Aryl piperidine compound as well as preparation method, pharmaceutical composition and application thereof

Abstract

The invention discloses an aryl piperidine compound as well as a preparation method, a pharmaceutical composition and application thereof. The structure of the compound is shown in a formula (I), the compound further comprises an isomer, a prodrug, a stable isotope derivative, pharmaceutically acceptable salt or a mixture of the isomer, the prodrug, the stable isotope derivative and the pharmaceutically acceptable salt, and the compound has a remarkable PRMT5 enzyme inhibition effect and is higher in inhibition activity in the presence of MTA; meanwhile, the inhibitory activity on MTAP-deficient tumor cells is higher than that of a wild type, and selectivity is achieved; the compound has good activity at the molecular level and the cellular level and is wide in application; the preparation method is high in universality and easy for compound structure expansion. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to an aryl piperidine compound and a preparation method, a pharmaceutical composition and application thereof, and in particular to an aryl piperidine compound which can be prepared as an anti-tumor drug and a preparation method, a pharmaceutical composition and application thereof. Background Art

[0002] Epigenetic regulation of gene expression is an important biological determinant of protein production and cell differentiation and plays an important pathogenic role in many human diseases. Epigenetic regulation involves heritable modifications of genetic material without changing its nucleotide sequence. Epigenetic regulation is mediated by selective and reversible modifications of DNA and proteins to control conformational transitions between transcriptionally active and inactive states of chromatin. These covalent modifications can be regulated by enzymes such as methyltransferases such as PRMT5, many of which are associated with specific genetic alterations that can lead to human diseases.

[0003] Protein arginine methyltransferases (PRMTs) catalyze arginine methylation, transferring the methyl group on S-adenosylmethionine (SAM) to the guanidinium nitrogen atom of arginine residues in proteins, and regulate a variety of cellular processes such as cell growth, differentiation, and proliferation by affecting the activity of substrate proteins and protein-protein interactions. Currently, nine human PRMTs (PRMT1-9) have been discovered, which can be divided into type I (PRMT1-4, 6, and 8), type II (PRMT5 and 9), and type III (PRMT7) based on their monomethylation, asymmetric dimethylation (aDMA), and symmetric dimethylation functions (SDMA).

[0004] Studies have shown that knockout of PRMT5 leads to slower cell growth, while overexpression of PRMT5 leads to excessive cell proliferation. PRMT5 expression is upregulated in glioblastoma, lung cancer, lymphoma, breast cancer, and colorectal cancer. In addition, cancer cells with MTAP gene deletion are more sensitive to PRMT5 inhibition. MTAP is missing in 40% of glioblastomas, 25% of melanomas, urothelial carcinomas and pancreatic cancers, and 15% of non-small cell lung cancers. MTAP-deficient cells accumulate the metabolite MTA, which binds to PRMT5 to form a PRMT5-MTA complex, thereby inhibiting PRMT5 methyltransferase activity.

[0005] The first generation of PRMT5 inhibitors are mainly divided into SAM non-competitive and SAM competitive inhibitors. Representative drugs include GSK-3326595, JNJ-64619178, PF-06939999, PRT543, and PRT811. They all have non-selective inhibitory activity against PRMT5 and are also related to patients' dose-limiting thrombocytopenia, anemia, and neutropenia. They have the defects of large side effects and low therapeutic index, which limits the application of the first generation of PRMT5 inhibitors. The second generation of PRMT5 inhibitors can specifically act on MTAP-deficient tumors, thereby improving tumor selectivity and reducing toxic side effects. Summary of the invention

[0006] Objectives of the invention: The first objective of the present invention is to provide an aryl piperidine compound, the second objective is to provide a method for preparing the aryl piperidine compound, the third objective is to provide a pharmaceutical composition comprising the aryl piperidine compound, and the fourth objective is to provide an application of the aryl piperidine compound and the pharmaceutical composition thereof in the preparation of anti-tumor drugs.

[0007] Technical solution: The aryl piperidine compound of the present invention has a structure of formula (I), and also includes its isomers, prodrugs, stable isotope derivatives, pharmaceutically acceptable salts or mixtures thereof:

[0008]

[0009] in:

[0010] R 1 , R 2 are independently selected from hydrogen, substituted C1-C6 alkyl, substituted C1-C6 haloalkyl, substituted C6-C 10 aryl, substituted C5-C9 heteroaryl, substituted C3-C8 cycloalkyl or substituted C2-C6 heterocycloalkyl; the substituent is selected from at least one of hydrogen, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the C5-C9 heteroaryl, C2-C6 heterocycloalkyl contains 1-3 heteroatoms selected from N, O or S; when R 1 With R 2 When different, the carbon atom connected thereto is in racemic configuration, R configuration or S configuration;

[0011] Ring A is selected from substituted C6~C 10 An aromatic ring or a substituted C5-C9 heteroaromatic ring; the substituent is selected from hydrogen, halogen, C1-C6 alkyl or C1-C6 haloalkyl; the C5-C9 heteroaromatic ring contains 1-3 heteroatoms selected from N, O or S;

[0012] n is an integer selected from 0 to 3;

[0013] Ring B is selected from substituted 5-6 membered heteroaromatic rings or substituted 5-6 membered heterocycloalkane; the substituent is selected from at least one hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C2-C6 heterocycloalkyl, C1-C6 alkoxy, C1-C6 acyl, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the 5-6 membered heteroaromatic ring and 5-6 membered heterocycloalkane contain 1 to 3 heteroatoms selected from N, O or S;

[0014] X 1 , X 2 Each independently selected from CR 3 or N; R 3 Selected from hydrogen, halogen, amino, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy.

[0015] Preferably, in the structure:

[0016] R 1 is hydrogen;

[0017] R 2 Selected from hydrogen, substituted C6~C 10 aryl, substituted C5-C9 heteroaryl, substituted C3-C8 cycloalkyl or substituted C2-C6 heterocycloalkyl; the substituent is selected from at least one of hydrogen, halogen, cyano, methyl, ethyl, methoxy, trifluoromethyl, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the C5-C9 heteroaryl, C2-C6 heterocycloalkyl contains 1-3 heteroatoms selected from N, O or S; when R 1 With R 2 When different, the carbon atom connected thereto is in racemic configuration, R configuration or S configuration;

[0018] Ring A is selected from substituted C6~C 10 Aromatic ring or substituted C5-C9 heteroaromatic ring; the substituent is selected from hydrogen, halogen or C1-C6 alkyl; the C5-C9 heteroaromatic ring contains 1-3 heteroatoms selected from N, O or S;

[0019] n is selected from 1 or 2;

[0020] Ring B is selected from a substituted 5-membered heteroaromatic ring or a substituted 5-membered heterocycloalkane, wherein the substituent is selected from at least one of hydrogen, halogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, methoxy, cyclopropylmethoxy, trifluoromethoxy or 2,2-difluoroethyl; the 5-membered heteroaromatic ring or the 5-membered heterocycloalkane contains 1 to 3 heteroatoms selected from N, O or S;

[0021] X 1 , X 2 Each independently selected from CR 3 or N; R 3 is selected from hydrogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, methoxy, trifluoromethoxy or 2,2-difluoroethyl.

[0022] Preferably, in the structure:

[0023] R 1 is hydrogen;

[0024] R 2 is selected from substituted benzene ring, substituted pyridine, substituted pyridazine, substituted pyrazine, substituted furan, substituted thiophene, substituted oxazole, substituted pyrazole, substituted imidazole, substituted thiazole, substituted pyrimidine, substituted naphthyl ring, substituted quinoline, substituted indole, substituted indazole, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted piperazine, substituted piperidine, substituted pyrrole or substituted tetrahydrofuran; the substituent is selected from at least one hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, cyclopropyl, methoxy, trifluoromethoxy, cyclopropyloxy, morpholinyl, piperidinyl, tetrahydropyrrolidinyl, 2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl, hydroxyl, amino, methylamino, dimethylamino, acetamido, carboxyl, methoxycarbonyl or nitro; the carbon atom connected thereto is in racemic configuration;

[0025] Ring A is selected from substituted benzene ring, substituted thiophene, substituted thiazole, substituted pyrrole, substituted pyrazole, substituted imidazole, substituted furan, substituted oxazole or substituted isoxazole; the substituent is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl or isopropyl;

[0026] n is selected from 1;

[0027] Ring B is selected from substituted pyrrole, pyrazole, isoxazole, thiophene, thiazole, furan, dihydropyrrole or dihydrofuran, wherein the substituent is selected from at least one hydrogen, fluorine, chlorine, bromine, methyl, ethyl, isopropyl, trifluoromethyl, trifluoroethyl, methoxy, trifluoromethoxy;

[0028] X 1 , X 2 Each independently selected from CR 3or N; R 3 is selected from hydrogen, methyl, ethyl, trifluoromethyl, methoxy, trifluoromethoxy or 2,2-difluoroethyl.

[0029] Preferably, in the structure:

[0030] R 1 is hydrogen;

[0031] R 2 Selected from

[0032] The carbon atom to which it is attached is in a racemic configuration;

[0033] Ring A is selected from

[0034] n is taken from 1;

[0035] Ring B is selected from

[0036] X 1 , X 2 are independently selected from CH, CF, C-Cl, C-Br or N.

[0037] Preferably, the aryl piperidine compound is selected from any one of the following compounds:

[0038]

[0039]

[0040]

[0041] The present invention utilizes the synthetic lethal relationship between PRMT5 and MTAP to design a series of highly effective and selective PRMT5 inhibitors, which preferentially target PRMT5 in MTAP-deficient tumors while retaining PRMT5 activity in normal tissues.

[0042] Furthermore, the pharmaceutically acceptable salt is a salt formed by the arylpiperidine compound and any one of the following acids:

[0043] The acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, citric acid or ferulic acid.

[0044] The preparation method of the aryl piperidine compound of the present invention is selected from any one of the following methods:

[0045] (1) When R 1 =H, n=1, and ring B is a pyrazole ring. The preparation method of the aryl piperidine compound is as follows:

[0046]

[0047] Compound 2 is prepared by reacting compound 1 with pinacol diboron, the catalyst used is selected from [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl2), tetrakis(triphenylphosphine)palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2) or palladium acetate (Pd(OAc)2), preferably Pd(dppf)Cl2; the solvent used is selected from dichloromethane, tetrahydrofuran, toluene, 1,4-dioxane, ethyl acetate, acetone, N,N-dimethylformamide or a mixed solvent of any two of them, preferably 1,4-dioxane; the base used is selected from potassium hydroxide, potassium carbonate, potassium acetate, sodium carbonate, preferably potassium acetate; the reaction temperature is selected from 80 to 120°C, preferably 100°C.

[0048] Compound 4 is prepared from compound 3 via Sandmeyer reaction, the diazotizing agent used is selected from methyl nitrite, ethyl nitrite, amyl nitrite, isoamyl nitrite, isobutyl nitrite, tert-butyl nitrite, isopropyl nitrite, preferably isoamyl nitrite; the solvent and iodination agent used are selected from methyl iodide, diiodomethane, triiodomethane, ethyl iodide, diiodoethane or a mixed solvent of any two, preferably diiodomethane; the reaction temperature is selected from 60 to 100° C., preferably 80° C.

[0049] Compound 5 is prepared by reacting compound 4 with compound 2, wherein the solvent used is selected from a mixed solvent consisting of any two of toluene, 1,4-dioxane, N,N-dimethylformamide, n-butanol, and water, preferably a mixed solvent consisting of 1,4-dioxane and water; the base used is selected from potassium hydroxide, potassium carbonate, potassium acetate, sodium carbonate, cesium carbonate, sodium bicarbonate, preferably cesium carbonate; the catalyst used is selected from [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl2), tetrakis(III) (Triphenylphosphine) palladium (Pd(PPh3)4), bis(triphenylphosphine) palladium dichloride (Pd(PPh3)2Cl2), palladium acetate (Pd(OAc)2) and chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (SPhospdG2), preferably tetrakis(triphenylphosphine)palladium (Pd(PPh3)4); the reaction temperature is selected from 60 to 100°C, preferably 80°C.

[0050] Compound 6 is prepared by aromatization reaction of compound 5, the solvent used is selected from anhydrous toluene, anhydrous tetrahydrofuran, anhydrous 1,4-dioxane, anhydrous N,N-dimethylformamide or a mixed solvent of any two, preferably anhydrous toluene; the base used is selected from sodium amide, potassium hydroxide, potassium carbonate, potassium acetate, sodium carbonate, preferably sodium amide; the reaction temperature is selected from 80 to 120°C, preferably 100°C.

[0051] Compound 7 is prepared by hydrolysis reaction of compound 6, the base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, preferably lithium hydroxide, the solvent used is selected from methanol, ethanol, tetrahydrofuran, dioxane, water, or a mixed solvent thereof, preferably a mixed solvent of methanol:tetrahydrofuran:water = 1:1:1; the reaction temperature is selected from 50 to 90°C, preferably 60°C.

[0052] Compound 10 is prepared by reacting compound 8 with compound 9, and the solvent used is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, preferably dichloromethane. When 10 is a carboxylic acid, the condensing agent used is selected from carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or hexafluorophosphate benzotriazole-1-yl-oxytripyrrolidinophosphine (PyBOP), preferably HATU; the acid binding agent used is selected from N,N-diisopropylethylamine or triethylamine, preferably N,N-diisopropylethylamine. When 10 is an acid chloride, the base used is selected from N,N-diisopropylethylamine, triethylamine, pyridine, preferably triethylamine.

[0053] Compound 11 is prepared from compound 10 through a cyclization reaction, the solvent used is selected from 1,4-dioxane, acetonitrile, benzene, toluene, polyphosphoric acid, or a mixed solvent of any two, preferably acetonitrile, the catalyst used is selected from phosphorus oxychloride, phosphorus pentoxide, phosphorus trioxide, preferably phosphorus oxychloride, and the temperature used is selected from 50 to 90° C., preferably 60° C.

[0054] Compound 12 is prepared by reduction reaction of compound 11, the solvent used is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, preferably dichloromethane, and the reducing agent used is selected from sodium triacetoxyborohydride, sodium borohydride, sodium cyanoborohydride, preferably sodium borohydride.

[0055] Compound Ⅰ-A is prepared by reacting compound 12 with compound 7, wherein the solvent used is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably N,N-dimethylformamide; the condensing agent used is selected from carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP), preferably HATU; the acid binding agent used is selected from N,N-diisopropylethylamine or triethylamine, preferably N,N-diisopropylethylamine.

[0056] (2) When R 1 =H, n=1, ring B is The preparation method of the aryl piperidine compound is as follows:

[0057]

[0058] Compound 15 is prepared by reacting compound 13 with compound 14, the base used is selected from sodium hydride, potassium carbonate, sodium carbonate, cesium carbonate, preferably sodium hydride; the solvent used is selected from anhydrous toluene, anhydrous tetrahydrofuran, anhydrous 1,4-dioxane, anhydrous N,N-dimethylformamide or a mixed solvent of any two, preferably anhydrous tetrahydrofuran; the temperature used is selected from 50 to 80°C, preferably 65°C.

[0059] Compound 16 is prepared from compound 15, the solvent used is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably dichloromethane; the base used is selected from N,N-diisopropylethylamine or triethylamine, preferably N,N-diisopropylethylamine; the electrophilic reagent used is selected from trifluoromethanesulfonic anhydride and trifluoromethanesulfonic acid, preferably trifluoromethanesulfonic anhydride.

[0060] Compound 17 is prepared by reacting compound 16 with compound 2, wherein the solvent used is selected from a mixed solvent consisting of any two of toluene, 1,4-dioxane, N,N-dimethylformamide, n-butanol, and water, preferably a mixed solvent of 1,4-dioxane and water; the base used is selected from potassium hydroxide, potassium carbonate, potassium acetate, sodium carbonate, sodium bicarbonate, preferably potassium carbonate; the catalyst used is selected from [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (Pd(dppf)Cl2), tetra(tris(II)-iodine, The reaction mixture is prepared from palladium (Pd(PPh3)4), bis(triphenylphosphine)palladium dichloride (Pd(PPh3)2Cl2), palladium acetate (Pd(OAc)2) and chloro(2-dicyclohexylphosphino-2',6'-dimethoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (SPhospdG2), preferably tetrakis(triphenylphosphine)palladium (Pd(PPh3)4); the reaction temperature is selected from 60 to 100°C, preferably 80°C.

[0061] Compound 18 is prepared by hydrolysis reaction of compound 17, the base used is selected from lithium hydroxide, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, preferably lithium hydroxide, the solvent used is selected from methanol, ethanol, tetrahydrofuran, dioxane, water, or a mixed solvent thereof, preferably a mixed solvent of methanol:tetrahydrofuran:water = 1:1:1; the reaction temperature is selected from 50 to 90°C, preferably 60°C.

[0062] Compound Ⅰ-B is prepared by reacting compound 18 with compound 12, wherein the solvent used is selected from dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably N,N-dimethylformamide; the condensing agent used is selected from carbonyldiimidazole (CDI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) or benzotriazole-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP), preferably HATU; the acid binding agent used is selected from N,N-diisopropylethylamine or triethylamine, preferably N,N-diisopropylethylamine.

[0063] Where: R 2 , A ring, X 1 , X 2 The definition of is as mentioned above;

[0064] The pharmaceutically acceptable salt is obtained by salifying the corresponding acid with the aryl piperidine compound prepared by the above method.

[0065] The pharmaceutical composition of the present invention comprises the arylpiperidine compound and a pharmaceutically acceptable carrier. Specifically, the compound can be added with common pharmaceutical excipients and pharmaceutically acceptable carriers such as flavorings, sweeteners, liquid / solid fillers, diluents, etc. to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions or injections.

[0066] The aryl piperidine compound or the pharmaceutical composition thereof described in the present invention is used in the preparation of PRMT5 inhibitor drugs, specifically anti-tumor drugs, for treating glioblastoma, lung cancer, lymphoma, breast cancer, colorectal cancer, etc.

[0067] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0068] This class has significant PRMT5 enzyme inhibition, and the inhibitory activity is stronger in the presence of MTA (IC 50 The optimal value is less than 10 nM); at the same time, the inhibitory activity against MTAP-deficient tumor cells (most of the IC 50 values ​​below 100 nM) are stronger than the wild type (most IC 50 The value is higher than 1μM), which is selective; it has good activity at both the molecular level and the cellular level and is widely used; the preparation method is highly versatile and easy to expand the compound structure. DETAILED DESCRIPTION

[0069] The technical solution of the present invention is further described below in conjunction with embodiments.

[0070] Example 1: Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(5-(trifluoromethyl)pyridin-2-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (IA-1)

[0071] 1. Synthesis of methyl 4-amino-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (2)

[0072] Methyl 4-amino-3-bromobenzoate 1 (4.00 g, 17.4 mmol), bipyraclostrobin (8.83 g, 34.8 mmol) and 60 mL of 1,4-dioxane were added to a 250 mL eggplant-shaped bottle in sequence. Potassium acetate (5.12 g, 52.2 mmol) and Pd(dppf)Cl2 (1.42 g, 1.74 mmol) were added under stirring at room temperature. The reaction system was refluxed at 100 °C for 18 h under N2 protection. TLC (V 石油醚 :V 乙酸乙酯=1:1) monitor the reaction to be complete, cool to room temperature, filter with suction, wash the filter cake with dichloromethane, concentrate the filtrate to obtain a semi-solid residue. Dissolve it with a small amount of dichloromethane, filter with suction, collect the filter cake, concentrate the filtrate, repeat the operation once, combine the two filter cakes, and dry to obtain 3.00g of off-white solid, with a yield of 62.2%.

[0073] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 8.30 (d, J = 2.2Hz, 1H), 7.87 (dd, J = 8.6, 2.2Hz, 1H), 6.55 (d, J = 8.6Hz, 1H), 3.84 (s, 3H), 1.34 (s, 12H).

[0074] 2. Synthesis of 5-iodo-1-methyl-1H-pyrazole-4-carbonitrile (4)

[0075] 5-Amino-1-methyl-1H-pyrazole-4-carbonitrile 3 (5.00 g, 40.9 mmol) and 90 mL CH2I2 were added to a 250 mL three-necked flask in sequence, heated to 80°C, and isoamyl nitrite (11.0 mL, 81.9 mmol) was added dropwise. After the addition, heating was continued at 80°C for 1 h. TLC (V 石油醚 :V 乙酸乙酯 =10:1) to monitor the reaction completion, cool to room temperature, extract the reaction solution with concentrated HCl (30 mL × 2), combine the concentrated HCl layers, adjust the pH of the water layer to 1 with saturated NaOH aqueous solution, extract with ethyl acetate (30 mL × 2), combine the ethyl acetate layers, evaporate the solvent under reduced pressure, and the residue is purified by column chromatography (V 石油醚 :V 乙酸乙酯 =20:1) to obtain 3.0 g of a white solid with a yield of 31.5%.

[0076] 1 HNMR(400MHz, CDCl3-d)δ(ppm):7.81(s,1H),3.99(s,3H).

[0077] 3. Synthesis of methyl 4-amino-3-(4-cyano-1-methyl-1H-pyrazol-5-yl)benzoate (5)

[0078] To a 250 mL eggplant-shaped flask, intermediate 2 (3.92 mg, 14.2 mmol), intermediate 4 (3.00 g, 12.9 mmol), Pd(PPh3)4 (744 mg, 0.644 mmol), cesium carbonate (12.6 g, 38.7 mmol), 80 mL 1,4-dioxane and 8 mL H2O were added in sequence. N2 was used as protection and the temperature was raised to 80 °C for 2 h. TLC (V 石油醚 :V 乙酸乙酯=2:1) ​​to monitor the reaction completion, cool to room temperature, evaporate half of the solvent under reduced pressure, extract with ethyl acetate (40 mL×2), combine the ethyl acetate layers and dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and purify the residue by column chromatography (V 石油醚 :V 乙酸乙酯 =2:1+TEA) to give 2.0 g of a yellow solid with a yield of 60.4%.

[0079] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 7.99 (d, J = 8.6 Hz, 1H), 7.89 (s, 1H), 7.83 (s, 1H), 6.82 (d, J = 8.6 Hz, 1H), 3.87 (s, 3H), 3.77 (s, 3H).

[0080] 4. Synthesis of 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid methyl ester (6)

[0081] To a 25 mL eggplant-shaped flask, intermediate 5 (1.00 g, 3.90 mmol), 10 mL of anhydrous toluene and NaNH2 (304 mg, 7.80 mmol) were added in sequence. The reaction system was heated at 100°C under N2 protection for 2 h. TLC (V 二氯甲烷 :V 甲醇 =20:1+TEA) to monitor the completion of the reaction, cool the reaction solution to below 5°C, add a small amount of ice water, and then evaporate the solvent under reduced pressure. The residue is slurried with ice water, filtered, and the filter cake is collected and dried in vacuo to obtain 800 mg of a white solid with a yield of 80.0%.

[0082] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.80 (s, 1H), 8.28 (s, 1H), 8.02 (d, J = 8.8Hz, 1H), 7.60 (d, J = 8.8Hz, 1H), 7.38 (s, 2H), 4.42 (s, 3H), 3.89 (s, 3H).

[0083] 5. Synthesis of 4-amino-1-methyl-1H-pyrazolo[4,3-c]quinoline-8-carboxylic acid (7)

[0084] In a 25 mL eggplant-shaped flask, intermediate 6 (800 mg, 3.1 mmol), 3 mL THF, 3 mL MeOH and 3 mL H2O were added in sequence, and LiOH (150 mg, 6.24 mmol) was added under stirring. The temperature was raised to 70 °C for 2 h. TLC (V 二氯甲烷 :V 甲醇=4:1+TEA) to monitor the completion of the reaction, after distilling off THF and MeOH under reduced pressure, 6 mL of H2O was added to the residue, and the pH was adjusted to 3 with 1.5 mol / L HCl. Solid precipitated and was filtered off with suction. The filter cake was washed with acetone, and the filter cake was collected and dried in vacuo to obtain 750 mg of a white solid with a yield of 99.0%.

[0085] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.83 (d, J = 1.8 Hz, 1H), 8.73 (s, 1H), 8.25 (dd, J = 8.6, 1.8 Hz, 1H), 7.90 (d, J = 8.6 Hz, 1H), 4.49 (s, 3H).

[0086] 6. Synthesis of N-(2-(thiophen-2-yl)ethyl)-5-(trifluoromethyl)picolinamide (10-1)

[0087] 2-(Thienyl)ethyl-1-amine 8-1 (1.33 g, 10.5 mmol), 5-(trifluoromethyl)picolinic acid 9-1 (2.00 g, 10.5 mmol), HATU (4.00 g, 10.5 mmol) and 20 mL of DCM were added to a 100 mL eggplant-shaped bottle in sequence, and DIPEA (4.32 mL, 26.2 mmol) was added under stirring. The reaction system was reacted under N2 protection at 25 °C for 6 h. TLC (V 石油醚 :V 乙酸乙酯 =10:1) to monitor the reaction completion, column chromatography purification (V 石油醚 :V 乙酸乙酯 =3:1) to give 2.4 g of a white solid with a yield of 76.4%.

[0088] 1 H NMR (400MHz, CDCl3-d) δ (ppm): 8.80 (s, 1H), 8.34 (d, J = 8.2Hz, 1H), 8.10 (dd, J = 7.7, 2.3Hz, 1H), 7.18 (dd, J = 5 .1,1.1Hz,1H),6.96(dd,J=5.1,3.4Hz,1H),6.91–6.87(m,1H),3.78(q,J=6.7Hz,2H),3.18(t,J=6.8Hz,2H).

[0089] 7. Synthesis of 4-(5-(trifluoromethyl)pyridin-2-yl)-6,7-dihydrothieno[3,2-c]pyridine (11-1)

[0090] The intermediate 10-1 (2.00 g, 6.66 mmol) and 20 mL of acetonitrile were added to a 50 mL eggplant-shaped bottle, and POCl3 (2.50 mL, 26.6 mmol) was slowly added dropwise under stirring. The reaction system was reacted at 60 °C under N2 protection for 12 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) to monitor the reaction completion, cool to room temperature, evaporate the solvent under reduced pressure, dissolve the residue in ethyl acetate, wash with saturated sodium bicarbonate aqueous solution, water and saturated brine in sequence, evaporate the solvent under reduced pressure, and purify by column chromatography (V 石油醚 :V 乙酸乙酯 =50:1) to give 644 mg of a yellow solid with a yield of 34.3%.

[0091] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 8.95–8.91 (m, 1H), 8.18 (d, J = 8.3Hz, 1H), 8.04 (dd, J = 8.6, 2. 5Hz,1H),7.56(d,J=5.2Hz,1H),7.14(d,J=5.2Hz,1H),4.08–4.01(m,2H),3.01–2.93(m,2H).

[0092] 8. Synthesis of 4-(5-(trifluoromethyl)pyridin-2-yl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (12-1)

[0093] The intermediate 11-1 (644 mg, 2.28 mmol) and 10 mL of anhydrous methanol were added to a 25 mL eggplant-shaped flask in sequence, and NaBH4 (173 mg, 4.56 mmol) was slowly added under ice bath conditions. The reaction system was moved to room temperature for 2 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) to monitor the reaction completion, evaporate the solvent under reduced pressure, and dissolve the residue in 10 mL of ethyl acetate, then wash with saturated brine, and purify by column chromatography (V 石油醚 :V 乙酸乙酯 =3:1) to give 520 mg of light yellow liquid, with a yield of 80.2%.

[0094] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.93 (s, 1H), 8.17 (d, J = 8.3Hz, 1H), 7.64 (d, J = 8.3Hz, 1H), 7.19 (d, J = 5. 2Hz,1H),6.61(d,J=5.2Hz,1H),5.15(s,1H),3.23–3.15(m,1H),3.04-2.94(m,1H),2.92–2.71(m,2H).

[0095] 9. Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(5-(trifluoromethyl)pyridin-2-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (IA-1)

[0096] To a 25 mL eggplant-shaped flask, intermediate 12-1 (44.0 mg, 0.155 mmol), intermediate 7 (38.0 mg, 0.155 mmol), HATU (76.0 mg, 0.201 mmol) and 1.5 mL DMF were added in sequence, and DIPEA (128 μL, 0.774 mmol) was added under stirring. The reaction system was reacted at room temperature for 16 h. TLC (V 二氯甲烷 :V 甲醇 =10:1) to monitor the reaction completion, add 6 mL of water to dilute the reaction solution, extract with ethyl acetate (5 mL×3), combine the organic phases, dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and column chromatography (V 二氯甲烷 :V 氨甲醇(7mmol / l) =150:1) to obtain 25.0 mg of a white solid with a yield of 31.7%.

[0097] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.97 (s, 1H), 8.28 (s, 3H), 7.95 (s, 1H), 7.60 (s, 2H), 7.37 (d, J = 5.2Hz, 1H), 7.27(s,2H),7.02(d,J=5.2Hz,1H),6.68(s,1H),4.53–3.82(m,5H),3.22-3.05(m,1H),2.97-2.93(m,1H).

[0098] Example 2: Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-yl)methanone (IA-7)

[0099] 1. Synthesis of N-(2-(thiophen-2-yl)ethyl)-4-(trifluoromethyl)benzamide (10-2)

[0100] Compound 8-1 (2.00 g, 15.7 mmol), 30 mL DCM and TEA (1.75 mg, 17.3 mmol) were added to a 50 mL eggplant-shaped bottle in sequence, and 4-(trifluoromethyl)benzoyl chloride 10-2 (3.44 g, 16.5 mmol) was slowly added dropwise under ice bath conditions. After 5 min, the reaction system was moved to room temperature for 5 h. TLC (V石油醚 :V 乙酸乙酯 =10:1) to monitor the reaction completion, add water to quench, separate the organic layer, evaporate the solvent under reduced pressure, and purify by column chromatography (V 石油醚 :V 乙酸乙酯 =3:1), to give 4.50 g of a white solid with a yield of 95.6%.

[0101] 1 H NMR (400MHz, CDCl3-d) δ (ppm): 7.82 (d, J = 8.2Hz, 2H), 7.68 (d, J = 8.2Hz, 2H), 7.19 (d, J = 5.0Hz, 1H) ,6.97(dd,J=5.1,3.4Hz,1H),6.88(d,J=3.2Hz,1H),3.75(q,J=6.2Hz,2H),3.17(t,J=6.5Hz,2H).

[0102] 2. Synthesis of 4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]pyridine (11-2)

[0103] Intermediate 10-2 (4.50 g, 14.4 mmol) was used as the raw material and the operation was the same as that of intermediate 11-1 to obtain 3.80 g of light yellow liquid with a yield of 93.7%.

[0104] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 7.79 (d, J = 8.2 Hz, 2H), 7.69 (d, J = 8.2 Hz, 2H), 7.1 2(d,J=5.2Hz,1H),6.95(d,J=5.2Hz,1H),4.04–3.93(m,2H),3.01–2.88(m,2H).

[0105] 3. Synthesis of 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (12-2)

[0106] Intermediate 11-2 (3.80 g, 13.5 mmol) was used as the starting material and the operation was the same as that of intermediate 12-1 to obtain 2.30 g of a white solid with a yield of 60.1%.

[0107] 1H NMR (300MHz, DMSO-d6) δ (ppm): 7.66 (d, J = 8.3Hz, 2H), 7.49 (d, J = 8.2Hz, 2H), 7.16 (d, J = 5.1Hz, 1 H), 6.38 (d, J = 5.2Hz, 1H), 4.98 (s, 1H), 3.14–3.03 (m, 1H), 2.98–2.79 (m, 2H), 2.79–2.67 (m, 1H).

[0108] 4. Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-yl)methanone (IA-7)

[0109] Using intermediate 12-2 (44.0 mg, 0.155 mmol) and intermediate 7 (38.0 mg, 0.155 mmol) as raw materials, the operation was the same as IA-1 to obtain 30.0 mg of white solid with a yield of 38.1%.

[0110] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 8.25 (s, 2H), 7.77 (d, J = 8.3Hz, 2H), 7.69–7.50 (m, 5H), 7.46 (d, J = 5.0Hz ,1H),7.21(s,2H),6.94(s,1H),4.39(s,3H),4.06-3.92(m,1H),3.32-3.04(m,2H),3.01-2.86(m,1H).

[0111] Example 3: Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrofuro[3,2-c]pyridin-5(4H)-yl)methanone (IA-10)

[0112] 1. Synthesis of N-(2-(furan-2-yl)ethyl)-4-(trifluoromethyl)benzamide (10-3)

[0113] 2-(Furan-2-yl)ethan-1-amine 8-2 (250 mg, 2.25 mmol) and 10 mL DCM were added to a 25 mL eggplant-shaped bottle, and compound 9-2 (493 mg, 2.36 mmol) was slowly added dropwise under ice bath conditions. After 5 min, the reaction system was transferred to room temperature for 2 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) to monitor the reaction completion, column chromatography purification (V 石油醚 :V 乙酸乙酯=5:1), to give 400 mg of a yellow solid, with a yield of 62.8%.

[0114] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 7.83 (d, J = 8.1Hz, 2H), 7.69 (d, J = 8.6Hz, 2H), 7.38-7.3 5(m,1H),6.34-6.31(m,1H),6.13(s,1H),3.76(q,J=6.2Hz,2H),2.98(t,J=6.4Hz,2H).

[0115] 2. Synthesis of 4-(4-(trifluoromethyl)phenyl)-6,7-dihydrofuro[3,2-c]pyridine (11-3)

[0116] P2O5 (585 mg, 4.12 mmol), 10 mL toluene and POCl3 (648 μL, 6.95 mmol) were added to a 50 mL eggplant-shaped bottle in sequence. The reaction system was heated to 100 °C and refluxed. Intermediate 10-3 (400 mg, 1.41 mmol) was added and the reaction system was refluxed for 2 h. TLC (V 石油醚 :V 乙酸乙酯 =3:1) to monitor the reaction completion, cool to room temperature, pour the reaction solution into crushed ice, adjust the pH of the water layer to 10 with saturated K2CO3 aqueous solution, extract the water layer with toluene (20mL×3), collect the organic phase, wash with water, dry with anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and purify by column chromatography (V 石油醚 :V 乙酸乙酯 =7.5:1), to obtain 47.0 mg of oily liquid, with a yield of 12.5%.

[0117] 1 H NMR (400MHz, CDCl3-d) δ (ppm): 7.86 (d, J = 8.4Hz, 2H), 7.70 (d, J = 8.4Hz, 2H), 7.38 (d ,J=2.3Hz,1H),6.47(d,J=2.1Hz,1H),4.11(t,J=8.8Hz,2H),2.89(t,J=8.8Hz,2H).

[0118] 3. Synthesis of 4-(4-(trifluoromethyl)phenyl)-4,5,6,7-tetrahydrofuro[3,2-c]pyridine (12-3)

[0119] The intermediate 11-3 (47.0 mg, 0.177 mmol) and 2 mL of anhydrous dichloromethane were added to a 25 mL eggplant-shaped flask in sequence, and NaBH(OAc)3 (126 mg, 0.594 mmol) was slowly added under stirring. The reaction system was reacted at room temperature for 12 h. TLC (V石油醚 :V 乙酸乙酯 =1:1) to monitor the reaction completion, dilute the reaction solution with dichloromethane, cool to 0°C, slowly add 10% NaOH aqueous solution, separate the organic layer, dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and purify by column chromatography (V 石油醚 :V 乙酸乙酯 =5:1+TEA) to give 30.0 mg of a colorless oily liquid with a yield of 63.4%.

[0120] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 7.59 (d, J = 8.3Hz, 2H), 7.44 (d, J = 8.1Hz, 2H), 7.28(s,1H),5.99(s,1H),4.98(s,1H),3.33–3.05(m,2H),2.90–2.61(m,2H).

[0121] 4. Synthesis of (4-amino-1-methyl-1H-pyrazolo[4,3-c]quinolin-8-yl)(4-(4-(trifluoromethyl)phenyl)-6,7-dihydrofuro[3,2-c]pyridin-5(4H)-yl)methanone (IA-10)

[0122] Using intermediate 12-3 (30.0 mg, 0.112 mmol) and intermediate 7 (27.0 mg, 0.112 mmol) as raw materials, the operation was the same as IA-1 to obtain 23.0 mg of white solid with a yield of 42.0%.

[0123] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.25 (s, 2H), 7.78 (d, J = 8.5Hz, 2H), 7.67 (d, J = 8.4Hz, 3H), 7.60 (t, J = 9.3Hz, 2H) ,7.21(s,2H),6.76(s,1H),6.56(s,1H),4.40(s,3H),4.04-3.91(m,1H),3.07-2.95(m,1H),2.77-2.62(m,2H).

[0124] Example 4: Synthesis of (4-amino-1,3-dihydrofuro[3,4-c]quinolin-8-yl)(4-(5-(trifluoromethyl)pyridin-2-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (IB-1)

[0125] 1. Synthesis of 4-oxotetrahydrofuran-3-carbonitrile (15)

[0126] 60% NaH (1.52 g, 63.3 mmol) and 90 mL of anhydrous THF were added to a 250 mL three-necked flask in sequence. Under N2 protection, butyl glycolate 13 (5.00 g, 63.3 mmol) dissolved in 5 mL of THF was slowly added dropwise to the reaction system, heated to 65°C and refluxed, and then acrylonitrile 14 (2.03 g, 63.3 mmol) dissolved in 5 mL of THF was slowly added dropwise. The reaction system continued to reflux for 2 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) monitor the reaction completion, evaporate the solvent under reduced pressure, dissolve the residue with 80mL 2mol / LNaOH aqueous solution, extract with ether (80mL×3), adjust the pH of the aqueous layer to 1 with concentrated hydrochloric acid, extract the aqueous layer with DCM (80mL×3), combine the DCM layers, and evaporate the solvent under reduced pressure to obtain 500mg of a yellow oil with a yield of 7.10%.

[0127] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 4.62 (t, J = 9.0Hz, 1H), 4.23 (t, J = 9.7Hz, 1H), 4.15 (d, J = 17.6Hz, 1H), 3.99 (d, J = 17.4Hz, 1H), 3.60 (t, J = 9.1Hz, 1H).

[0128] Synthesis of 2,4-cyano-2,5-dihydrofuran-3-yl trifluoromethanesulfonate (16)

[0129] To a 25 mL three-necked flask, intermediate 15 (290 mg, 2.61 mmol) and 3 mL DCM were added in sequence. The reaction system was cooled to -78 °C under N2 protection. DIPEA (546 μL, 3.13 mmol) was slowly added under stirring and stirred for 30 min. Tf2O (439 μL, 2.61 mmol) was added dropwise within 1 min and stirred for 1 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) monitor the reaction completion, slowly add water to the reaction solution to dilute, warm to room temperature, separate the organic layer, dry over anhydrous Na2SO4, filter, collect the filtrate, and evaporate the solvent under reduced pressure to obtain 520 mg of black liquid, with a yield of 82.0%.

[0130] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 4.85 (s, 1H), 3.68 (s, 1H), 3.09 (s, 1H), 2.50 (s, 1H).

[0131] 3. Synthesis of methyl 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylate (17)

[0132] To a 25 mL eggplant-shaped bottle, intermediate 16 (520 mg, 2.14 mmol), 10 mL of dioxane and 1 mL of water were added in sequence. Under stirring conditions, intermediate 2 (474 ​​mg, 1.71 mmol), K2CO3 (885 mg, 6.40 mmol), and Pd(PPh3)4 (248 mg, 0.214 mmol) were added in sequence. The reaction system was refluxed at 80°C under N2 protection for 16 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) to monitor the reaction completion, evaporate the solvent under reduced pressure, dilute the residue with 5 mL of ethyl acetate and 5 mL of water, stir at room temperature for 30 min, solid precipitates, filter, wash the filter cake with 5 mL of ethyl acetate and 5 mL of DCM containing 2% MeOH in turn, and purify by column chromatography (V 二氯甲烷 :V 甲醇 =25:1), to give 90.0 mg of dark white solid, with a yield of 22.0%.

[0133] 1 H NMR(300MHz,DMSO-d6)δ(ppm):8.09(d,J=2.0Hz,1H),7.98(dd,J=8.8,2.1Hz,1H),7.58( d,J=8.9Hz,1H),6.94(s,2H),5.38(t,J=3.5Hz,2H),5.01(t,J=3.5Hz,2H),3.85(s,3H).

[0134] 4. Synthesis of 4-amino-1,3-dihydrofurano[3,4-c]quinoline-8-carboxylic acid (18)

[0135] In a 25 mL eggplant-shaped flask, intermediate 17 (80.0 mg, 0.328 mmol) and 3 mL of a mixed solvent of THF+MeOH+H2O in equal proportions were added. LiOH (31.4 mg, 1.31 mmol) was added to the reaction solution under stirring, and the reaction system was refluxed at 75°C for 3 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1) monitor the reaction completion, evaporate MeOH and THF under reduced pressure, dilute the residue with a small amount of water, adjust the pH of the aqueous layer to 6.0 with 1.5 mol / L HCl, solid precipitates, filter and collect the filter cake to obtain 50.0 mg of off-white solid, yield 66.0%, which is directly used for the next step without purification.

[0136] 5. Synthesis of (4-amino-1,3-dihydrofurano[3,4-c]quinolin-8-yl)(4-(5-(trifluoromethyl)pyridin-2-yl)-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl)methanone (IB-1)

[0137] To a 25 mL eggplant-shaped bottle, intermediate 12-1 (62.0 mg, 0.217 mmol), intermediate 18 (50.0 mg, 0.217 mmol), HATU (107 mg, 0.282 mmol) and 2 mL DMF were added in sequence, and DIPEA (180 uL, 1.086 mmol) was added under stirring. The reaction system was reacted at room temperature for 16 h. TLC (V 石油醚 :V 乙酸乙酯 =1:1+TEA) to monitor the reaction completion, the reaction solution was diluted with 6 mL of water, extracted with EA (5 mL×3), the ethyl acetate layers were combined, dried over anhydrous sodium sulfate, the solvent was evaporated under reduced pressure, and column chromatography (V 石油醚 :V 乙酸乙酯 =1:1+TEA) to obtain 15.0 mg of a white solid with a yield of 14.0%.

[0138] 1 H NMR (400MHz, DMSO-d6) δ (ppm): 8.95 (s, 1H), 8.26 (dd, J=8.6, 2.4Hz, 1H), 7.91 (s, 1H), 7.68–7.43 (m, 3H), 7.36 (d, J= 4.9Hz,1H),6.98(s,1H),6.71(s,2H),6.65(s,1H),5.34(s,2H),5.01(s,2H),4.16–3.12(m,2H),3.17–2.88(m,2H).

[0139] Example 5: PRMT5 inhibitory activity test of some compounds of the present invention

[0140] 1. Experimental reagents

[0141] PRMT5(Active Motif,Cat.No.31921)[ 3 H]-SAM(PerkinElmer,Cat.No.NET155V001MC)SAM(Sigma,Cat.No.A7007-100MG)SAH(Sigma,Cat.No.A9384-25MG)MTA(Sigma,D5011-100MG)OptiPlate(384-well,white)(Perkin Elmer, Cat. No. 6007299) 384-well Flashplate (Perkin Elmer, Cat. No. SMP410A001PK)

[0142] 2. Experimental methods

[0143] (1) Prepare 1x detection buffer (modified Tris buffer);

[0144] (2) Compound serial dilution: Compounds were transferred to the test plate by Echo in 100% DMSO, and the final concentration fraction of DMSO was 1%;

[0145] (3) PRMT5+MTA: Prepare enzyme in 1x assay buffer. 3 H]-SAM and MTA mixed solution;

[0146] (4) Prepare substrate solution in 1x assay buffer;

[0147] (5) PRMT5: 15 μL enzyme and [ 3 H]-SAM mixed solution was transferred to the test plate (PRMT5+MTA: 15 μL enzyme, [ 3 H]-SAM and MTA mixed solution was transferred to the test plate);

[0148] (6) Add 10 μL of substrate solution to each well to start the reaction and 10 μL of 1x detection buffer as a blank control;

[0149] (7) Incubate at room temperature for 90 min;

[0150] (8) Add cold SAM to 1x detection buffer to make a stop solution;

[0151] (9) Add 5 μL of stop solution to each well to stop the reaction;

[0152] (10) Transfer 25 μL per well to Flashplate;

[0153] (11) Incubate at room temperature for at least 1 h;

[0154] (12) Flashplate detection through Microbeta;

[0155] (13) Data processing: Fit the data in Excel and use formula (1): inh% = (Max-Signal) / (Max-Min)*100 to obtain the inhibition value; use formula (2): Y = Bottom + (Top-Bottom) / (1 + (IC 50 / X)*HillSlope) to get IC 50 Values ​​where Y is the inhibition rate and X is the compound concentration.

[0156] 3. Experimental results

[0157] Edit number <![CDATA[PRMT5-MTA,IC 50 (nM) a ]]> IA-1 B IA-7 C IA-10 B IB-1 A

[0158] a :A:IC 50 <10nM, B:10nM≤IC 50 <100nM, C:100nM≤IC 50 <1.0μM

[0159] Example 6: In vitro cell SDMA activity test of some compounds of the present invention

[0160] 1. Experimental reagents

[0161] Protease inhibitors were purchased from MCE; phosphatase inhibitor II was purchased from MCE; BCA kit was purchased from Beyotime Co., Ltd.; PVDF membrane was purchased from Millipore, USA; ECL was purchased from Nanjing Novezan Biotechnology Co., Ltd.; SDMA antibody was purchased from CST, catalog number 13222S; β-Tubulin was purchased from CST, catalog number 2146S; HRP-conjugated secondary antibody was purchased from CST; PRMT5 antibody was purchased from Abcam, catalog number ab109451.

[0162] 2. Experimental methods

[0163] Compounds were administered at the expected concentration and for the expected time in the MV-4-11 cell line. After washing twice with PBS, cells were lysed with RIPA buffer, protease inhibitors, and phosphatase inhibitor II for 1 h. The mixture was centrifuged at 12,000 rpm for 15 min. Total protein content was quantified by BCA kit, protein was diluted to a uniform concentration, 6× Loading Buffer was added, protein was denatured at 100°C, and then transferred to -80°C for storage until analysis. Equivalent protein was electrophoresed by 8-10% SDS-PAGE and then transferred to PVDF membrane. Subsequently, the membrane was blocked with 5% (w / v) skim milk for 1-2 h. Proteins were detected with primary antibodies, followed by HRP-conjugated secondary antibodies and visualized with ECL as HRP substrate. Target blots were detected using a chemiluminescence system. Grayscale analysis was performed using Image J software.

[0164] 3. Experimental results

[0165]

[0166]

[0167] a :A:IC 50 <100 nM, B:100 nM≤IC 50 <1000 nM; b :A:IC 50 >10μM, B:1μM≤IC 50 <10μM.

Claims

1. An aryl piperidine compound, characterized in that: Having the structure of formula (I), also includes isomers, prodrugs, stable isotope derivatives, pharmaceutically acceptable salts or mixtures thereof: in: R 1 , R 2 are independently selected from hydrogen, substituted C1-C6 alkyl, substituted C1-C6 haloalkyl, substituted C6-C 10 aryl, substituted C5-C9 heteroaryl, substituted C3-C8 cycloalkyl or substituted C2-C6 heterocycloalkyl; the substituent is selected from at least one of hydrogen, halogen, cyano, C1-C3 alkyl, C1-C3 haloalkyl, C1-C3 alkoxy, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the C5-C9 heteroaryl, C2-C6 heterocycloalkyl contains 1-3 heteroatoms selected from N, O or S; when R 1 With R 2 At different times, the carbon atom connected thereto is in racemic configuration, R configuration or S configuration; Ring A is selected from substituted C6~C 10 An aromatic ring or a substituted C5-C9 heteroaromatic ring; the substituent is selected from hydrogen, halogen, C1-C6 alkyl or C1-C6 haloalkyl; the C5-C9 heteroaromatic ring contains 1-3 heteroatoms selected from N, O or S; n is an integer selected from 0 to 3; Ring B is selected from substituted 5-6 membered heteroaromatic rings or substituted 5-6 membered heterocycloalkane; the substituent is selected from at least one hydrogen, halogen, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, C2-C6 heterocycloalkyl, C1-C6 alkoxy, C1-C6 acyl, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the 5-6 membered heteroaromatic ring and 5-6 membered heterocycloalkane contain 1 to 3 heteroatoms selected from N, O or S; X 1 , X 2 Each independently selected from CR 3 or N; R 3 Selected from hydrogen, halogen, amino, cyano, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy or C1-C6 haloalkoxy.

2. The aryl piperidine compound according to claim 1, characterized in that In the structure: R 1 is hydrogen; R 2 Selected from hydrogen, substituted C6~C 10 aryl, substituted C5-C9 heteroaryl, substituted C3-C8 cycloalkyl or substituted C2-C6 heterocycloalkyl; the substituent is selected from at least one of hydrogen, halogen, cyano, methyl, ethyl, methoxy, trifluoromethyl, hydroxyl, amino, methylamino, dimethylamino, acetylamino, carboxyl, methoxycarbonyl or nitro; the C5-C9 heteroaryl, C2-C6 heterocycloalkyl contains 1-3 heteroatoms selected from N, O or S; when R 1 With R 2 At different times, the carbon atom connected thereto is in racemic configuration, R configuration or S configuration; Ring A is selected from substituted C6~C 10 Aromatic ring or substituted C5-C9 heteroaromatic ring; the substituent is selected from hydrogen, halogen or C1-C6 alkyl; the C5-C9 heteroaromatic ring contains 1-3 heteroatoms selected from N, O or S; n is selected from 1 or 2; Ring B is selected from a substituted 5-membered heteroaromatic ring or a substituted 5-membered heterocycloalkane, wherein the substituent is selected from at least one of hydrogen, halogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, trifluoroethyl, methoxy, cyclopropylmethoxy, trifluoromethoxy or 2,2-difluoroethyl; the 5-membered heteroaromatic ring or the 5-membered heterocycloalkane contains 1 to 3 heteroatoms selected from N, O or S; X 1 , X 2 Each independently selected from CR 3 or N; R 3 is selected from hydrogen, methyl, ethyl, isopropyl, cyclopropyl, trifluoromethyl, methoxy, trifluoromethoxy or 2,2-difluoroethyl.

3. The aryl piperidine compound according to claim 1, characterized in that In the structure: R 1 is hydrogen; R 2 is selected from substituted benzene ring, substituted pyridine, substituted pyridazine, substituted pyrazine, substituted furan, substituted thiophene, substituted oxazole, substituted pyrazole, substituted imidazole, substituted thiazole, substituted pyrimidine, substituted naphthyl ring, substituted quinoline, substituted indole, substituted indazole, substituted cyclopropyl, substituted cyclobutyl, substituted cyclopentyl, substituted cyclohexyl, substituted piperazine, substituted piperidine, substituted pyrrole or substituted tetrahydrofuran; the substituent is selected from at least one hydrogen, fluorine, chlorine, bromine, cyano, methyl, ethyl, trifluoromethyl, difluoromethyl, cyclopropyl, methoxy, trifluoromethoxy, cyclopropyloxy, morpholinyl, piperidinyl, tetrahydropyrrolidinyl, 2-(N,N-dimethylamino)ethyl, 2-(N,N-diethylamino)ethyl, hydroxyl, amino, methylamino, dimethylamino, acetamido, carboxyl, methoxycarbonyl or nitro; the carbon atom connected thereto is in racemic configuration; Ring A is selected from substituted benzene ring, substituted thiophene, substituted thiazole, substituted pyrrole, substituted pyrazole, substituted imidazole, substituted furan, substituted oxazole or substituted isoxazole; the substituent is selected from hydrogen, fluorine, chlorine, bromine, methyl, ethyl or isopropyl; n is selected from 1; Ring B is selected from substituted pyrrole, pyrazole, isoxazole, thiophene, thiazole, furan, dihydropyrrole or dihydrofuran, wherein the substituent is selected from at least one hydrogen, fluorine, chlorine, bromine, methyl, ethyl, isopropyl, trifluoromethyl, trifluoroethyl, methoxy, trifluoromethoxy; X 1 , X 2 Each independently selected from CR 3 or N; R 3 is selected from hydrogen, methyl, ethyl, trifluoromethyl, methoxy, trifluoromethoxy or 2,2-difluoroethyl.

4. The aryl piperidine compound according to claim 1, characterized in that: In the structure: R 1 is hydrogen; R 2 Selected from The carbon atom to which it is attached is in a racemic configuration; Ring A is selected from n is taken from 1; Ring B is selected from X 1 , X 2 are independently selected from CH, CF, C-Cl, C-Br or N.

5. The aryl piperidine compound according to claim 1, characterized in that: Select any of the following compounds:

6. The aryl piperidine compound according to claim 1, characterized in that: The pharmaceutically acceptable salt is a salt formed by the arylpiperidine compound and any one of the following acids: The acid is hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, citric acid or ferulic acid.

7. A method for preparing an arylpiperidine compound according to claim 1, characterized in that: Choose from any of the following methods: (1) When R 1 =H, n=1, and ring B is a pyrazole ring. The preparation method of the aryl piperidine compound is as follows: (2) When R 1 =H, n = 1, ring B is The preparation method of the aryl piperidine compound is as follows: Where: R 2 , A ring, X 1 , X 2 The definition of is as in claim 1; The pharmaceutically acceptable salt is obtained by salifying the corresponding acid with the aryl piperidine compound prepared by the above method.

8. A pharmaceutical composition, characterized in that The invention comprises the arylpiperidine compound according to claim 1 and a pharmaceutically acceptable carrier.

9. Use of the arylpiperidine compound according to claim 1 or the pharmaceutical composition according to claim 8 in the preparation of a PRMT5 inhibitor drug.

10. The use according to claim 9, characterized in that: The drug is an anti-tumor drug.

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