical stage. This drug has shown significant anti-tumor effects in the MC38 mouse symbiotic cancer model, increasing the number of CD8 ⁺ T cells and natural killer T cells in the tumor microenvironment and synergizing with PD-1 antibodies for enhanced efficacy. Furthermore, PTT-3213 has provided efficacy comparable to twice-weekly injections 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 antitumor pathway, particularly in hematologic malignancies including leukemia, lymphoma, and multiple myeloma.

Recent research indicates that abnormal activation or overexpression of GPR65 is closely related to the occurrence of behaviors such as seizures, fear, and anxiety, which are among the most common symptoms of epileptic seizures. For example, Lauren et al. found that exposing mice to a closed environment with high concentrations of _CO2 caused systemic carbon dioxide poisoning as CO2 binds to water molecules in the mice. This resulted in the continuous activation of GPR65 in microglia by high concentrations of H ⁺ , further promoting the release of the pro-inflammatory cytokine interleukin-1β (IL-1β), thereby stimulating abnormal firing of neurons in the subfornical organ and ultimately inducing seizures such as panic, fear, and anxiety in the mice. Katherine et al.'s research indicated that compared to wild-type mice, gene knockout mice (GPR65-/-) showed significant relief of fear and anxiety symptoms in experiments. Furthermore, a clinical study showed that compared to healthy volunteers, patients with panic disorder had significantly higher levels of GPR65 mRNA expression in peripheral blood mononuclear cells, and this expression 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 epileptic symptoms such as seizures, fear, and anxiety, suggesting that targeting and antagonizing GPR65 may be a potential new anti-epileptic therapy.

Given 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 against GPR65.

Summary of the Invention

The purpose of this invention is to provide a GPR65 small molecule regulator, its preparation method, and its application.

This invention provides compounds of Formula I, or their stereoisomers, or their pharmaceutically acceptable salts, or their solvates, or their crystal forms, or their prodrugs, or their metabolites, or their deuterated derivatives:

Wherein, ^X1 is selected from _CR1 and N, ^X2 is selected from _CR2 and N, ^X3 is selected from _CR3 and N, and at least one of ^X1 , ^X2 and ^X3 is N;

_R1 , _R2 , and _R3 are each independently selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, amino, and nitro groups;

R ₄ is selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, _C1-6 alkylthio, halogen-substituted _C1-6 alkylthio, amino, phenyl, 5-6 membered heteroaryl ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclic group;

Alternatively, _R2 and _R4 can be linked together to form a benzene ring;

L is selected from none, O, C _1-4 alkylene; n is selected from 0, 1, 2, 3, 4;

The C ring is selected from benzene rings, 5-6 membered heteroaromatic rings, 3-8 membered saturated cycloalkyl groups, and 3-8 membered saturated heterocyclic groups;

Each _Rc is independently selected from hydrogen, _C1-6 alkyl groups (unsubstituted or substituted with one or more _Rc1) , _C1-6 alkoxy groups (unsubstituted or substituted with one or more _Rc1) , _C2-10 alkenyl groups (unsubstituted or substituted with one or more _Rc2) , _C2-10 alkynyl groups (unsubstituted or substituted with one or more _Rc2) , cyano, halogen, nitro, amino, phenyl groups (unsubstituted or substituted with one or more _Rc3 ), 5-6 heteroaryl groups (unsubstituted or substituted with one or more _Rc3) , 3-8 saturated cycloalkyl groups (unsubstituted or substituted with one or more _Rc3) , 3-8 saturated heterocyclic groups (unsubstituted or substituted with one or more _Rc3) , fused ring groups (unsubstituted or substituted with one or more _Rc3) , _L0 COOL _{1 R6} , _L0 NR ₈ COOL _{1 R6} , L0 CONR _{8 L1 R 6} , OR ₇ , COR ₉ ;

R _{_c1} are each independently selected from hydroxyl, halogen, and C _{_1-6} alkyl groups;

R _{_c2} are each independently selected from hydroxyl, halogen, and C _{_1-6} alkyl groups;

R _{_c3} are each independently selected from halogens, C _{_1-6} alkyl groups, hydroxyl groups or halogen-substituted C _{_1-6} alkyl groups, C _{_1-6} alkoxy groups, hydroxyl groups or halogen-substituted C _{_1-3} alkoxy groups, 3-8 membered saturated heterocyclic groups;

_L0 is selected from none, _C2-4 alkenyl groups;

_L1 is selected from none and _C1-4 alkylene groups;

_R6 is selected from _C1-6 alkyl groups, unsubstituted or substituted with one or more _R6a groups of the following: 5-6 membered heteroaryl, phenyl, 3-8 membered saturated heterocyclic group, 3-8 membered saturated cycloalkyl, fused ring; _R6a is independently selected from halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, COOR _6b , hydroxyl;

R _6b is selected from _C1-6 alkyl groups;

_R8 is selected from hydrogen, _C1-6 alkyl, 3-8 membered saturated cycloalkyl, and 3-8 membered saturated heterocyclic groups;

R ₇ is selected from phenyl or benzyl;

_R9 is selected from unsubstituted or substituted rings with one or more _R9a , and _R9a is selected from halogens and _C1-6 alkyl groups.

Alternatively, among m R_{_c} , two adjacent R_{_c} are connected to form a ring;

The _R5 is selected _{from L2NR13L3L4R10} , phenyl _{groups that} are not substituted or are substituted _by one or more _R11 , _{and L5L6R14 ;}

_L2 is selected from non-membered, 3-6 membered saturated nitrogen heterocycles;

_L3 is selected from _C1-6 alkylene groups that are non-alkyl, C1-6 alkylene groups, or _C1-6 alkylene groups that are hydroxyl-substituted;

_L4 is selected from none.

R ₁₃ is selected from hydrogen and _C1-6 alkyl groups;

_R10 is selected from amino protecting groups, _C1-6 alkyl groups, NR _{a1 Ra2} , OR _a3 , CONHL _{7 Ra4} , and the following groups that are unsubstituted or substituted by one or more _R12 groups: 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclic, 5-6 membered heteroaryl, phenyl, fused ring, spirocyclic; _Ra1 is a benzene ring, _Ra2 is selected from hydrogen, _C1-5 alkyl, _Ra3 is selected from _C1-6 alkyl, phenyl, benzyl; _Ra4 is selected from 3-6 membered saturated cycloalkyl; _L7 is selected from none, _C1-4 alkylene; _R12 is independently selected from halogen, hydroxyl, _C1-6 alkoxy, _C1-6 alkyl, cyano, COOR _b1 , and R _b1 is selected from _C1-6 alkyl;

_R11 is selected from halogens, _C1-6 alkoxy groups, _C1-6 alkyl groups, and hydroxyl groups;

_L5 is selected from 3-6 membered saturated nitrogen heterocycles that are unsubstituted, unsubstituted, or substituted by one or more _R5a , where each _R5a is independently selected from hydroxyl groups or halogens.

_L6 is selected from none and _C1-4 alkylene groups;

R ₁₄ is selected from The following groups are not substituted or are substituted by one or more R _14a : benzene ring, 3-6 membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, fused ring; R _14a are each independently selected from halogen, halogen-substituted C _1-6 alkyl, C _1-6 alkyl; R ₁₄ is selected from amino protecting groups;

The compound is not

Furthermore, the aforementioned Choose from one of the following structures:

Furthermore, _R3 is derived from hydrogen, halogen, _C1-6 alkyl, or halogen-substituted _C1-6 alkyl;

_R4 is selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, _C1-6 alkylthio, halogen-substituted _C1-6 alkylthio, amino, phenyl, and 3-6 member saturated cycloalkyl.

Furthermore, the L is selected from none, O, n is selected from 0, 1, 2, 3, and 4;

The C ring is selected from benzene rings, 5-6 membered nitrogen-containing aromatic rings, 3-6 membered saturated cycloalkyl groups, and 3-6 membered saturated heterocyclic groups;

m is selected from 0, 1, 2, 3, and 4;

Each _Rc is independently selected from hydrogen, _C1-4 alkyl groups (unsubstituted or substituted with one or more _Rc1) , _C1-4 alkoxy groups (unsubstituted or substituted with one or more _Rc1) , _C2-4 alkenyl groups (unsubstituted or substituted with one or more _Rc2) , _C2-4 alkynyl groups (unsubstituted or substituted with one or more _Rc2) , cyano, halogen, nitro, amino, phenyl groups (unsubstituted or substituted with one or more _Rc3) , 5-6 membered heteroaryl groups (unsubstituted or substituted with one or more _Rc3) , 3-6 membered saturated cycloalkyl groups (unsubstituted or substituted with one or more _Rc3) , 3-6 membered saturated heterocyclic groups (unsubstituted or substituted with one or more _Rc3) , 5-membered nitrogen-containing heterocyclic rings and 5-6 membered saturated heterocycles (unsubstituted or substituted with one or more _Rc3 ), and other unsubstituted or substituted Rc3 groups. _{C3- substituted} 5- _membered nitrogen heteroaromatic rings and _5-6 - _{membered heteroaromatic} rings, _L0COOL1R6 , _L0NR8COL1R6 , _{L0CONR8L1R6 , OR7 , COR9 ;}

R _{_c1} are each independently selected from hydroxyl, halogen, and C _{_1-4} alkyl groups;

R _{_c2} are each independently selected from hydroxyl, halogen, and C _{_1-4} alkyl groups;

R _c3 are each independently selected from halogen, _C1-4 alkyl, hydroxyl or halogen-substituted _C1-4 alkyl, _C1-4 alkoxy, hydroxyl or halogen-substituted _C1-4 alkoxy, 3-6 membered saturated heterocyclic groups;

_L0 is selected from none, _C2-3 alkenyl groups;

_L1 is selected from none and _C1-3 alkylene groups;

_R6 is selected from _C1-4 alkyl groups, unsubstituted or substituted with one or more _R6a groups of the following: 5-6 membered heteroaryl, phenyl, 5-6 membered saturated heterocyclic group, 4-6 membered saturated cycloalkyl, benzocyclopentadienyl 5-6 membered saturated heterocyclic group; _R6a is independently selected from halogen, _C1-4 alkyl, halogen-substituted _C1-4 alkyl, _C1-4 alkoxy, halogen-substituted _C1-4 alkoxy, COOR _6b , hydroxyl;

R _6b is selected from _C1-4 alkyl groups;

_R8 is selected from hydrogen, _C1-4 alkyl, 3-6 membered saturated cycloalkyl, and 3-6 membered saturated heterocyclic groups;

R ₇ is selected from phenyl or benzyl;

_R9 is selected from 5-6 membered saturated nitrogen heterocyclic benzo[a] that is unsubstituted or substituted by one or more _R9a , and _R9a is selected from halogens and _C1-4 alkyl groups.

Alternatively, among the m _Rcs , two adjacent _Rcs are connected to form a ring, wherein the ring is selected from 5-6 nucleotide saturated oxyheterocycles or benzene rings.

_Further , _R5 is selected _{from L2NR13R10} , _NR13L3L4R10 , phenyl _groups that are not substituted or _are substituted _by one or more _R11 , _{and L5L6R14 ;}

_L2 is selected from non-membered, 4-6 membered saturated nitrogen heterocycles;

_L3 is selected from _C1-5 alkylene groups that are non-alkyl, C1-5 alkylene groups, or _C1-5 alkylene groups that are hydroxyl-substituted;

_L4 is selected from none.

R ₁₃ is selected from hydrogen and _C1-5 alkyl groups;

_R10 is selected from amino protecting groups, _C1-5 alkyl groups, NR _{a1 Ra2} , OR _a3 , CONHL _{7 Ra4} , and the following groups that are unsubstituted or substituted by one or more _R12 groups: 3-6 membered saturated cycloalkyl groups, 3-6 membered saturated heterocyclic groups, 5-6 membered heteroaryl groups, phenyl groups, 5-6 membered saturated carbocyclic benzophenone rings, etc. _Ra1 is a benzene ring; _Ra2 is selected from hydrogen, _C1-5 alkyl; _Ra3 is selected from _C1-5 alkyl, phenyl, benzyl; _Ra4 is selected from 3-6 membered saturated cycloalkyl; _L7 is selected from none, _C1-2 alkylene; _R12 is independently selected from halogen, hydroxyl, _C1-5 alkoxy, _C1-5 alkyl, cyano, COOR _b1 , and _Rb1 is selected from _C1-5 alkyl;

_R11 is selected from halogens, _C1-5 alkoxy groups, _C1-5 alkyl groups, and hydroxyl groups;

_L5 is selected from 4-6 membered saturated nitrogen heterocycles that are unsubstituted, unsubstituted, or substituted by one or more _R5a , where each _R5a is independently selected from hydroxyl groups or halogens.

_L6 is selected from non- _C1-2 alkylene groups;

R ₁₄ is selected from The following groups are not substituted or are substituted by one or more R _14a : benzene ring, 5-membered saturated nitrogen heterocycle, 5-6-membered heteroaryl, 5-membered heteroaryl fused benzene ring, where each R _14a is independently selected from halogen, halogen-substituted _C1-5 alkyl, or _C1-5 alkyl; R ₁₄ is selected from amino protecting groups.

Furthermore, the structure of the compound is as shown in Formula II or Formula III:

Wherein, _R3 is selected from hydrogen, halogen, _C1-3 alkyl, and halogen-substituted _C1-3 alkyl;

_R4 is selected from hydrogen, halogen, _C1-3 alkyl, and halogen-substituted _C1-3 alkyl;

R _x1 is selected from halogens, _C1-3 alkyl groups, and halogen-substituted _C1-3 alkyl groups;

R _x2 is selected from halogens, _C1-3 alkyl groups, and halogen-substituted _C1-3 alkyl groups;

R _x3 is selected from halogens, _C1-3 alkyl groups, halogen-substituted _C1-3 alkyl groups, 5-membered nitrogen heteroaromatic rings with 5-6-membered saturated heterocycles, and 5-membered nitrogen heteroaromatic rings with 5-6-membered heteroaromatic rings;

f is selected from 0, 1, and 2;

_R15 are each independently selected from hydrogen, halogens, and _C1-3 alkyl groups.

Furthermore, the compound is selected from one of the following compounds:

The present invention also provides a pharmaceutical composition comprising a formulation prepared by adding pharmaceutically acceptable excipients to compounds listed in the table above, their stereoisomers, their pharmaceutically acceptable salts, their solvates, their crystal forms, their prodrugs, their metabolites or their deuterated derivatives as active ingredients.

The present invention also provides the use of compound 9, its stereoisomers, its pharmaceutically acceptable salts, its solvates, its crystal forms, its prodrugs, its metabolites or their deuterated derivatives, or the compounds in the above table, their stereoisomers, their pharmaceutically acceptable salts, their solvates, their crystal forms, their prodrugs, their metabolites or their deuterated derivatives, in the preparation of G protein-coupled receptor antagonists.

Furthermore, the G protein-coupled receptor antagonist is a G protein-coupled receptor 65 antagonist.

Furthermore, the G protein-coupled receptor antagonist is a drug for the prevention and/or treatment of G protein-coupled receptor-related diseases.

Furthermore, the G protein-coupled receptor-related diseases are epilepsy or cancer.

Furthermore, the cancers mentioned are 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.

The present invention also provides the use of the above-mentioned compounds, their stereoisomers, their pharmaceutically acceptable salts, their solvates, their crystal forms, their prodrugs, their metabolites or their deuterated derivatives, in combination with immune checkpoint inhibitors in the preparation of medicaments for treating cancer; said compounds are compound 9 or compounds in the table above.

Furthermore, the immune checkpoint inhibitor is a PD-1 antibody.

Regarding the definition of terms used in this invention: Unless otherwise stated, the initial definitions provided for groups or terms herein apply to the groups or terms used throughout this specification; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and context.

The minimum and maximum carbon atom content in hydrocarbon groups are indicated by prefixes. For example, the prefix C _{a to C b} alkyl indicates any alkyl group containing "a" to "b" carbon atoms. For instance, C _{1 to C 6} alkyl refers to straight-chain or branched alkyl groups containing 1, 2, 3, 4, 5, or 6 carbon atoms; C 1 to _{C 4} alkylene refers to straight-chain or branched alkylene groups containing 1, 2, 3, or 4 carbon atoms; and so on.

"Heteroaryl" or "heteroary ring" refers to a heteroaryl group containing one or more heteroatoms. Heteratoms referred to here include, but are not limited to, oxygen, sulfur, and nitrogen.

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

"Nitrogen heterocycle" refers to a heterocycle containing nitrogen atoms. The heteroatom in the heterocycle may be only a nitrogen atom, or it may include nitrogen atoms and other heteroatoms, including but not limited to oxygen and sulfur.

"Parallel rings" refers to polycyclic rings with two rings sharing two adjacent ring atoms.

"Spiral ring" refers to a polycyclic ring with two rings sharing a single atom.

Amino protecting groups are chemical groups used to protect amino ( _-NH2 ) groups, such as tert-butoxycarbonyl, trifluoromethanesulfonyl, benzyloxycarbonyl, dibenzoyl, methanesulfonyl, etc.

Halogens are fluorine, chlorine, and bromine.

Experimental results show that the compounds of this invention exhibit excellent antagonistic activity against human G protein-coupled receptor 65 and can be used to prepare G protein-coupled receptor 65 antagonists. As is well known to those skilled in the art, G protein-coupled receptor 65 antagonists are effective against epilepsy and tumors; therefore, the compounds of this invention have broad application prospects in the preparation of drugs for anti-epileptic and anti-tumor purposes.

Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following embodiments. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention.

Attached Figure Description

Figure 1 shows the antitumor effect of compound 121: (A) subcutaneous tumor in mice; (B) tumor weight; (C) day-tumor volume curve; (D) day-body weight curve.

Figure 2 shows the anti-epileptic effect of compound 121: (A) is the latency period of the first seizure; (B) is the total seizure time; (C) is the highest seizure severity; and (D) is the number of seizures.

Figure 3 shows the anti-tumor effect of compound 300: (A) subcutaneous tumor in mice; (B) tumor weight; (C) day-tumor volume curve; (D) day-body weight curve.

Figure 4 shows the anti-epileptic effect of compound 300: (A) is the latency period of the first seizure; (B) is the number of seizures; and (C) is the highest seizure severity.

Detailed Implementation

The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

Example 1 Preparation of compound 10

Step a: Preparation of intermediate 1 (4-chloro-5-fluoro-6-(methoxy)-2-methylpyrimidine).

The starting material, 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol), was dissolved in acetonitrile. Then, 2,4,6-trimethylphenol (136 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 224 mg of intermediate 1, with a yield of 80%. MS (ESI) m/z: 281.1 [M+H] ⁺ .

Step b: Preparation of product 10 5-fluoro-6-(methtrioxy)-2-methyl-N-(pent-3-yl)pyrimidine-4-amine.

Intermediate 1 (112 mg, 0.4 mmol) and 3-aminopentane (44 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, and N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added. The mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain product 10, a white solid with a yield of 53%. ¹ H NMR (400MHz, CDCl ₃ )δ6.87(s,2H),4.54(d,J＝8.4Hz,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 = 7.4Hz, 6H). ¹³ C NMR (101MHz, CDCl ₃ )δ160.36,160.25,152.61,152.55,152.06,151.98,146.62,133.46,130.01,129 .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 FN ₃ O[M+H] ⁺ 332.2133, found 332.2126.

Example 2 Preparation of compound 21

Step a: Same as step a in Example 1.

Step b: Preparation of product 21, 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methoxy)-2-methylpyrimidine.

Intermediate 1 (112 mg, 0.4 mmol), (4-fluoro-2-isopropoxyphenyl)boronic acid (79.2 mg, 0.4 mmol), 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (29.2 mg, 0.04 mmol), and potassium carbonate (110.4 mg, 0.8 mmol) were weighed into a round-bottom flask and dissolved in dioxane/water (10 ml: 1 ml). The mixture was then incubated overnight at 100°C under nitrogen protection. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. Product 21 was obtained by column chromatography as a white solid with a yield of 78%. MS (ESI) m/z: 399.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.50–7.38(m,1H),6.83(s,2H),6.71–6.65(m,1H),6.61(d,J＝11.1Hz,1H),4 .54–4.44(m,1H),2.44(s,3H),2.23(s,3H),2.03(s,6H),1.24(d,J=5.7Hz,6H).

Example 3 Preparation of compound 62

Step a: Same as step a in Example 1.

Step b: Preparation of product GP142 5-fluoro-6-(mestrioxy)-2-methyl-N-(1-phenylcyclobutyl)pyrimidine-4-amine.

Intermediate 1 (112 mg, 0.4 mmol) and 1-phenylcyclobutylamine hydrochloride (91.8 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, and N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added. The mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 156.5 mg of product 62 as a white solid, with a yield of 66%. ¹ H NMR (400MHz, CDCl ₃ )δ7.54(d,J＝7.4Hz,1H),7.33(t,J＝7.6Hz,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). ¹³ C NMR (101MHz, CDCl ₃ )δ160.89,160.78,153.78,153.71,151.55,151.48,147.59,146.06,134.56,131.56,130.61, 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 FN ₃ O[M+H] ⁺ 392.2133,found392.2126.

Example 4 Preparation of compound 90

Step a: Same as step a in Example 1.

Step b: Preparation of product 90 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methoxy)-2-methylpyrimidine.

Intermediate 1 (112 mg, 0.4 mmol) and o-benzylhydroxylamine (61.2 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, and N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added. The mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 63 mg of product 90, a grayish-white solid, with a yield of 43%. MS (ESI) m/z: 368.2 [M+H] ⁺ .

Example 5 Preparation of compound 93

Step a: Preparation of intermediate 1, 6-chloro-5-fluoro-2-methyl-N-(1-phenylcyclobutyl)pyrimidine-4-amine.

The starting materials 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol) and 1-phenylcyclobutylamine hydrochloride (192.9 mg, 1.05 mmol) were dissolved in 10 mL of acetonitrile. N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added, and the mixture was reacted overnight at 80 °C. After the reaction was completed, the mixture was concentrated under reduced pressure, mixed with crude silica gel, and separated by column chromatography to obtain 259 mg of intermediate 1, a white solid, with a yield of 89%. MS (ESI) m/z: 292.1 [M+H] ⁺ .

Step b: Preparation of product 93 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methoxy)-2-methylpyrimidine.

Intermediate 1 (146 mg, 0.5 mmol) and 2-cyclohexylethylamine (63.5 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide. N,N-diisopropylethylamine (260 μL, 1.2 mmol) was added, and the mixture was reacted overnight at 120 °C. After the reaction was complete, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain product 93, a white solid with a yield of 59%. MS (ESI) m/z: 383.3 [M+H] ⁺ .

Example 6 Preparation of compound 94

Step a: Same as step a in Example 1.

Step b: Preparation of intermediate 2, methyl 1-((5-fluoro-6-(methoxy)-2-methylpyrimidin-4-yl)amino)cyclobutane-1-carboxylate.

Intermediate 1 (112 mg, 0.4 mmol) and methyl 1-aminocyclobutane-1-carboxylate (51.6 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide. N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added, and the mixture was reacted overnight at 120 °C. After the reaction was complete, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 86.6 mg of intermediate 1 as a white solid, with a yield of 58%. MS (ESI) m/z: 374.2 [M+H] ⁺ .

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

Dissolve intermediate 2 obtained in the previous step in 5 ml of methanol, add 2 ml of lithium hydroxide solution (2 mol/L), react at 50 degrees Celsius for 2 h. After the reaction is complete, directly evaporate the reaction solution to dryness, then use dilute hydrochloric acid to adjust the pH to 4, extract with EA, and evaporate to dryness to obtain intermediate 3, which can be directly used in the next step of the reaction.

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

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

Example 7 Preparation of compound 95

Step a: Same as step a in Example 5.

Step b: Preparation of product 95 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methoxy)-2-methylpyrimidine.

Intermediate 1 (146 mg, 0.5 mmol) and m-aminobenzonitrile (59 g, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide. N,N-diisopropylethylamine (325 μL, 1.2 mmol) was added, and the mixture was reacted overnight at 120 °C. After the reaction was complete, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain product 95, a white solid with a yield of 40%. MS (ESI) m/z: 374.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.55–7.50(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).

Example 8 Preparation of compound 121

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

The starting material, 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol), was dissolved in acetonitrile. Then, 4-fluoro-2,6-dimethylphenol (140 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 218 mg of intermediate 1, with a yield of 77%. MS (ESI) m/z: 285.0 [M+H] ⁺ .

Step b: Preparation of product 121 5-fluoro-4-(4-fluoro-2-isopropoxyphenyl)-6-(methoxy)-2-methylpyrimidine.

Intermediate 1 (113.6 mg, 0.4 mmol) and 1-phenylcyclobutylamine hydrochloride (91.8 mg, 0.5 mmol) were dissolved in 5 mL of dimethyl sulfoxide, and N,N-diisopropylethylamine (209 μL, 1.2 mmol) was added. The mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 115.6 mg of product 121, a white solid, with a yield of 73%. ¹ H NMR (400MHz, CDCl ₃ )δ7.54(d,J＝7.4Hz,2H),7.34(t,J＝7.7Hz,2H),7.23(t,J＝7.3Hz,1H),6.74(d,J＝8.9Hz,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). ¹³ C NMR (101MHz, CDCl ₃ )δ160.89,160.78,158.38,153.45,153.39,151.61,151.54,145.96,145.54,145.52,132.93,132.84,131. 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 F ₂ N ₃ O[M+H] ⁺ 396.1882,found 396.1877.

Example 9 Preparation of compound 133

Step a: Same as step a in Example 1.

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

Intermediate 1 (112 mg, 0.4 mmol) and 6-fluoro-3-piperidin-4-yl-1,2-benzisazole 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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain product 133, a white solid with a yield of 70%. MS (ESI) m/z: 465.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.71–7.65(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).

Example 10 Preparation of compound 137

Step a: Same as step a in Example 5.

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

Intermediate 1 (112 mg, 0.4 mmol), (4-propylphenyl)boronic acid (65.6 mg, 0.4 mmol), 1,1-bis(diphenylphosphine)ferrocene palladium dichloride (29.2 mg, 0.04 mmol), and potassium carbonate (110.4 mg, 0.8 mmol) were weighed into a round-bottom flask and dissolved in dioxane/water (10 ml: 1 ml). The mixture was then incubated overnight at 100°C under nitrogen protection. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The product 137 was obtained by column chromatography as a white solid (129 mg), with a yield of 86%. MS (ESI) m/z: 376.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.80(d,J＝6.8Hz,2H),7.61–7.52(m,2H),7.33(t,J＝7.7Hz,2H),7.26–7.19(m,3H),5.54(s,1H),2.80–2.6 6(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＝7.3Hz,3H).

Example 11 Preparation of compound 149

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

The starting material, 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol), was dissolved in acetonitrile. Then, 2,6-dimethyl-4-nitrophenol (167 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 245 mg of intermediate 1, with a yield of 79%. MS (ESI) m/z: 312.0 [M+H] ⁺ .

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

Intermediate 1 (180 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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 116 mg of intermediate 2, with a yield of 55%. MS (ESI) m/z: 423.2 [M+H] ⁺ .

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

Intermediate 2 (116 mg, 0.275 mmol) was dissolved in 10 mL of ethanol/water (v/v = 10:1), and iron powder (61 mg, 1.1 mmol) and ammonium chloride (29.4 mg, 0.55 mmol) were added. The reaction was carried out at 80 °C for 2 h under TLC monitoring. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure and separated by column chromatography to obtain 94 mg of intermediate 3, with a yield of 88%. MS (ESI) m/z: 393.2 [M+H] ⁺ .

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.

Intermediate 3 (94 mg, 0.242 mmol), o-fluorobenzoic acid (34 mg, 0.242 mmol), HATU (137.9 mg, 0.363 mmol), and DIPEA (126 μL, 0.726 mmol) were weighed into a round-bottom flask, dissolved in dichloromethane, and reacted at room temperature for 2 h. The reaction was monitored by TLC. After the reaction was completed, the solution was concentrated under reduced pressure and separated by column chromatography to obtain 93 mg of product 149, with a yield of 75%. MS (ESI) m/z: 515.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ8.36(d,J＝15.3Hz,1H),8.15(t,J＝7.5Hz,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).

Example 12 Preparation of compound 151

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

The starting material, 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol), was dissolved in acetonitrile. Then, 4-bromo-2,6-dimethylphenol (167 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 300 mg of intermediate 1, with a yield of 87%. MS (ESI) m/z: 347.0 [M+H] ⁺ .

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

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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 102 mg of intermediate 2, with a yield of 45%. MS (ESI) m/z: 456.1 [M+H] ⁺ .

Step c: Preparation of product GP403 5-fluoro-6-((3'-fluoro-3,5-dimethyl-[1,1'-biphenyl]-4-yl)oxy)-2-methyl-N-(1-phenylcyclobutyl)pyrimidine-4-amine.

Intermediate 2 (102 mg, 0.225 mmol), m-fluorophenylboronic acid (32 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 and dissolved in dioxane/water (10 ml: 1 ml). The mixture was then incubated overnight at 100°C under nitrogen protection. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. 80 mg of product 151, a white solid, was obtained by column chromatography, with a yield of 75%. MS (ESI) m/z: 472.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.57–7.55(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).

Example 13 Preparation of compound 184

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

The starting material, 4,6-dichloro-5-fluoro-2-methylpyrimidine (180 mg, 1 mmol), was dissolved in acetonitrile. Methyl 4-hydroxy-3,5-dimethylbenzoate (180 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was complete, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 259 mg of intermediate 1, with a yield of 80%. MS (ESI) m/z: 325.1 [M+H] ⁺ .

Step b: Preparation of intermediate methyl 2,4-((5-fluoro-2-methyl-6-((1-phenylcyclobutyl)amino)pyrimidin-4-yl)oxy)-3,5-dimethylbenzoate.

Intermediate 1 (162 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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 131 mg of intermediate 2, with a yield of 60%. MS (ESI) m/z: 436.2 [M+H] ⁺ .

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

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

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.

Intermediate 3 (84 mg, 0.2 mmol), (S)-N-methyl-1-phenylethane-1-amine (32 mg, 0.24 mmol), HATU (114 mg, 0.3 mmol), and DIPEA (104 μL, 0.6 mmol) were weighed into a round-bottom flask, dissolved in dichloromethane, and reacted at room temperature for 2 h. The reaction was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain 86 mg of product 184, with a yield of 82%. MS (ESI) m/z: 539.3 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.50–7.43(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).

Example 14 Preparation of compound 212

Step a: Same as step a in Example 12.

Step b: Same as step b in Example 12.

Step c: Preparation of product 212 6-(2,6-dimethyl-4-(pyrazolo[1,5-a]pyrimidin-3-yl)phenoxy)-5-fluoro-2-methyl-N-(1-phenylcyclobutyl)pyrimidin-4-amine.

Intermediate 2 (102 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)pyrazol[1,5-a]pyrimidine (55 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 and dissolved in dioxane/water (10 ml: 1 ml). The reaction was carried out under nitrogen protection at 100 °C overnight. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. 74 mg of product 212, a white solid, was obtained by column chromatography, with a yield of 66%. MS (ESI) m/z: 495.2 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO) δ9.16(d,J＝7.1Hz,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＝8.0Hz,2H),7.32( t,J＝6.8Hz,2H),7.18(t,J＝7.4Hz,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 (101MHz, DMSO) δ160.49,160.39,156.03,155.95,151.07,151.03,149.49,147.92,144.09,143.29,132.72,131.63,131.14,129.13,128. 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.

Example 15 Preparation of compound 220

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

The starting material 2,4,6-trichloro-5-fluoropyrimidine (201 mg, 1 mmol) was dissolved in acetonitrile, and 4-bromo-2,6-dimethylphenol (201 mg, 1 mmol) and potassium carbonate (276 mg, 2 mmol) were added sequentially. The reaction was carried out at 80 °C for 6 hours. The reaction was monitored by TLC. After the reaction was completed, the mixture was filtered, the filtrate was evaporated to dryness, mixed with silica gel, and subjected to column chromatography to obtain 310 mg of intermediate 1, with a yield of 85%. MS (ESI) m/z: 366.9 [M+H] ⁺ .

Step b: Preparation of intermediate 2,6-(4-bromo-2,6-dimethylphenoxy)-2-chloro-5-fluoro-N-(1-phenylcyclobutyl)pyrimidine-4-amine.

Intermediate 1 (183 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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 253 mg of intermediate 2, with a yield of 53%. MS (ESI) m/z: 478.0 [M+H] ⁺ .

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.

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

-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, CDCl ₃ )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＝6.1 Hz,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).

Example 16 Preparation of compound 262

Step a: Same as step a in Example 12.

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.

Intermediate 1 (183 mg, 0.5 mmol) and 3-(pyrrolidone-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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 135 mg of intermediate 2, with a yield of 59%. MS (ESI) m/z: 457.1 [M+H] ⁺ .

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

Intermediate 2 (103 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-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 and dissolved in dioxane/water (10 ml: 1 ml). The reaction was carried out under nitrogen protection at 100 °C overnight. After the reaction was completed, the mixture was filtered, and the filtrate was concentrated under reduced pressure. 92 mg of product 262, a white solid, was obtained by column chromatography, with a yield of 82%. MS (ESI) m/z: 499.3 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.64(s,1H),7.21–7.15(m,2H),7.05(s,2H),6.99(t,J＝8.7Hz,2H),5.44–5.37(m,1H),4.19(t,J＝6.1Hz,2H),4.09–4.02(m,1H),3.86– 3.79(m,1H),2.95(t,J=6.3Hz,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).

Example 17 Preparation of Compound 265

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

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

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

Intermediate 1 (183 mg, 0.5 mmol) and 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 mixture was reacted overnight at 120 °C. After the reaction was completed, the mixture 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, concentrated under reduced pressure, and separated by column chromatography to obtain 152 mg of intermediate 2, with a yield of 63%. MS (ESI) m/z: 482.0 [M+H] ⁺ .

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-methylpyrimidine-4-amine).

Intermediate 2 (109 mg, 0.225 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)-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 and dissolved in dioxane/water (10 ml: 1 ml). The reaction was carried out under nitrogen protection at 100 °C overnight. After the reaction was complete, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The product 265 was obtained by column chromatography as a white solid with a yield of 76%. MS (ESI) m/z: 522.2 [M+H] ⁺ . ¹ H NMR (400MHz, CDCl ₃ )δ7.68(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＝9.0Hz,1H),5.65(s,1H), 4.23(t,J＝6.1Hz,2H),2.98(t,J＝6.4Hz,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).

Example 18 Preparation of Compound 300

Step a: Preparation of intermediate 1-tert-butyl(S)-4-[(tert-butyldimethylsilyl)oxy]-2-oxopyrrolidine-1-carboxylic acid ester

At 0 °C, tert-butyldimethylchlorosilane (7.8 g, 52 mmol) and imidazole (5.1 g, 25.74 mmol) were added to an ultradry dimethylformamide (25 mL) solution of (S)-4-hydroxy-2-pyrrolidone (5.0 g, 49.5 mmol). The reaction was heated to room temperature and stirred for 3 hours. The mixture was poured into water to obtain a precipitate, which was extracted with ethyl acetate, ensuring that no water residue remained, to give 2(S)-4-[(tert-butyldimethylsilyl)oxy]pyrrolidone-2-one. A solution of 2 (10.9 g, 50.7 mmol) in ultra-dry acetonitrile (100 mL) was added at 0 °C under a nitrogen atmosphere. 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) were added. The mixture was heated 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 brine, dried over sodium sulfate, filtered, and concentrated to give tert-butyl(S)-4-[(tert-butyldimethylsilyl)oxy]-2-oxopyrrolidine-1-carboxylic acid ester. [M+H] ⁺ = 316.2.

Step b: Preparation of intermediate 2-tert-butylN-[(2S)-2-[(tert-butyldimethylsilyl)oxy]-4-hydroxy-4-phenylbutyl]carbamate

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

Step c: Preparation of intermediate 3(4S)-4-[(tert-butyldimethylsilyl)oxy]-2-phenylpyrrolidine-1-carboxylic acid tert-butyl ester

Under a nitrogen atmosphere, triethylamine (TEA, 16 mL, 114.6 mmol) and methanesulfonyl chloride (MsCl, 3.3 mL, 42.0 mmol) were added to an ultra-dry dichloromethane (120 mL) solution of intermediate 2 (15.8 g, 38.2 mmol) at -60 °C. The resulting mixture was stirred at -60 °C for 1 hour. The reaction solution 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 an 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.

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

At room temperature, tetrabutylammonium fluoride (TBAF, 50 mL, 1.0 M tetrahydrofuran solution, 49.7 mmol) was added to an ultra-dry tetrahydrofuran (76 mL) solution of intermediate 3 (18.1 g, 38.2 mmol). The mixture was stirred at room temperature for 2 hours and 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 an 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.

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

At -78°C, diethylaminosulfur trifluoride (DAST, 2.5 mL, 19.2 mmol) was added to an ultra-dry dichloromethane (25 mL) solution of intermediate 4 (2.7 g, 9.6 mmol). The mixture was stirred at -78°C for 2 hours, then slowly heated to room temperature and stirred overnight. At 0°C, an aqueous sodium bicarbonate solution was added dropwise to the mixture, followed by extraction with dichloromethane. The combined organic layers were washed with saturated brine, dried over sodium sulfate, filtered, and concentrated. 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 an 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.

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

Intermediate 5 was dissolved in dichloromethane, and 5 equivalents of dioxane hydrochloride solution were added. The mixture was reacted for 4 hours, evaporated to dryness, dissolved in sodium bicarbonate, extracted with ethyl acetate, and the organic layers were combined, filtered, and concentrated to give the product (2S,4R)-4-fluoro-2-phenylpyrrolidine. [M+H] ⁺ = 166.1.

Step g: Preparation of intermediate 7 4-(4-bromo-2,6-dimethylphenoxy)-5-fluoro-6-[(2S,4R)-4-fluoro-2-phenylpyrrolidone-1-yl]-2-methylpyrimidine

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

Step h: Preparation of intermediate 8-tert-butyl 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2-phenylpyrrolidine-1-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid ester

Dissolve 9 in dioxane, add 3 mL of water, 1 equivalent of 1-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. React overnight at 100°C under a nitrogen atmosphere. Purify the mixture by silica gel column chromatography using an ethyl acetate/hexane gradient as eluent to give the product tert-butyl3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2-phenylpyrrolidine-1-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylic acid ester. [M+H] ⁺ = 612.3.

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

Intermediate 8 was dissolved in dichloromethane, and 5 equivalents of dioxane hydrochloride solution were added. The mixture was reacted for 4 hours, evaporated to dryness, dissolved in sodium bicarbonate, extracted with ethyl acetate, and the organic layers were combined, filtered, and concentrated. The mixture was purified by silica gel column chromatography with a methanol/dichloromethane gradient as the eluent to give compound 300 3-(4-((5-fluoro-6-((2S,4R)-4-fluoro-2-phenylpyrrolidine-1-yl)-2-methylpyrimidin-4-yl)oxy)-3,5-dimethylphenyl)-1H-pyrrolo[2,3-b]pyridine. ¹ H NMR (400MHz, DMSO) δ11.86 (s, 1H), 8.31 (d, J = 8.1Hz, 1H), 8.28–8.23 (m, 1H), 7.83 (s, 1H), 7.43 (s, 2H), 7.37–7.29 (m, 4H), 7.26–7.1 9(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). ^13C NMR (101MHz, DMSO) δ160.59,160.48,157.12,157.02,151.57,149.51,148.01,143.30,139.65,132.82,132.23,131.16,129.72,128. 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

Referring to the synthesis methods of Examples 1-16 above, and in conjunction with conventional methods in the art, the remaining compounds in Table 1 of this invention were prepared, and their corresponding characterization data are shown in Table 2.

Table 2. Numbering and characterization data of GPR65 antagonists

The following specific experimental examples demonstrate the beneficial effects of the present invention.

Experimental Example 1: Study on the pharmacological effects of the compounds of this invention

1. Experimental Methods

1.1 Luciferase-based activity assay system

GloSensor is a modified luciferase that exhibits low catalytic activity and weak luminescence when not bound to cAMP, but shows significantly enhanced catalytic activity and enhanced luminescence upon substrate addition when bound to cAMP.

When HEK293T cell density reached 60%-70%, pcDNA3.1-Flag-GPR65 and pGloSensor ^™ 22F plasmid (mass ratio 1:3) were transfected into the cells. After 24 hours, the cells were resuspended at a concentration of 2.5 × ^10⁴ cells/mL in sterile white 96-well plates coated with L-poly-L-lysine (100 ng/mL). After cell adhesion, the supernatant was discarded, and 40 μL of HBSS solution containing 30 μM D-fluorescein potassium salt (containing 20 mM TAPS solution, pH = 8.40) was added to each well. The cells were then incubated at 37°C with ₅ % CO₂ for 90 minutes to equilibrate. The test drug was diluted with HBSS solution containing 100 μM IBMX and 0.1% BSA (containing 20 mM HEPES solution, pH = 7.20) to a final drug screening concentration of 10 μM. Remove the equilibrated plate, discard the supernatant, and add 45 μL of the test solution to each well. Use a pH 7.20 buffer as the positive well and a pH 8.40 buffer as the negative well. After incubation at room temperature for 1 hour, detect the bioluminescent signal using a multi-mode microplate reader. Antagonism rate (%) = (Test drug signal value - pH 8.40 signal value) / (pH 7.20 signal value - pH 8.40 signal value) × 100%. Compounds with an antagonism rate <50% are further tested, with a three-fold dilution to 30 μM (the highest concentration). Each concentration has at least three replicates. Plot a concentration-antagonism rate curve and perform activity fitting using Graphpad 8.3.

1.2 Pharmacodynamic evaluation of compound 121 against MC38 tumors

Male C57BL/6 mice, weighing 20-22g, were housed in an SPF-grade animal facility. MC38 cells were cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin antibiotics) under contamination-free conditions. MC38 cells in the logarithmic growth phase were collected, washed three times with sterile PBS, and then resuspended in DMEM medium to a concentration of 1× ^10⁷ cells/mL. 100 μL of cell suspension per mouse was injected subcutaneously into the right axilla. Compound 121 was dissolved in 10% DMSO and diluted with 30% PEG400, 5% HS-15, and 55% physiological saline to a 3 mg/mL solution, which was then allowed to clarify. PD-1 monoclonal antibody was diluted to 0.6 mg/mL with a specific buffer and kept on ice until needed. Mice with tumors approximately 100 ^cm³ were randomly divided into four groups: a solvent group, a PD-1 monoclonal antibody group (6 mg/kg), a compound 121 group (30 mg/kg), and a combination of compound 121 and PD-1 monoclonal antibody (6 mg/kg PD-1 and 30 mg/kg compound 121). Mice were orally administered compound 121 twice daily (morning and evening ⁾ . Tumor volume was measured every other day, calculated as (length × ^width² )/2. Once the PD-1 monoclonal antibody group reached 200 ^cm³ , mice in the PD-1 monoclonal antibody group and the combination therapy group were intraperitoneally injected with PD-1 monoclonal antibody solution every two days. The experiment was terminated when the tumor volume in the solvent group reached 1500 ^cm³ , and the subcutaneous tumors were fixed in 4% paraformaldehyde.

1.3 Pharmacodynamic evaluation of compound 121 for antiepileptic purposes

Male SD rats, weighing 220-250g, were housed in an SPF-grade animal facility. Compounds 121 and 284 were dissolved in 10% DMSO and then diluted to 1 and 3 mg/mL solutions with 30% PEG400, 5% HS-15, and 55% physiological saline, respectively. The solutions were clear. Pentylenetetrazol (PTZ) is a central nervous system stimulant that can induce acute epilepsy. Pentylenetetrazol was prepared to 18 mg/mL with physiological saline and kept on ice. Rats were randomly divided into four groups: solvent + PTZ group, 121 (5 mg/kg) + PTZ group, 121 (10 mg/kg) + PTZ group, and 284 (10 mg/kg) + PTZ group, with 284 serving as a negative control. Rats were administered the corresponding drug solutions via intraperitoneal injection. One hour later, they were injected intraperitoneally with 65 mg/kg PTZ solution. The rats' condition was continuously observed for one hour, and relevant indicators were recorded. The seizures of each rat were graded according to the Racine scoring system, specifically: Grade 1, stuttering and facial twitching; Grade 2, head twitching; Grade 3, limb lifting and twitching; Grade 4, falling to the ground and limb twitching; Grade 5, rolling over, strong generalized convulsions accompanied by limb rigidity.

1.4 Pharmacodynamic evaluation of compound 300 against CT26 tumor

Because the compound exhibited significant differences in activity between humans and mice (human _IC50 = 0.009 μM, mouse _IC50 > 30 μM), Gpr65 humanized mice were used for efficacy evaluation.

Humanized female Gpr65-Balbc mice, aged 4-5 weeks, were housed in an SPF-grade animal facility. CT26 cells were cultured in DMEM complete medium (containing 10% FBS and 1% penicillin-streptomycin antibiotics) under strict contamination-free conditions. CT26 cells in the logarithmic growth phase were collected, washed three times with sterile PBS, and then resuspended in DMEM medium to a concentration of 5 × ^10⁶ cells/mL. 100 μL of cell suspension per mouse was injected subcutaneously into the right axilla. Compound 300 was dissolved in 10% DMSO and diluted with 30% PEG400, 5% HS-15, and 55% physiological saline to a 1 mg/mL solution, which was then allowed to clarify. PD-1 monoclonal antibody was diluted to 0.6 mg/mL with a specific buffer and kept on ice until needed. Mice with tumors approximately 100 ^cm³ were randomly divided into four groups: a solvent group, a PD-1 monoclonal antibody group (6 mg/kg), a compound 300 group (10 mg/kg), and a combination of compound 300 and PD-1 monoclonal antibody (6 mg/kg PD-1 and 10 mg/kg compound 300). Mice were given compound 300 orally once daily. Tumor volume was measured every other day, calculated as (length × ^width² )/ ² . When the PD-1 monoclonal antibody group reached 200 ^cm³ , mice in the PD-1 monoclonal antibody group and the combination therapy group were administered PD-1 monoclonal antibody solution intraperitoneally every two days. The experiment was terminated when the tumor volume in the solvent group reached 1500 ^cm³ , and the subcutaneous tumors were fixed in 4% paraformaldehyde.

1.5 Pharmacodynamic evaluation of compound 300 for antiepileptic purposes

Because the compound exhibited significant differences in activity between humans and mice (human _IC50 = 0.009 μM, mouse _IC50 > 30 μM), Gpr65 humanized mice were used for efficacy evaluation.

Humanized Gpr65-Balb/c mice and ordinary wild-type Balb/c mice, both male, 8-9 weeks old, were housed in an SPF-grade animal facility. Compound 300 was dissolved in 10% DMSO, and a 1 mg/mL drug solution was prepared by adding 30% PEG400, 5% HS-15, and 55% physiological saline. The solution was clear. Pentylenetetrazol (PTZ) is a central nervous system stimulant that can induce acute epilepsy. Pentylenetetrazol was prepared into an 8 mg/mL solution with physiological saline and kept on ice. Humanized and wild-type mice were randomly divided into two groups: the solvent + PTZ group and the 300 (1 mg/kg) + PTZ group. Mice were given the corresponding drug solution via intraperitoneal injection, followed by a subcutaneous injection of 80 mg/kg PTZ solution 30 minutes later. The mice's condition was continuously observed for 1 hour, and relevant indicators were recorded. The seizures of each mouse were graded according to the Racine scoring system, specifically: Grade 1, stuttering and facial twitching; Grade 2, head twitching; Grade 3, limb lifting and twitching; Grade 4, falling to the ground and limb twitching; Grade 5, rolling over, strong generalized convulsions accompanied by limb rigidity.

2. Experimental Results

2.1 GPR65 antagonistic activity of the compounds of this invention

All compounds in this invention were detected by the cAMP-Glosensor method. Concentration-antagonism rate curves were plotted, and activity was fitted using Graphpad 8.3. The activities are listed in Table 3.

Table 3. Antagonistic effect of the compounds of this invention on hGPR65

2.2 Compound 121 has antitumor activity.

Compared to the solvent group, compound 121 alone and in combination with PD-1 monoclonal antibody showed significant antitumor effects, manifested as a reduction in tumor volume (Fig. 1A, Fig. 1C) and a decrease in tumor dry weight (Fig. 1B). Furthermore, the 15-day administration did not affect the body weight of mice, and no adverse effects were observed (Fig. 1D).

2.3 Compound 121 has anti-epileptic effects.

Compared with the solvent group and compound 284, compound 121 (10 mg/kg, i.p.) can alleviate PTZ-induced acute epilepsy in rats, specifically by prolonging the latency of the first seizure (Fig. 2A), reducing the seizure severity (Fig. 2C), and decreasing the total seizure time (Fig. 2B) and the number of seizures (Fig. 2D).

2.4 Compound 300 has antitumor activity.

In Gpr65 humanized mice, compared to the solvent group, the PD-1 monoclonal antibody did not show significant antitumor effects. Compound 300, both alone and in combination with the PD-1 monoclonal antibody, showed significant antitumor effects, manifested as a reduction in tumor volume (Figure 3A) and a decrease in tumor dry weight (Figure 3B). However, in wild-type mice, compound 300 lost its efficacy (Figures 3C and 3D), indicating that the compound exerts its antitumor effect through antagonism of GPR65. This is consistent with the results of cell activity experiments, showing that compound 300 exhibits good targeting.

Compound 2.5 has anti-epileptic effects.

In Gpr65-humanized mice, compound 300 (10 mg/kg, i.p.) alleviated PTZ-induced acute epilepsy compared to the solvent group, specifically by prolonging the latency to the first seizure (Fig. 4A), reducing the frequency of seizures (Fig. 4B), and lowering the seizure severity (Fig. 4C). However, in wild-type mice, compound 300 lost its efficacy, indicating that its antiepileptic effect is dependent on antagonizing GPR65. This is consistent with the results of cell activity experiments, demonstrating good targeting of compound 300.

Experimental results show that the compounds of this invention have excellent antagonistic activity against human G protein-coupled receptor 65, and have broad application prospects in the preparation of drugs for anti-epileptic and anti-tumor purposes.

Claims (15)

Compounds represented by Formula I, or their stereoisomers, pharmaceutically acceptable salts, solvates, crystal forms, prodrugs, metabolites, or deuterated derivatives thereof:
Wherein, ^X1 is selected from _CR1 and N, ^X2 is selected from _CR2 and N, ^X3 is selected from _CR3 and N, and at least one of ^X1 , ^X2 and ^X3 is N; _R1 , _R2 , and _R3 are each independently selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, amino, and nitro groups; R ₄ is selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, _C1-6 alkylthio, halogen-substituted _C1-6 alkylthio, amino, phenyl, 5-6 membered heteroaryl ring, 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclic group; Alternatively, _R2 and _R4 can be linked together to form a benzene ring; L is selected from none, O, C _1-4 alkylene; n is selected from 0, 1, 2, 3, 4; The C ring is selected from benzene rings, 5-6 membered heteroaromatic rings, 3-8 membered saturated cycloalkyl groups, and 3-8 membered saturated heterocyclic groups; Each _Rc is independently selected from hydrogen, _C1-6 alkyl groups (unsubstituted or substituted with one or more _Rc1) , _C1-6 alkoxy groups (unsubstituted or substituted with one or more _Rc1) , _C2-10 alkenyl groups (unsubstituted or substituted with one or more _Rc2) , _C2-10 alkynyl groups (unsubstituted or substituted with one or more _Rc2) , cyano, halogen, nitro, amino, phenyl groups (unsubstituted or substituted with one or more _Rc3 ), 5-6 heteroaryl groups (unsubstituted or substituted with one or more _Rc3) , 3-8 saturated cycloalkyl groups (unsubstituted or substituted with one or more _Rc3) , 3-8 saturated heterocyclic groups (unsubstituted or substituted with one or more _Rc3) , fused ring groups (unsubstituted or substituted with one or more _Rc3) , _L0 COOL _{1 R6} , _L0 NR ₈ COOL _{1 R6} , L0 CONR _{8 L1 R 6} , OR ₇ , COR ₉ ; R _{_c1} are each independently selected from hydroxyl, halogen, and C _{_1-6} alkyl groups; R _{_c2} are each independently selected from hydroxyl, halogen, and C _{_1-6} alkyl groups; R _{_c3} are each independently selected from halogens, C _{_1-6} alkyl groups, hydroxyl groups or halogen-substituted C _{_1-6} alkyl groups, C _{_1-6} alkoxy groups, hydroxyl groups or halogen-substituted C _{_1-3} alkoxy groups, 3-8 membered saturated heterocyclic groups; _L0 is selected from none, _C2-4 alkenyl groups; _L1 is selected from none and _C1-4 alkylene groups; _R6 is selected from _C1-6 alkyl groups, unsubstituted or substituted with one or more _R6a groups of the following groups: 5-6 membered heteroaryl, phenyl, 3-8 membered saturated heterocyclic group, 3-8 membered saturated cycloalkyl, fused ring; _R6a is independently selected from halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, COOR _6b , hydroxyl; R _6b is selected from _C1-6 alkyl groups; _R8 is selected from hydrogen, _C1-6 alkyl, 3-8 saturated cycloalkyl, and 3-8 saturated heterocyclic groups; R ₇ is selected from phenyl or benzyl; _R9 is selected from unsubstituted or substituted rings with one or more _R9a , and _R9a is selected from halogens and _C1-6 alkyl groups. Alternatively, among m R_{_c} , two adjacent R_{_c} are connected to form a ring; The _R5 is selected _{from L2NR13L3L4R10} , phenyl _{groups that} are not substituted or are substituted _by one or more _R11 , _{and L5L6R14 ; L2} is selected from non-membered, 3-6 membered saturated nitrogen heterocycles; _L3 is selected from _C1-6 alkylene groups that are non-alkyl, C1-6 alkylene groups, or _C1-6 alkylene groups that are hydroxylated; _L4 is selected from none. R ₁₃ is selected from hydrogen and _C1-6 alkyl groups; _R10 is selected from amino protecting groups, _C1-6 alkyl groups, NR _{a1 Ra2} , OR _a3 , CONHL _{7 Ra4} , and the following groups that are unsubstituted or substituted by one or more _R12 groups: 3-8 membered saturated cycloalkyl, 3-8 membered saturated heterocyclic, 5-6 membered heteroaryl, phenyl, fused ring, spirocyclic; _Ra1 is a benzene ring, _Ra2 is selected from hydrogen, _C1-5 alkyl, _Ra3 is selected from _C1-6 alkyl, phenyl, benzyl; _Ra4 is selected from 3-6 membered saturated cycloalkyl; _L7 is selected from none, _C1-4 alkylene; _R12 is independently selected from halogen, hydroxyl, _C1-6 alkoxy, _C1-6 alkyl, cyano, COOR _b1 , and R _b1 is selected from _C1-6 alkyl; _R11 is selected from halogens, _C1-6 alkoxy groups, _C1-6 alkyl groups, and hydroxyl groups; _L5 is selected from 3-6 membered saturated nitrogen heterocycles that are unsubstituted, unsubstituted, or substituted by one or more _R5a , where each _R5a is independently selected from hydroxyl groups or halogens. _L6 is selected from none and _C1-4 alkylene groups; R ₁₄ is selected from The following groups are not substituted or are substituted by one or more R _14a : benzene ring, 3-6 membered saturated nitrogen heterocycle, 5-6 membered heteroaryl, fused ring; R _14a are each independently selected from halogen, halogen-substituted C _1-6 alkyl, C _1-6 alkyl; R ₁₄ is selected from amino protecting groups; The compound is not The compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its crystal form, or its prodrug, or its metabolite, or its deuterated derivative, according to claim 1, is characterized in that... Choose from one of the following structures: The compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its crystal form, or its prodrug, or its metabolite, or its deuterated derivative according to claim 2, is characterized in that _R3 is derived from hydrogen, halogen, _C1-6 alkyl, or halogen-substituted _C1-6 alkyl. _R4 is selected from hydrogen, halogen, _C1-6 alkyl, halogen-substituted _C1-6 alkyl, _C1-6 alkoxy, halogen-substituted _C1-6 alkoxy, _C1-6 alkylthio, halogen-substituted _C1-6 alkylthio, amino, phenyl, and 3-6 member saturated cycloalkyl. The compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its crystal form, or its prodrug, or its metabolite, or its deuterated derivative according to any one of claims 1-3, is characterized in that the L is selected from none, O, n is selected from 0, 1, 2, 3, and 4; The C ring is selected from benzene rings, 5-6 membered nitrogen-containing heteroaromatic rings, 3-6 membered saturated cycloalkyl groups, and 3-6 membered saturated heterocyclic groups; m is selected from 0, 1, 2, 3, and 4; Each _Rc is independently selected from hydrogen, _C1-4 alkyl groups (unsubstituted or substituted with one or more _Rc1) , _C1-4 alkoxy groups (unsubstituted or substituted with one or more _Rc1) , _C2-4 alkenyl groups (unsubstituted or substituted with one or more _Rc2) , _C2-4 alkynyl groups (unsubstituted or substituted with one or more _Rc2) , cyano, halogen, nitro, amino, phenyl groups (unsubstituted or substituted with one or more _Rc3) , 5-6 membered heteroaryl groups (unsubstituted or substituted with one or more _Rc3) , 3-6 membered saturated cycloalkyl groups (unsubstituted or substituted with one or more _Rc3) , 3-6 membered saturated heterocyclic groups (unsubstituted or substituted with one or more _Rc3) , 5-membered nitrogen-containing heterocyclic rings and 5-6 membered saturated heterocycles (unsubstituted or substituted with one or more _Rc3 ), and other unsubstituted or substituted Rc3 groups. _{C3- substituted} 5- _membered nitrogen heteroaromatic rings and _5-6 - _{membered heteroaromatic} rings, _L0COOL1R6 , _L0NR8COL1R6 , _{L0CONR8L1R6 , OR7 , COR9 ;} R _{_c1} are each independently selected from hydroxyl, halogen, and C _{_1-4} alkyl groups; R _{_c2} are each independently selected from hydroxyl, halogen, and C _{_1-4} alkyl groups; R _{_c3} are 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 heterocyclic groups; _L0 is selected from none, _C2-3 alkenyl groups; _L1 is selected from none and _C1-3 alkylene groups; _R6 is selected from _C1-4 alkyl groups, unsubstituted or substituted with one or more _R6a groups of the following: 5-6 membered heteroaryl, phenyl, 5-6 membered saturated heterocyclic group, 4-6 membered saturated cycloalkyl, benzocyclopentadienyl 5-6 membered saturated heterocyclic group; _R6a is independently selected from halogen, _C1-4 alkyl, halogen-substituted _C1-4 alkyl, _C1-4 alkoxy, halogen-substituted _C1-4 alkoxy, COOR _6b , hydroxyl; R _6b is selected from _C1-4 alkyl groups; _R8 is selected from hydrogen, _C1-4 alkyl, 3-6 membered saturated cycloalkyl, and 3-6 membered saturated heterocyclic groups; R ₇ is selected from phenyl or benzyl; _R9 is selected from 5-6 membered saturated nitrogen heterocyclic benzo[a] that is unsubstituted or substituted by one or more _R9a , and _R9a is selected from halogens and _C1-4 alkyl groups. Alternatively, among the m _Rcs , two adjacent _Rcs are connected to form a ring, wherein the ring is selected from 5-6 nucleotide saturated oxyheterocycles or benzene rings. The compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its crystal form, or its prodrug, or its metabolite, or its deuterated derivative, according to any one of claims 1-3, characterized in that _R5 is selected from _{L2 NR13 R10} , _{NR13 L3 L4 R10} , an _{unsubstituted} or substituted phenyl group, or _{L5 L6 R14} ; _L2 is selected from non-membered, 4-6 membered saturated nitrogen heterocycles; _L3 is selected from _C1-5 alkylene groups that are non-alkyl, C1-5 alkylene groups, or _C1-5 alkylene groups that are hydroxylated; _L4 is selected from none. R ₁₃ is selected from hydrogen and _C1-5 alkyl groups; _R10 is selected from amino protecting groups, _C1-5 alkyl groups, NR _{a1 Ra2} , OR _a3 , CONHL _{7 Ra4} , and the following groups that are unsubstituted or substituted by one or more _R12 groups: 3-6 membered saturated cycloalkyl groups, 3-6 membered saturated heterocyclic groups, 5-6 membered heteroaryl groups, phenyl groups, 5-6 membered saturated carbocyclic benzophenone rings, etc. _Ra1 is a benzene ring; _Ra2 is selected from hydrogen, _C1-5 alkyl; _Ra3 is selected from _C1-5 alkyl, phenyl, benzyl; _Ra4 is selected from 3-6 membered saturated cycloalkyl; _L7 is selected from none, _C1-2 alkylene; _R12 is independently selected from halogen, hydroxyl, _C1-5 alkoxy, _C1-5 alkyl, cyano, COOR _b1 , and _Rb1 is selected from _C1-5 alkyl; _R11 is selected from halogens, _C1-5 alkoxy groups, _C1-5 alkyl groups, and hydroxyl groups; _L5 is selected from 4-6 membered saturated nitrogen heterocycles that are unsubstituted, unsubstituted, or substituted by one or more _R5a , where each _R5a is independently selected from hydroxyl groups or halogens. _L6 is selected from non- _C1-2 alkylene groups; R ₁₄ is selected from The following groups are not substituted or are substituted by one or more R _14a : benzene ring, 5-membered saturated nitrogen heterocycle, 5-6-membered heteroaryl, 5-membered heteroaryl fused benzene ring, where each R _14a is independently selected from halogen, halogen-substituted _C1-5 alkyl, or _C1-5 alkyl; R ₁₄ is selected from amino protecting groups. The compound, or its stereoisomer, or its pharmaceutically acceptable salt, or its solvate, or its crystal form, or its prodrug, or its metabolite, or its deuterated derivative, according to any one of claims 1-5, is characterized in that the structure of the compound is as shown in Formula II or Formula III:

Wherein, _R3 is selected from hydrogen, halogen, _C1-3 alkyl, and halogen-substituted _C1-3 alkyl; _R4 is selected from hydrogen, halogen, _C1-3 alkyl, or halogen-substituted _C1-3 alkyl; R _x1 is selected from halogens, _C1-3 alkyl groups, and halogen-substituted _C1-3 alkyl groups; R _x2 is selected from halogens, _C1-3 alkyl groups, and halogen-substituted _C1-3 alkyl groups; R _x3 is selected from halogens, _C1-3 alkyl groups, halogen-substituted _C1-3 alkyl groups, 5-membered nitrogen heteroaromatic rings with 5-6-membered saturated heterocycles, and 5-membered nitrogen heteroaromatic rings with 5-6-membered heteroaromatic rings; f is selected from 0, 1, and 2; R ₁₅ are each independently selected from hydrogen, halogens, and _C1-3 alkyl groups. According to claim 1, the compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate, its crystal form, its prodrug, its metabolite, or its deuterated derivative thereof, characterized in that: the compound is selected from one of the following compounds:















A pharmaceutical composition, characterized in that the pharmaceutical composition is a formulation prepared by adding pharmaceutically acceptable excipients to a compound as an active ingredient, 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, according to any one of claims 1-7. Use of compound 9, its stereoisomer, its pharmaceutically acceptable salt, its solvate, its crystal form, its prodrug, its metabolite or its deuterated derivative, or any compound of claims 1-6, its stereoisomer, its pharmaceutically acceptable salt, its solvate, its crystal form, its prodrug, its metabolite or its deuterated derivative in the preparation of a G protein-coupled receptor antagonist;
The use according to claim 9 is characterized in that: the G protein-coupled receptor antagonist is a G protein-coupled receptor 65 antagonist. The use according to claim 9 or 10 is characterized in that: the G protein-coupled receptor antagonist is a drug for the prevention and/or treatment of G protein-coupled receptor-related diseases. The use according to claim 11 is characterized in that: the G protein-coupled receptor-related disease is epilepsy or cancer. According to the use described in claim 12, the cancer is 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. Use of a compound, its stereoisomer, its pharmaceutically acceptable salt, its solvate, its crystal form, its prodrug, its metabolite or its deuterated derivative, in combination with an immune checkpoint inhibitor in the preparation of a medicament for treating cancer; said compound is compound 9 or the compound of any one of claims 1-6. According to the use described in claim 14, the immune checkpoint inhibitor is a PD-1 antibody.