Pyridine-containing polycyclic derivative as well as preparation method and application thereof

Abstract

The invention relates to a pyridine-containing polycyclic derivative as well as a preparation method and application thereof. Particularly, the invention relates to a compound as shown in a general formula (I), a preparation method thereof and a pharmaceutical composition containing the compound, the invention relates to a general formula (I) and application of the general formula (I) as a biological regulator in preparation of drugs for treating autoimmune diseases, chronic inflammatory diseases, acute inflammatory diseases, autoinflammatory diseases, atherosclerosis, diabetes, fibrotic diseases, metabolic diseases, cancers, tumors, leukemia and lymphoma, wherein substituents in the general formula (I) are as defined in the specification. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a pyridine-containing polycyclic derivative, a preparation method and an application thereof. Background Art

[0002] MK2 (MAPK activated protein kinase 2) is a protein kinase that is an important downstream molecule of the classic inflammatory signal transduction pathway MAPK and regulates the expression of inflammatory factors. After cells are stimulated by inflammatory factors, the activated p38 MAPK protein phosphorylates and activates MK2, leading to the phosphorylation of MK2 substrate TTP, promoting the stability of pro-inflammatory cytokine mRNA, thereby increasing the protein expression level of inflammatory cytokines.

[0003] In both mouse arthritis models and chondrocytes of osteoarthritis patients, MK2 is activated. Inhibition of the p38-MK2 pathway by genetic or pharmacological means can inhibit the expression of inflammatory factors, indicating that MK2 is involved in mediating the inflammatory regulation of arthritis. p38-MK2 inhibitors are expected to treat patients with relapsed, refractory rheumatoid arthritis who have poor response to existing therapies.

[0004] Currently, only Aclaris Therapeutics' p38-MK2 inhibitor ATI-450 has entered clinical stage IIb for rheumatoid arthritis. Preliminary clinical trial results show that ATI-450 is safe and tolerable in patients with rheumatoid arthritis, with outstanding and sustained efficacy. Other MK2 inhibitors in clinical trials include BMS's CC-99677 and Moerae Matrix's MMI-0100, both of which can directly inhibit the activity of MK2, with a different mechanism from ATI-450.

[0005] As drugs, p38-MK2 inhibitors have good application prospects in the pharmaceutical industry. p38-MK2 inhibitors provide a safer and more effective treatment for patients with rheumatoid arthritis who have relapsed, are refractory, and respond poorly to existing therapies; p38-MK2 inhibitors have a broad spectrum of anti-inflammatory effects (TNF / IL-6 / IL-1), and can theoretically be used to treat a variety of inflammatory diseases and autoimmune diseases; p38-MK2 inhibitors are small molecule inhibitors, which have advantages over large molecule drugs such as TNF monoclonal antibodies, such as oral convenience and good patient compliance. At the same time, p38-MK2 inhibitors have not been reported to have the toxic side effects of JAK inhibitors such as infection, tumors, thrombosis, and cardiac toxicity. Therefore, there is a huge clinical demand for the development of new p38-MK2 inhibitors. Summary of the invention

[0006] The object of the present invention is to provide a compound described by general formula (I) or (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof, wherein the compound structure represented by general formula (I) or (II) is as follows:

[0007]

[0008] in:

[0009] M1, M2 and M3 are each independently selected from carbon or oxygen, and are not oxygen at the same time;

[0010] M4, M5 and M6 are each independently selected from carbon or oxygen, and are not oxygen at the same time;

[0011] R1 is selected from deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, C 2-6 Alkenyl or C 2-6 The alkynyl group may be further substituted with deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, C 2-6 Alkenyl and C 2-6 substituted by one or more substituents in the alkynyl group;

[0012] R2 is selected from deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, C 2-6 Alkenyl or C 2-6 The alkynyl group may be further substituted with deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Haloalkoxy, C 2-6 Alkenyl and C 2-6 substituted by one or more substituents in the alkynyl group;

[0013] n is selected from 0, 1, 2 or 3; and

[0014] m is selected from 0, 1, 2 or 3.

[0015] In a preferred embodiment of the present invention, said M1 is selected from carbon or oxygen;

[0016] M2 and M3 are selected from carbon;

[0017] M4 is selected from carbon or oxygen;

[0018] M5 and M6 are selected from carbon;

[0019] R1 is selected from deuterium, halogen, oxo or C 1-3 alkyl;

[0020] R2 is selected from deuterium, halogen, oxo or C 1-3 alkyl; and

[0021] n is 2; and

[0022] m is 2.

[0023] In a preferred embodiment of the present invention, the structure of the compound is as follows:

[0024]

[0025] The present invention further provides a compound represented by the general formula (M-1), a stereoisomer thereof or a pharmaceutically acceptable salt thereof:

[0026]

[0027] in:

[0028] M1, M2, M3, R1 and n are as described above.

[0029] In a further preferred embodiment of the present invention, the compound of formula (M-1), its stereoisomers or pharmaceutically acceptable salts thereof are characterized in that they are selected from the following compounds:

[0030]

[0031] The present invention further provides a method for preparing the compound of the aforementioned general formula (I) or its stereoisomers and pharmaceutically acceptable salts thereof, comprising the following steps:

[0032]

[0033] The compound represented by the general formula (M-1) is reacted to obtain the target compound represented by the general formula (I);

[0034] in,

[0035] M1, M2, M3, R1 and n are as described above. The present invention further relates to a pharmaceutical composition, which comprises a therapeutically effective dose of the compound, its stereoisomer or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable carriers, diluents or excipients; further, the compound, its stereoisomer or a pharmaceutically acceptable salt thereof, accounts for 0.1% to 95% by weight in the composition, preferably 0.5% to 85%, more preferably 1% to 60%, further preferably 10% to 50%, further preferably 15-40%, further preferably 20-30%, further preferably 20-25% (based on the total weight of the pharmaceutical composition).

[0036] The present invention further relates to the use of the compound, its stereoisomer or its pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of drugs for treating diseases mediated by p38 kinase.

[0037] The present invention further relates to the use of the compound, its stereoisomer or its pharmaceutically acceptable salt, or its pharmaceutical composition in the preparation of drugs for treating autoimmune diseases, metabolic diseases and tumors, etc., wherein the autoimmune diseases are selected from chronic inflammatory diseases, acute inflammatory diseases, autoinflammatory diseases and atherosclerosis, etc.; the metabolic diseases are selected from diabetes and fibrotic diseases, etc.; the tumors are selected from leukemia and lymphoma, etc. The present invention also relates to a method for treating, preventing and / or treating autoimmune diseases, metabolic diseases and tumors, etc., which comprises administering to a patient a therapeutically effective dose of the compound of the present invention, its stereoisomer or its pharmaceutically acceptable salt, or its pharmaceutical composition; wherein the autoimmune diseases are selected from chronic inflammatory diseases, acute inflammatory diseases, autoinflammatory diseases and atherosclerosis, etc.; the metabolic diseases are selected from diabetes and fibrotic diseases, etc.; the tumors are selected from leukemia and lymphoma, etc.

[0038] The present invention also provides methods of using the compounds or pharmaceutical compositions of the present invention to treat disease conditions, including but not limited to conditions associated with p38 kinase-mediated diseases.

[0039] Detailed description of the invention

[0040] Unless stated otherwise, the terms used in the specification and claims have the following meanings.

[0041] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 8 carbon atoms, further preferably an alkyl group containing 1 to 6 carbon atoms, and most preferably an alkyl group containing 1 to 3 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 4-heptyl, 1-propylbutyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2-Methyl-2-ethyl-pentyl, 2-methyl-3-ethyl-pentyl, n-nonyl, 2-methyl-2-ethyl-hexyl, 2-methyl-3-ethyl-hexyl, 2,2-diethyl-pentyl, n-decyl, 3,3-diethyl-hexyl, 2,2-diethyl-hexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, 4-heptyl, 1-propylbutyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like.The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate groups. Methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl are preferred in the present invention.

[0042] The term "alkylene" refers to a divalent alkyl group formed by further replacing one of the hydrogen atoms of an alkyl group, wherein alkyl is as defined above. For example, "methylene" refers to -CH2-, "ethylene" refers to -(CH2)2-, "propylene" refers to -(CH2)3-, "butylene" refers to -(CH2)4-, etc. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. The alkylene group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available attachment point. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo, carboxyl or carboxylate groups. Methyl, ethyl, isopropyl, tert-butyl, haloalkyl, deuterated alkyl, alkoxy-substituted alkyl and hydroxy-substituted alkyl are preferred in the present invention.

[0043] The term "alkenyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon double bond, which is a straight or branched group comprising 2 to 20 carbon atoms, preferably an alkenyl group containing 2 to 12 carbon atoms, more preferably an alkenyl group containing 2 to 8 carbon atoms, further preferably an alkenyl group containing 2 to 6 carbon atoms, and most preferably an alkenyl group containing 2 to 4 carbon atoms. For example, vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl or 3-butenyl, etc. Alkenyl can be substituted or unsubstituted, and when substituted, substituents are preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0044] The term "alkenylene" refers to a divalent alkenyl group formed by further replacing one hydrogen atom of an alkenyl group, wherein the alkenyl group is as defined above, for example, vinylene, propenylene, butenylene, etc. The alkenylene group may be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0045] The term "alkynyl" refers to an alkyl group as defined above consisting of at least two carbon atoms and at least one carbon-carbon triple bond, which is a straight or branched group containing 2 to 20 carbon atoms, preferably an alkynyl group containing 2 to 12 carbon atoms, more preferably an alkynyl group containing 2 to 8 carbon atoms, further preferably an alkynyl group containing 2 to 6 carbon atoms, and most preferably an alkynyl group containing 2 to 4 carbon atoms. For example, ethynyl, propynyl, 1-butynyl, 2-butynyl or 3-butynyl, etc. Alkynyl can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0046] The term "alkynylene" refers to a divalent alkynyl group formed by further replacing one of the hydrogen atoms of an alkynyl group, wherein the alkynyl group is as defined above. For example, ethynylene, propynylene, butynylene, etc. Alkynylidene can be substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups, which are independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0047] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, carboxyl or carboxylate.

[0048] "Haloalkyl" refers to an alkyl group substituted with one or more halogens, wherein alkyl is as defined above. Non-limiting examples of haloalkyl include: trifluoromethyl or -CH2CF3.

[0049] "Haloalkoxy" refers to an alkoxy group substituted with one or more halogens, wherein alkoxy is as defined above.

[0050] "Hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein alkyl is as defined above.

[0051] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0052] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0053] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are replaced independently of each other by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and the skilled person can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogens may be unstable when combined with carbon atoms with unsaturated (e.g. olefinic) bonds.

[0054] The hydrogen atoms described in the present invention can be replaced by their isotope deuterium, and any hydrogen atom in the example compounds of the present invention can also be replaced by a deuterium atom.

[0055] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0056] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention, which are safe and effective when used in mammals and have the desired biological activity. DETAILED DESCRIPTION

[0057] The present invention is further described below in conjunction with examples, but these examples are not intended to limit the scope of the present invention.

[0058] Example

[0059] The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or / and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shift (δ) is given in parts per million (ppm). The NMR is measured using a Bruker AVANCE-400 nuclear magnetic spectrometer, and the measurement solvent is deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD3OD), deuterated chloroform (CDCl3) or deuterated water (D2O), and the internal standard is tetramethylsilane (TMS).

[0060] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 Infinity Series mass spectrometer, and HPLC was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6 mm column).

[0061] Thin layer chromatography silica gel plates use Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates. The specifications used for TLC are 0.15mm-0.20mm, and the specifications used for thin layer chromatography separation and purification products are 0.4mm-0.5mm. Column chromatography generally uses Yantai Huanghai silica gel 200-300 mesh silica gel as the carrier.

[0062] The starting materials in the examples of the present invention are known and can be purchased on the market, or can be synthesized by or according to methods known in the art.

[0063] Unless otherwise specified, all reactions of the present invention are carried out under continuous magnetic stirring in a dry nitrogen or argon atmosphere, the solvent is a dry solvent, and the reaction temperature is in degrees Celsius.

[0064] The eluent system of silica gel column chromatography and the developing solvent system of thin layer chromatography used for the intermediates and purified compounds in the examples include: A: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: dichloromethane and acetone system. The volume ratio of the solvent is adjusted according to the polarity of the compound, and a small amount of alkaline or acidic reagents such as triethylamine and acetic acid can also be added for adjustment.

[0065] Unless otherwise specified, in the embodiments of the present invention, the ratios in the mobile phases in the HPLC chiral separation conditions and HPLC chiral analysis conditions are volume ratios.

[0066] The chiral separation conditions and chiral analysis methods used in the embodiments of the present invention are as follows:

[0067] Condition one:

[0068] HPLC chiral separation conditions:

[0069]

[0070] Method 1:

[0071] HPLC chiral analysis method:

[0072]

[0073] Condition two:

[0074] SFC chiral separation conditions:

[0075]

[0076]

[0077] Method 2:

[0078] SFC chiral analysis method:

[0079]

[0080] Condition three:

[0081] HPLC chiral separation conditions:

[0082]

[0083] Method 3:

[0084] HPLC chiral analysis method:

[0085]

[0086] Intermediate 1

[0087] 2,11-Dichloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-bipyrido

[0088] [1,2-a:3',4'-g]Azine-12-one

[0089]

[0090] Step 1: Under nitrogen protection, 1A (4 g, 27.3 mmol) was dissolved in anhydrous acetonitrile (60 mL), N-iodosuccinimide (7.37 g, 32.8 mmol) and p-toluenesulfonic acid (235 mg, 1.36 mmol) were added, and the reaction was heated to 80 ° C and stirred for 16 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (100 mL), and then washed with saturated sodium sulfite solution (80 mL), water (80 mL) and saturated sodium chloride solution (80 mL) in sequence, dried, concentrated, and the residue was purified by silica gel column chromatography (elution system A) to obtain 2-chloro-3-fluoro-5-iodo-pyridin-4-amine 1B (6.5 g), yield: 87.4%.

[0091] MS m / z(ESI):272.9[M+H] + ;

[0092] 1 H NMR (400MHz, DMSO-d6) δ8.10 (s, 1H), 6.69 (s, 2H).

[0093] Step 2: Under nitrogen protection, 1B (4 g, 14.7 mmol) was dissolved in 1,4-dioxane (60 mL) and water (12 mL), and potassium trifluoroborate (2.36 g, 17.6 mmol), 1,1-bis(diphenylphosphino)ferrocenepalladium dichloride (533 mg, 0.734 mmol) and sodium carbonate (3.89 g, 36.7 mmol) were added, and the reaction was heated to 80°C and stirred for 4 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (100 mL), washed with water (60 mL) and saturated sodium chloride solution (60 mL) in sequence, dried, filtered, concentrated, and the residue was purified by silica gel column chromatography (elution system B) to obtain 2-chloro-3-fluoro-5-vinyl-pyridine-4-amine 1C (2.1 g), with a yield of 82.9%.

[0094] MS m / z(ESI):173.0[M+H] + ;

[0095] 1 H NMR (400MHz, DMSO-d6) δ7.98(s,1H),6.82(dd,1H),6.62(s,2H),5.78(dd,1H),5.33(dd,1H).

[0096] Step 3: 1C (1 g, 5.79 mmol) was dissolved in 1,4-dioxane (15 mL), 1D (1.30 g, 5.79 mmol) was added, and the reaction was heated to 100°C and stirred for 2 hours. Concentrated sulfuric acid (1.42 g, 0.772 mL) was added to the reaction solution, and the reaction was continued to stir at 100°C for 0.5 hours. The reaction solution was cooled to room temperature, diluted with ethyl acetate (50 mL), washed with water (50 mL) and saturated sodium chloride solution (50 mL) in sequence, dried, concentrated, and the residue was purified by silica gel column chromatography (elution system B) to obtain 6-(but-3-ene-1-yl)-2'-chloro-3'-fluoro-4-hydroxy-5'-vinyl-2H-[1,4'-bipyridyl]-2-one 1E (1.1 g), yield: 59.2%.

[0097] MS m / z(ESI):321.0[M+H] + ;

[0098] 1 H NMR(400MHz,DMSO-d6)δ11.13(s,1H),8.80(s,1H),6.29–6.22(m,1H),6.08-6.00( m,2H),5.67-5.58(m,3H),4.95-4.89(m,2H),2.19-2.16(m,2H),2.13-2.09(m,2H).

[0099] Step 4: Dissolve 1E (100 mg, 0.312 mmol) in N,N-dimethylformamide (3 mL), add 2-(chloromethyl-d2)-3,5-difluoropyridine (77 mg, 0.468 mmol), 18-crown-6 (124 mg, 0.468 mmol) and potassium carbonate (129 mg, 0.935 mmol), and heat the reaction to 75 °C and stir for 2 hours. The reaction solution was cooled to room temperature and extracted with ethyl acetate (50 mL). The organic phase was washed with water (30 mL×2) and saturated sodium chloride solution (30 mL) in turn, dried and concentrated. The residue was purified by silica gel column chromatography (elution system B) to obtain 6-(but-3-en-1-yl)-2'-chloro-4-((3,5-difluoropyridin-2-yl)methoxy-d2)-3'-fluoro-5'-vinyl-2H-[1,4'-bipyridine]-2-one 1F (110 mg) with a yield of 78.4%.

[0100] MS m / z(ESI):450.1[M+H] + ;

[0101] Step 5: 1F (110 mg, 0.245 mmol) and Grubbs second generation catalyst (21 mg, 0.0245 mmol) were dissolved in 1,2-dichloroethane (80 mL), and the reaction was heated to 40°C and stirred for 1 hour under nitrogen protection. The reaction solution was cooled to room temperature and concentrated, and the residue was purified by silica gel column chromatography (elution system B) to obtain (Z)-2-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-7,8-dihydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one 1G (95 mg), yield: 92.1%.

[0102] MS m / z(ESI):422.1[M+H] + ;

[0103] 1 H NMR(400MHz,DMSO-d6)δ8.59(d,1H),8.32(s,1H),8.10-8.05(m,1H),6.43(d,1H),6.19(d,1 H),6.04(d,1H),6.03–5.98(m,1H),2.72-2.64(m,2H),2.59–2.52(m,1H),2.47-2.41(m,1H).

[0104] Step 6: 1G (95 mg, 0.225 mmol) and platinum dioxide (5 mg, 0.0225 mmol) were dissolved in tetrahydrofuran (3 mL), the reaction was replaced with hydrogen three times, and then stirred at room temperature for 2 hours under a hydrogen atmosphere. The reaction solution was filtered and concentrated, and the residue was purified by silica gel column chromatography (elution system B) to obtain 2-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one 1H (90 mg), yield: 94.3%.

[0105] MS m / z(ESI):424.1[M+H] + ;

[0106] Step 7: 1H (90 mg, 0.212 mmol) and N-chlorosuccinimide (34 mg, 0.255 mmol) were dissolved in 1,2-dichloroethane (9 mL) and isopropanol (3 mL), and the reaction was heated to 60°C for 2 hours. The reaction solution was cooled to room temperature, washed with water (20 mL) and saturated sodium chloride solution (20 mL), dried, and concentrated. The residue was purified by silica gel column chromatography (elution system B) to obtain 2,11-dichloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one intermediate 1 (90 mg), yield: 92.5%.

[0107] MS m / z(ESI):458.0[M+H] + ;

[0108] 1 H NMR(400MHz, CDCl3)δ8.39(d,1H),8.26(s,1H),7.35-7.30(m,1H),6.43(s,1H),2.93–2 .88(m,1H),2.80-2.75(m,1H),2.28-2.17(m,3H),2.05-1.95(m,1H),1.62-1.45(m,2H).

[0109] Intermediate 2

[0110] 2-Bromo-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-bipyrido

[0111] [1,2-a:3',4'-g]Azine-12-one

[0112]

[0113] Intermediate 2 was synthesized by referring to the synthesis method of intermediate 1.

[0114] MS m / z(ESI):502.0[M+H] + ;

[0115] 1H NMR(400MHz,DMSO-d6)δ8.60(d,1H),8.47(s,1H),8.12-8.07(m,1H),6.85(s,1H),2.99- 2.94(m,1H),2.84-2.78(m,1H),2.14-1.98(m,4H),1.58–1.49(m,1H),1.40-1.31(m,1H).

[0116] Example 1

[0117] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)pyrimidin-4-yl-6-d)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0118]

[0119] Step 1: Dissolve N,N-dimethylformamide dimethyl acetal (3g, 25.18mmol) in mesitylene (2mL) at room temperature, add deuterated methanol-d4 (3.63g, 100.7mmol) and stir for 16 hours. Distill the reaction solution at atmospheric pressure to remove most of the deuterated methanol. Add deuterated methanol-d4 (3.63g, 100.7mmol) to the reaction solution again, and stir the reaction at room temperature for 16 hours. Distill the reaction solution at atmospheric pressure to remove most of the deuterated methanol to obtain the crude product 1,1-dimethoxy-N,N-dimethylformamide-d 1a (3.6g, content about 20%), which is used directly in the next step without purification.

[0120] Step 2: Under nitrogen protection, intermediate 1 (90 mg, 0.196 mmol), tributyl (1-ethoxyethylene) tin (75 mg, 0.206 mmol) and bistriphenylphosphine palladium dichloride (14 mg, 0.019 mmol) were dissolved in 1,4-dioxane (2 mL), and the reaction was heated to 125 ° C by microwave and stirred for 2 hours. The reaction solution was cooled to room temperature, and dilute hydrochloric acid (0.2 M, 1 mL) was added and stirred for 1 hour. After the reaction solution was diluted with ethyl acetate (50 mL), the organic phase was washed with saturated sodium bicarbonate solution (30 mL) and saturated sodium chloride solution (30 mL) in sequence, dried and concentrated, and the residue was purified by silica gel column chromatography (elution system B) to obtain 2-acetyl-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 1b (90 mg) with a yield of 98.4%.

[0121] MS m / z(ESI):466.1[M+H] + ;

[0122] Step 3: 1b (50 mg, 0.107 mmol) was dissolved in the solution of 1a (1 mL), and the reaction was heated to 70°C and stirred for 2 hours. The reaction solution was cooled to room temperature and concentrated to obtain a crude product 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-2-(3-(dimethylamino)acryloyl-3-d)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 1c (55 mg), which was used directly in the next step without purification.

[0123] MS m / z(ESI):522.1[M+H] + ;

[0124] Step 4: 1c (50 mg, 95.80 μmol), 2-hydroxy-2-methylpropionamidine (49 mg, 0.479 mol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (73 mg, 0.479 mmol) were dissolved in a mixture of carbon tetrachloride (1 mL) and pyridine (1 mL), and the reaction was heated to 85 °C and stirred for 5 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed successively with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)pyrimidin-4-yl-6-d)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 1 (30 mg), yield: 55.8%.

[0125] MS m / z(ESI):561.2[M+H] + ;

[0126] 1 H NMR (400MHz, DMSO-d6) δ8.78(s,1H),8.60(d,1H),8.11-8.05(m,1H),8.03(s,1H),6.87(s,1H),5.10(s,1H),3. 09-3.04(m,1H),2.88-2.82(m,1H),2.21-2.10(m,3H),2.05-1.99(m,1H),1.62-1.53(m,7H),1.45-1.37(m,1H).

[0127] Example 1 Resolution of (S)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)pyrimidine-4-yl)-

[0128] 6-d)-5,6,7,8-tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one and (R)-11-chloro-10-

[0129] ((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl-6-d)-

[0130] 5,6,7,8-Tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one

[0131]

[0132] Example 1 (30 mg, 0.053 mmol) was subjected to chiral separation (IB column) to give 1-1 (14.0 mg, RT = 4.930 min), yield: 46.2%; 1-2 (13.8 mg, RT = 8.327 min), yield: 45.5%.

[0133] Example 1-1: MS m / z (ESI): 561.2 [M+H] + ;

[0134] Example 1-2: MS m / z (ESI): 561.2 [M+H] + ;

[0135] HPLC chiral separation conditions are the same as condition one; chiral separation method is the same as method one.

[0136] Example 2

[0137] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-methylpyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0138]

[0139] Step 1: Under nitrogen protection, 1b (50 mg, 0.107 mmol) was dissolved in N,N-dimethylformamide (1 mL), 1,1-dimethoxy-N,N-dimethylethylamine (143 mg, 1.07 mmol) was added, and the reaction was heated to 80°C and stirred for 16 hours. The reaction solution was cooled to room temperature and concentrated to obtain 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-2-(3-(dimethylamino)but-2-enoyl)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 2a (55 mg), which was directly used in the next step.

[0140] MS m / z(ESI):535.1[M+H] + ;

[0141] Step 2: Under nitrogen protection, 2a (55 mg, 0.102 mmol), 2-hydroxy-2-methylpropionamidine (32 mg, 0.308 mmol) and potassium carbonate (71 mg, 0.514 mmol) were dissolved in N,N-dimethylacetamide (1 mL), and the reaction was heated to 85 ° C and stirred for 6 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (30 mL × 2). The organic phases were combined, washed successively with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-methylpyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one (3.9 mg), yield: 6.6%.

[0142] MS m / z(ESI):574.2[M+H] + ;

[0143] 1 H NMR(400MHz,DMSO-d6)δ8.77(s,1H),8.60(d,1H),8.12-8.06(m,1H),7.91(s,1H),6.87(s,1H),5.04(s,1H), 3.09-3.04(m,1H),2.88-2.82(m,1H),2.61(s,3H),2.21–2.12(m,3H),2.04-1.98(m,1H),1.58-1.40(m,8H).

[0144] Decomposition of Example 2

[0145] (S)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-methylpyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocin-12-one and (R)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-methylpyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocin-12-one

[0146]

[0147] Example 2 (15 mg, 0.026 mmol) was subjected to chiral separation (IB column) to give 2-1 (6.8 mg, RT = 4.567 min), yield: 45.3%; 2-2 (6.4 mg, RT = 8.477 min), yield: 42.7%.

[0148] Example 2-1: MS m / z (ESI): 574.2 [M+H] + ;

[0149] Example 2-2: MS m / z (ESI): 574.2 [M+H] + ;

[0150] HPLC chiral separation conditions are the same as condition one; chiral separation method is the same as method one.

[0151] Example 3

[0152] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-carbonyl-1,6-dihydropyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0153]

[0154] Step 1: Dissolve intermediate 2 (0.55 g, 1.09 mmol), [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium (0.1 g, 20% wt) and triethylamine (332 mg, 3.28 mmol) in methanol (5 mL), replace with carbon monoxide gas 3 times under stirring, maintain the pressure at 0.3 MPa, heat the reaction to 100 ° C and stir for 16 hours. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated to obtain a crude product. The crude product was dissolved in tetrahydrofuran (5 mL) at room temperature, and a solution of lithium hydroxide (0.15 g, 3.28 mmol) in water (2.5 mL) was added dropwise under stirring, and the reaction was stirred at room temperature for 1 hour. Hydrochloric acid (1N) was added dropwise to the reaction solution to adjust the pH to 3-5. The aqueous phase was extracted with ethyl acetate (10 mL), the organic phase was dried and concentrated, and the residue was purified by silica gel column chromatography (elution system A) to give 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydro-5H-dipyrido[1,2-a:3',4'-g]azocine-2-carboxylic acid 3a (0.4 g), yield: 78.2%.

[0155] MS m / z(ESI):466.1[MH] - ;

[0156] Step 2: Dissolve 3a (170 mg, 0.363 mmol) and N,N'-carbonyldiimidazole (88 mg, 0.545 mmol) in acetonitrile (2 mL) at room temperature and stir for 2 hours to obtain solution A. Dissolve ethyl malonate potassium salt (136 mg, 0.799 mmol), magnesium chloride (87 mg, 0.908 mmol) and triethylamine (110 mg, 1.09 mmol) in acetonitrile (3 mL) at room temperature and stir for 2 hours to obtain solution B. Add solution A dropwise to solution B under stirring, and then heat the reaction to 80 ° C and stir for 6 hours. The reaction solution is cooled to room temperature, poured into water (50 mL), and the pH is adjusted to 6 with dilute hydrochloric acid. The aqueous phase was extracted with ethyl acetate (30 mL×2), and the organic phases were combined, washed with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated to give 3-(11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydro-5H-dipyrido[1,2-a:3',4'-g]azocine-2-yl)-3-carbonylpropionic acid ethyl ester 3b (110 mg), which was used in the next step without purification.

[0157] MS m / z(ESI):538.1[M+H] + ;

[0158] Step 3: 3b (110 mg, 0.204 mmol), 2-hydroxy-2-methylpropionamidine (63 mg, 0.613 mmol) and 1,8-diazobispiro[5.4.0]undec-7-ene (124 mg, 0.818 mmol) were dissolved in trifluoroethanol (3 mL), heated to 90 °C and stirred for 6 hours. The reaction solution was cooled to room temperature, poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (30 mL×2). The organic phases were combined, washed successively with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-carbonyl-1,6-dihydropyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 3 (55 mg), yield: 46.7%.

[0159] MS m / z(ESI):576.2[M+H] + ;

[0160] 1 H NMR(400MHz, CDCl3)δ8.57(s,1H),8.39(d,1H),7.35-7.26(m,1H),7.05(s,1H),6.45(s,1H),3 .00-2.95(m,1H),2.82-2.76(m,1H),2.31-2.20(m,3H),2.10–2.01(m,1H),1.64-1.46(m,8H).

[0161] Example 3 Resolution of (S)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-carbonyl-1,6-dihydropyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocin-12-one and (R)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)-6-carbonyl-1,6-dihydropyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocin-12-one

[0162]

[0163] Example 3 (54 mg, 0.053 mmol) was subjected to SFC chiral separation (AS column) to give 3-1 (14.0 mg, RT = 4.930 min), yield: 46.2%; 3-2 (13.8 mg, RT = 8.327 min), yield: 45.5%.

[0164] Example 3-1: MS m / z (ESI): 576.2 [M+H] + ;

[0165] Example 3-2: MS m / z (ESI): 576.2 [M+H] + ;

[0166] The SFC chiral separation conditions are the same as condition 2; the chiral separation method is the same as method 2.

[0167] Example 4

[0168] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0169]

[0170] Step 1: Under nitrogen protection, methylmagnesium bromide (3M, 1.80 mL) was added dropwise to a solution of methyl 6-chloro-3-fluoropyridine-2-carboxylate 4a (410 mg, 2.16 mmol) in tetrahydrofuran (8 mL) at -78°C and stirred for 2 hours. The reaction temperature was raised to 0°C, and a saturated ammonium chloride solution was added dropwise to the reaction solution. The aqueous phase was extracted with ethyl acetate (20 mL×2), and the organic phases were combined, dried, and concentrated. The residue was separated by silica gel column chromatography (elution system B) to obtain 2-(6-chloro-3-fluoropyridin-2-yl)propan-2-ol 4b (344 mg), with a yield of 83.88%.

[0171] MS m / z(ESI):190.1[M+H] + ;

[0172] Step 2: Under nitrogen protection, 4b (185 mg, 0.98 mmol), hexamethyltin (416 mg, 1.27 mmol) and [1,1'-bis(di-tert-butylphosphino)ferrocene] palladium dichloride (63.00 mg, 0.098 mmol) were dissolved in toluene (7.00 mL), and the reaction was heated to 110°C and stirred for 3 hours. The reaction solution was filtered and the filtrate was concentrated to obtain the crude product 2-(3-fluoro-6-(trimethylstannyl)pyridin-2-yl)propan-2-ol 4c (310 mg), which was used directly in the next step without further purification.

[0173] MS m / z(ESI):320.1[M+H] + ;

[0174] Step 3: Under nitrogen protection, intermediate 2 (85 mg, 0.17 mmol), 4c (97 mg, 0.3 mmol), bistriphenylphosphine palladium dichloride (12 mg, 0.017 mmol) and cuprous iodide (32 mg, 0.17 mmol) were dissolved in 1,4-dioxane (3 mL), and the reaction was heated to 115 °C and stirred for 16 hours. The reaction solution was filtered, the filtrate was concentrated, and the residue was separated by silica gel column chromatography (elution system A) to give 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 4 (105 mg), yield: 73.19%.

[0175] MS m / z(ESI):577.2[M+H] + ;

[0176] Example 4 Resolution of (S)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one and (R)-11-chloro-10-

[0177] ((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-

[0178] 5,6,7,8-Tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one

[0179]

[0180] Example 4 (105 mg, 0.18 mmol) was subjected to chiral separation (OJ column) to give the title product 4-1 (27.5 mg, RT = 4.093 min), yield: 26.19%; 4-2 (24.9 mg, RT = 5.250 min), yield: 23.71%.

[0181] Example 4-1, MS m / z (ESI): 577.2 [M+H] + ;

[0182] 1 H NMR(400MHz,DMSO-d6)δ8.71(s,1H),8.61(d,1H),8.20-8.05(m,2H),7.94-7.83(m,1H),6.86(s,1H),5.3 7(s,1H),3.09-2.97(m,1H),2.91-2.78(m,1H),2.25-1.93(m,4H),1.65-1.47(m,7H),1.47-1.36(m,1H).

[0183] Example 4-2, MS m / z (ESI): 577.2 [M+H] + ;

[0184] 1 H NMR(400MHz,DMSO-d6)δ8.71(s,1H),8.61(d,1H),8.20-8.05(m,2H),7.94-7.83(m,1H),6.86(s,1H),5.3 7(s,1H),3.09-2.97(m,1H),2.91-2.78(m,1H),2.25-1.93(m,4H),1.65-1.47(m,7H),1.47-1.36(m,1H).

[0185] HPLC chiral separation conditions are the same as condition three; chiral separation method is the same as method three.

[0186] Example 5

[0187] 11-Chloro-2-(5-chloro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0188]

[0189] Referring to the synthesis method of Example 4, the target product 11-chloro-2-(5-chloro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 5 was synthesized.

[0190] MS m / z(ESI):593.2[M+H] + ;

[0191] Example 5 Resolution of (S)-11-chloro-2-(5-chloro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one and (R)-11-chloro-2-(5-chloro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-

[0192] 5,6,7,8-Tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one

[0193]

[0194] Example 5 (75 mg, 0.202 mmol) was subjected to chiral separation (IB column) to give 5-1 (47.8 mg, RT = 5.010 min), yield: 39.83%; 5-2 (48 mg, RT = 6.903 min), yield: 40.00%.

[0195] Example 5-1, MS m / z (ESI): 593.2 [M+H] + ;

[0196] 1 H NMR(400MHz,DMSO-d6)δ8.73(s,1H),8.61(d,1H),8.17-8.04(m,3H),6.87(s,1H),5.63(s,1H), 3.09-2.99(m,1H),2.90-2.79(m,1H),2.24-1.93(m,4H),1.65-1.49(m,7H),1.48-1.37(m,1H).

[0197] Example 5-2, MS m / z (ESI): 593.2 [M+H] + ;

[0198] 1 H NMR(400MHz,DMSO-d6)δ8.73(s,1H),8.61(d,1H),8.17-8.04(m,3H),6.87(s,1H),5.63(s,1H), 3.09-2.99(m,1H),2.90-2.79(m,1H),2.24-1.93(m,4H),1.65-1.49(m,7H),1.48-1.37(m,1H).

[0199] The HPLC chiral separation conditions were the same as condition 1, wherein the mobile phase was Hexane:EtOH=70:30; the chiral separation method was the same as method 1, wherein the mobile phase was Hexane:EtOH=70:30.

[0200] Example 6

[0201] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(3-(2-hydroxypropane-2-yl)-1,2,4-triazine

[0202] -5-yl)-5,6,7,8-tetrahydro-12H-bipyrido[1,2-a:3',4'-g]azocine-12-one

[0203]

[0204] Referring to the synthesis method of Example 1, the target product 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(3-(2-hydroxypropane-2-yl)-1,2,4-triazine-5-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azine-12-one 6 was synthesized.

[0205] MS m / z(ESI):561.2[M+H] + .

[0206] 1 H NMR(400MHz,DMSO-d6)δ9.94(s,1H),8.87(s,1H),8.61(d,1H),8.14-8.05(m,1H),6.88(s,1 H),5.49(s,1H),3.15-3.04(m,1H),2.90-2.78(m,1H),2.22-1.93(m,4H),1.67-1.33(m,8H).

[0207] Example 7

[0208] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(3-hydroxy-3-methylcyclobutyl)pyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one

[0209]

[0210] Referring to the synthesis method of Example 1, the target product 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(3-hydroxy-3-methylcyclobutyl)pyrimidin-4-yl)-5,6,7,8-tetrahydro-12H-dipyrido[1,2-a:3',4'-g]azocine-12-one 7 was synthesized.

[0211] MS m / z(ESI):586.2[M+H] + .

[0212] Example 8

[0213] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazin-12(6H)-one

[0214]

[0215] Step 1: Under nitrogen protection, 2-bromo-3-fluoro-5-iodopyridin-4-amine 8a (4.9 g, 15.5 mmol), biboronic acid pinacol ester (7.85 g, 30.9 mmol), 1,1-bis(diphenylphosphine)diferronichloridopalladium (1.13 g, 1.55 mmol) and potassium acetate (3.03 g, 30.9 mmol) were dissolved in a mixture of 1,4-dioxane (60 mL) and N,N-dimethylformamide (20 mL), heated to 100 ° C and stirred for 4 hours. The reaction solution was cooled to room temperature, poured into water (100 mL), adjusted to neutral pH with dilute hydrochloric acid, and the aqueous phase was extracted with dichloromethane (50 mL × 2). The organic phases were combined, washed with water (50 mL) and saturated sodium chloride solution (50 mL), dried, concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give (4-amino-6-bromo-5-fluoropyridin-3-yl)boronic acid 8b (1.3 g) with a yield of 35.8%.

[0216] MS m / z(ESI):234.9[M+H] + ;

[0217] Step 2: Under nitrogen protection, 8b (1.3 g, 5.54 mmol) was dissolved in tetrahydrofuran (30 mL) and cooled to 0°C. 30% hydrogen peroxide solution (4.39 g, 38.7 mmol) was added dropwise to the reaction solution under stirring, and the reaction temperature was raised to room temperature and stirred for 1 hour. The reaction solution was diluted with ethyl acetate (50 mL), and the organic phase was washed with saturated sodium sulfite solution (50 mL), water (50 mL) and saturated sodium chloride solution (50 mL) in sequence, dried, concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to obtain 4-amino-6-bromo-5-fluoropyridin-3-ol 8c (1.0 g), with a yield of 87.3%.

[0218] MS m / z(ESI):206.9[M+H] + ;

[0219] Step 3: 8c (2.0 g, 9.66 mmol), 3-bromopropylene (1.75 g, 14.5 mmol) and potassium carbonate (4.01 g, 29.0 mmol) were dissolved in N,N-dimethylformamide (20 mL), and the reaction was stirred at room temperature for 2 hours. The reaction solution was poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with water (50 mL × 2) and saturated sodium chloride solution (50 mL) in sequence, dried, concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to obtain 5-(allyloxy)-2-bromo-3-fluoropyridin-4-amine 8d (1.9 g), with a yield of 79.6%.

[0220] MS m / z(ESI):247.0[M+H] + ;

[0221] 1 H NMR (400MHz, DMSO-d6) δ7.64(s,1H),6.22(s,2H),6.09-5.99(m,1H),5.49-5.43(m,1H),5.29-5.25(m,1H),4.65-4.63(m,2H).

[0222] Step 4: Under nitrogen protection, 8d (1.9 g, 7.69 mmol) and 2,2-dimethyl-6-(2-oxopent-4-enyl)-1,3-dioxane-4-one (2.6 g, 12.4 mmol) were dissolved in 1'4-dioxane (20 mL), heated to 115 ° C and stirred for 3 hours. The reaction was cooled to 90 ° C, concentrated sulfuric acid (1.89 g, 19.2 mmol) was added, and stirring was continued for 30 minutes. The reaction solution was cooled to room temperature, poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with water (50 mL) and saturated sodium chloride solution (50 mL) in sequence, dried, concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to obtain 5'-(allyloxy)-2'-bromo-3'-fluoro-4-hydroxy-6-(prop-1-en-1-yl)-2H-[1,4'-bipyridine]-2-one 8e (600 mg), yield: 20.5%

[0223] MS m / z(ESI):381.0[M+H] + ;

[0224] Step 5: Under nitrogen protection, 8e (600 mg, 1.88 mmol), 18-crown-6 (747 mg, 2.83 mmol) and potassium carbonate (781 mg, 5.65 mmol) were dissolved in N,N-dimethylformamide (10 mL) and heated to 75 ° C. 2-(chloromethyl-d2)-3,5-difluoropyridine (624 mg, 3.77 mmol) was added dropwise to the reaction solution under stirring, and the reaction was continued to stir for 2 hours. The reaction solution was cooled to room temperature and poured into water (50 mL), and the aqueous phase was extracted with ethyl acetate (50 mL × 2). The organic phases were combined, washed with water (50 mL×2) and saturated sodium chloride solution (50 mL) in sequence, dried, concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to obtain 5'-(allyloxy)-2'-bromo-4-((3,5-difluoropyridin-2-yl)methoxy-d2)-3'-fluoro-6-(prop-1-en-1-yl)-2H-[1,4'-bipyridine]-2-one 8f (580 mg), yield: 78.8%.

[0225] MS m / z(ESI):510.0[M+H] + ;

[0226] 1H NMR(400MHz, CDCl3)δ8.41(d,1H),8.06(s,1H),7.32-7.26(m,1H),6.30–6,22(m,1H),6.17(d,1H) ,6.06(d,1H),5.93-5.83(m,1H),5.58(d,1H),5.33-5.26(m,2H),4.70-4.60(m,2H),1.73(dd,3H).

[0227] Step 6: Under nitrogen protection, 8f (580 mg, 1.14 mmol) was dissolved in 1,2-dichloroethane (100 mL) and heated to 45°C. A solution of Grubbs second-generation catalyst (194 mg, 0.228 mmol) in 1,2-dichloroethane (25 mL) was added dropwise to the reaction solution under stirring. After the addition was completed, the reaction was stirred at 45°C for 2 hours. The reaction solution was cooled to room temperature and concentrated. The residue was purified by reverse phase chromatography (formic acid system) to obtain a crude product of 2-bromo-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluorobipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one 8g (440 mg), which was directly used in the next step.

[0228] MS m / z(ESI):468.0[M+H] + ;

[0229] Step 7: Under nitrogen protection, 8g (360mg, 0.769mmol), toluenesulfonyl hydrazide (1.15g, 6.15mmol) and sodium acetate (631mg, 7.69mmol) were dissolved in a mixture of tetrahydrofuran (5mL) and water (5mL), and the reaction was heated to 80°C and stirred for 4 hours. The reaction solution was cooled to room temperature, poured into water (50mL), and the aqueous phase was extracted with ethyl acetate (30mL×2). The organic phases were combined, washed successively with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give 2-bromo-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one 8h (180 mg), yield: 49.8%.

[0230] MS m / z(ESI):470.0[M+H] + ;

[0231] 1H NMR(400MHz,DMSO-d6)δ8.59(d,1H),8.45(s,1H),8.10-8.05(m,1H),6.21(d,1H),6.06(d,1H),4.62-4.5 6(m,1H),3.95-3.88(m,1H),2.86-2.82(m,1H),2.29-2.23(m,1H),2.08-2.03(m,1H),1.93-1.88(m,1H).

[0232] Step 8: Under nitrogen protection, 8h (200 mg, 0.425 mmol) was dissolved in a mixture of 1,2-dichloroethane (15 mL) and isopropanol (5 mL) and heated to 60°C. N-chlorosuccinimide (68 mg, 0.510 mmol) was added to the reaction solution under stirring and continued to stir for 3 hours. The reaction solution was cooled to room temperature and poured into water (50 mL). The aqueous phase was extracted with dichloromethane (30 mL × 2). The organic phases were combined, washed successively with water (30 mL) and saturated sodium chloride solution (30 mL), dried and concentrated, and the residue was purified by reverse phase chromatography (formic acid system) to give 2-bromo-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one 8i (190 mg), yield: 88.5%.

[0233] MS m / z(ESI):503.9[M+H] + ;

[0234] Step 9: Referring to the third step of Example 4, the target product 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one 8 was synthesized.

[0235] MS m / z(ESI):579.1[M+H] + .

[0236] Example 8 Resolution of (S)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one and (R)-11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(5-fluoro-6-(2-hydroxypropane-2-yl)pyridin-2-yl)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one

[0237] Dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazin-12(6H)-one

[0238]

[0239] Example 8 (40 mg, 0.069 mmol) was subjected to chiral separation (IB column) to give 8-1 (15.8 mg, RT = 4.807 min), yield: 39.1%; 8-2 (15.4 mg, RT = 7.090 min), yield: 37.8%.

[0240] Example 8-1: MS m / z (ESI): 579.1 [M+H] + ;

[0241] 1 H NMR(400MHz,DMSO-d6)δ8.70(s,1H),8.61(d,1H),8.12–8.07(d,2H),7.88(dd,1H),6.90(s,1H),5.38(s,1H),4.65-4. 58(m,1H),4.04-3.99(m,1H),2.99–2.94(m,1H),2.43-2.37(m,1H),2.19–2.10(m,1H),2.01-1.95(m,1H),1.54(s,6H).

[0242] Example 8-2: MS m / z (ESI): 579.1 [M+H] + ;

[0243] 1H NMR (400MHz, DMSO-d6) δ8.70(s,1H),8.61(d,1H),8.12-8.07(m,2H),7.88(dd,1H),6.90(s,1H),5.38(s,1H),4.64-4. 60(m,1H),4.04-3.99(m,1H),2.99-2.94(m,1H),2.43-2.37(m,1H),2.18-2.12(m,1H),2.02-1.94(m,1H),1.54(s,6H).

[0244] HPLC chiral separation conditions are the same as condition one; chiral separation method is the same as method one.

[0245] Example 9

[0246] 11-Chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl-6-d)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazin-12(6H)-one

[0247]

[0248] Referring to the synthesis methods of Examples 1 and 8, the target product 11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-2-(2-(2-hydroxypropane-2-yl)pyrimidin-4-yl-6-d)-7,8-dihydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-12(6H)-one 9 was synthesized.

[0249] MS m / z(ESI):563.1[M+H] + ;

[0250] 1 H NMR(400MHz,DMSO-d6)δ8.75(s,1H),8.60(d,1H),8.12-8.07(m,1H),7.99(d,1H),6.91(s,1H),5.08(s,1H),4.63-4.5 9(m,1H),4.11-4.04(m,1H),2.99-2.94(m,1H),2.49-2.41(m,1H),2.19-2.16(m,1H),1.99-1.96(m,1H),1.53(s,6H).

[0251] Example 10

[0252] N-(2-(1-(11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydrodipyrido[3,4-b:1',2'-d][1,4]oxazin-2-yl)-4-fluoro-1H-pyrazol-3-yl)propan-2-yl)acetamide

[0253]

[0254] Step 1: Under nitrogen protection, intermediate 8i (146 mg, 0.29 mmol), N-(2-(4-fluoro-1H-pyrazol-3-yl)propan-2-yl)acetamide (87 mg, 0.47 mmol), cuprous iodide (56 mg, 0.29 mmol), N1,N2-dimethylcyclohexane-1,2-diamine (42 mg, 0.29 mmol) and cesium carbonate (191 mg, 0.59 mmol) were dissolved in 1,4-dioxane (2 mL), and the reaction was heated to 100 °C and stirred for 16 hours. Saturated sodium chloride solution was added to the reaction solution, and the mixture was extracted with ethyl acetate (25 mL×2). The organic phases were combined, dried, and concentrated. The residue was purified by reverse phase HPLC to give N-(2-(1-(11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydrodipyrido[3,4-b:1',2'-d][1,4]oxazocine-2-yl)-4-fluoro-1H-pyrazol-3-yl)propan-2-yl)acetamide 10 (22 mg) with a yield of 12.32%.

[0255] MS m / z(ESI):609.2[M+H] + ;

[0256] Example 10 Resolution of (S)-N-(2-(1-(11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydrobipyrido[3,4-b:1',2'-d][1,4]oxazocin-2-yl)-4-fluoro-1H-pyrazol-3-yl)propane-2-yl )acetamide and (R)-N-(2-(1-(11-chloro-10-((3,5-difluoropyridin-2-yl)methoxy-d2)-1-fluoro-12-carbonyl-6,7,8,12-tetrahydrobipyrido[3,4-b:1',2'-d][1,4]oxazin-2-yl)-4-fluoro-1H-pyrazol-3-yl)propan-2-yl)acetamide

[0257]

[0258] Example 10 (22 mg, 0.036 mmol) was subjected to chiral separation (IB column) to give the title product 10-1 (10.2

[0259] mg, RT = 4.310 min), yield: 46.36%; 10-2 (7.3 mg, RT = 6.120 min), yield: 33.18%.

[0260] Example 10-1, MS m / z (ESI): 609.2 [M+H] + ;

[0261] 1 H NMR(400MHz,DMSO-d6)δ8.60(d,1H),8.51(s,1H),8.39(d,1H),8.15-8.03(m,2H),6.89(s,1H),4.69-4.59(m,1H), 3.94(t,1H),2.99-2.88(m,1H),2.46-2.36(m,1H),2.18-2.06(m,1H),2.04-1.91(m,1H),1.79(s,3H),1.57(s,6H).

[0262] Example 10-2, MS m / z (ESI): 609.2 [M+H] + ;

[0263] 1 H NMR(400MHz,DMSO-d6)δ8.60(d,1H),8.51(s,1H),8.39(d,1H),8.15-8.03(m,2H),6.89(s,1H),4.69-4.59(m,1H), 3.94(t,1H),2.99-2.88(m,1H),2.46-2.36(m,1H),2.18-2.06(m,1H),2.04-1.91(m,1H),1.79(s,3H),1.57(s,6H).

[0264] HPLC chiral separation conditions are the same as condition one; chiral separation method is the same as method one.

[0265] Example Test Evaluation

[0266] The present invention is further described and explained below in conjunction with test examples, but these embodiments are not intended to limit the scope of the present invention.

[0267] Experimental instruments and reagents:

[0268] 1. Instrument

[0269] EnVision (PerkinElmer, ENVISION 2015 or 2105-0020); mass spectrometer (SCIEX QTRAP 5500, AB SCIEX); liquid phase (LC-30AD, Shimadzu); high performance liquid chromatograph (Vanquish, Thermo Scientific); vortexer (VG3S25, IKA); medical refrigerator (HYC-940, Qingdao Haier Co., Ltd.); centrifuge (460R, Hettich); electronic analytical balance (SQP, Sartorius); microplate fast oscillator (QB-9001, Haimen Qilin Bell Instrument Co., Ltd.); constant temperature micromixer (Confide-S202H, FINEPCR).

[0270] 2. Reagents

[0271] Active p38 alpha Protein (R&D systems, 5477-KS-010); Inactive MK2 (Thermo, PV3316); ULight-CREBtide (Perkinelmer, TRF0107); ATP Solution (Invitrogen, PV3227); DMSO (Sigma, D2650); HEPES (1M) (Gibco, 15630-080); MgCl2 (1M) (Invitrogen, AM9530G); Tween 20 (Sigma, P9416); EGTA (Alfa Aesar, J60767); DTT (1M) (Sigma, 43816); Europium-anti-phospho-CREB (PerkinElmer, TRF0200); EDTA (0.5M) (Invitrogen, AM9260G); 10X LANCE detection buffer (PerkinElmer, CR97-100); 384-well plate (Perkinelmer, 6007299); 96V bottom plate (PS) (Axygen, WIPP02280); U937 (ATCC, CRL-1593.2); Phospho-HSP27 (S78 / S82) DuoSet IC ELISA (R&D systems, DYC2314-5); DuoSet ELISA Ancillary Reagent Kit 2 (R&D systems, DY008); Phorbol 12-myristate 13-acetate (PMA) (Abcam, Ab120297); Lipopolysaccharides from Escherichia coli O111:B4 (Sigma-Aldrich, L4391); Human TNF-alpha DuoSet ELISA (R&D systems, DY210); DMSO (Sigma, 34869); PBS (Gibco, 10010-023); human liver S9 (XENOTECH, H0610); NADPH (Biodex, BD11658); acetonitrile (Fisher, L-33055); formic acid (TIC, F0654); isotonic percoll (Cytiva); HEPES buffer (Gibco); cell buffer (Gibco); fetal bovine serum (Sigma); human insulin (Gibco); dexamethasone (Sigma); Williams'E Medium (Gibco); L-15 Medium (Gibco); GlutaMAX (100x) (Gibco); human hepatocytes (BioIVT). .

[0272] Test Example 1: Determination of the inhibitory effect of the compounds of the present invention on p38α / MK2 enzyme activity

[0273] 1. Experimental purpose: The purpose of this test case is to measure the inhibitory ability of the compound on p38α / MK2 enzyme activity.

[0274] 2. Experimental methods:

[0275] p38 / MK2 enzyme activity assay method: In 1× enzyme reaction buffer (50mM HEPES pH 7.5, 1mM EGTA, 10mMMgCl2, 2mM DTT, 0.01% Tween-20), incubate 0.06nM p38α (Active p38 alpha Protein), 1.2nM MK2 (Inactive MK2) and gradient dilutions of compounds for 1 hour. Add 50nM ULight-CREBtide and 25μM ATP (ATP Solution) and react at room temperature for 1.5 hours. Add an equal volume of detection and termination mixture (1×LANCE detection buffer containing 20mMEDTA and 2nM Europium-anti-phospho-CREB), mix and centrifuge, apply sealing film, and react at room temperature for 1 hour. Read the plate using the TR-FRET program of the EnVision instrument and detect the fluorescence value of each well at 665nm / 615nm.

[0276] p38 enzyme activity assay method: Incubate 3nM p38α (Active p38 alpha Protein) and gradient dilutions of compounds for 1 hour in 1× enzyme reaction buffer (50mM HEPES pH 7.5, 1mM EGTA, 10mMMgCl2, 2mM DTT, 0.01% Tween-20). Add 50nM ULight-MBP and 50μM ATP (ATP 10mM Solution) and react at room temperature for 1.5 hours. Add an equal volume of detection and termination mixture (1×LANCE detection buffer containing 20mM EDTA and 2nM Europium-anti-P-MBP), mix and centrifuge, apply sealing film, and react at room temperature for 1 hour. Read the plate using the TR-FRET program of the EnVision instrument and detect the fluorescence value of each well at 665nm / 615nm.

[0277] 3. Experimental data processing method:

[0278] The raw data (665nm / 615nm reading ratio) was calculated according to the following formula to obtain the inhibition rate: Inhibition rate % = [(average value of positive control wells - sample wells) / (average value of positive control wells - average value of negative control wells)] × 100, where the positive control wells are reaction wells without compound enzymes, and the negative control wells are reaction wells without enzymes.

[0279] Use log(inhibitor) vs.response--Variable slope(four parameters) in GraphPad Prism 6 to fit the compound concentration and the corresponding inhibition rate, fit the curve and obtain the compound IC 50 value.

[0280] The fitting calculation equation is Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope))

[0281] 4. Experimental results:

[0282] Example <![CDATA[p38 / MK2 IC 50 (nM)]]> <![CDATA[p38 IC 50 (nM)]]> p38 / p38 / MK2 1-1 0.10 16.1 161 4-2 0.16 19.5 122 8-1 0.09 19.1 212 10-1 0.04 10.1 253

[0283] 5. Experimental conclusion:

[0284] The above data show that the compounds of the present invention have strong inhibitory ability on p38α / MK2 enzyme activity and good selectivity.

[0285] Test Example 2: Determination of the inhibitory effect of the compounds of the present invention on p-Hsp27 in U937 cells

[0286] 1. Experimental purpose: The purpose of this test example is to test the inhibitory ability of the compounds of the present invention on p-Hsp27 of U937 cells.

[0287] 2. Experimental methods:

[0288] Treat U937 with 20ng / ml PMA for differentiation overnight, change to fresh culture medium, and let U937 rest overnight. After differentiation, digest and count U937 cells, and seed 25,000 cells per well in a 96-well plate until the cells adhere to the wall. Prepare compound concentration gradient, pre-treat cells with compounds for 1 hour (the highest final concentration of the compound is 1μM, 3-fold concentration gradient dilution), and add 10ng / ml LPS (Lipopolysaccharides from Escherichia coli O111:B4) to stimulate for 0.5 hours. After washing the cells with PBS, the cells were lysed with lysis buffer. Use Phospho-HSP27 (S78 / S82) DuoSet IC ELISA kit to detect p-Hsp27 in cell lysate. For specific steps, refer to the standard steps provided by the kit manufacturer.

[0289] 3. Experimental data processing method:

[0290] Calculate the p-Hsp27 inhibition rate: inhibition rate percentage = [(average p-Hsp27 in positive control wells - average p-Hsp27 in sample wells) / (average p-Hsp27 in positive control wells - average p-Hsp27 in negative control wells)] × 100%, where the positive control wells are wells without compound (DMSO-treated wells) and the negative control wells are wells without LPS stimulation.

[0291] Dose response curve fitting: Use log(inhibitor) vs.response--Variable slope(four parameters) in GraphPad Prism 6 to fit the compound concentration and the corresponding inhibition rate, fit the curve and obtain the compound IC 50 value.

[0292] The fitting calculation equation is Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope))

[0293] 4. Experimental results:

[0294]

[0295] 5. Experimental conclusion:

[0296] The compounds of the present invention have strong inhibitory ability on p-Hsp27 of U937 cells.

[0297] Test Example 3: Determination of the inhibitory effect of the compounds of the present invention on the expression level of TNF-α in U937 cells

[0298] 1. Experimental purpose: The purpose of this test example is to measure the inhibitory ability of the compounds of the present invention on the expression level of TNF-α in U937 cells.

[0299] 2. Experimental methods:

[0300] U937 cells were treated with 20ng / ml PMA for differentiation overnight, and then fresh culture medium was replaced. U937 cells were rested overnight. U937 cells were digested and counted after differentiation, and 50,000 cells were seeded in 96-well plates per well until the cells adhered to the wall. Compound concentration gradients were prepared, and cells were pretreated with compounds for 1 hour (the highest final concentration of the compound was 1μM, and the concentration gradient was diluted 3 times). After adding 10ng / ml LPS (Lipopolysaccharides from Escherichia coli O111:B4) for stimulation for 4 hours, the culture supernatant was collected by centrifugation for subsequent ELISA detection of TNF-α concentration in the supernatant. TNF-α concentration detection was performed according to the standard steps provided by the ELISA kit manufacturer.

[0301] 3. Experimental data processing method:

[0302] The standard curve of TNF-α standard was drawn by linear regression method, and the TNF-α concentration of the sample was converted according to the standard curve.

[0303] Calculate the inhibition rate: inhibition rate percentage = [(average TNF-α concentration in positive control wells - average TNF-α concentration in sample wells) / (average TNF-α concentration in positive control wells - average TNF-α concentration in negative control wells)] × 100%, where the positive control wells are compound-free wells (DMSO-treated wells) and the negative control wells are culture medium wells.

[0304] Dose response curve fitting: Use log(inhibitor) vs.response--Variable slope(four parameters) in GraphPad Prism 6 to fit the compound concentration and the corresponding inhibition rate, fit the curve and obtain the compound IC 50 The fitting calculation equation is Y = Bottom + (Top-Bottom) / (1 + 10^((LogIC 50 -X)*HillSlope)).

[0305] 4. Experimental results:

[0306]

[0307] 5. Experimental conclusion:

[0308] The above data show that the compounds of the present invention have a strong inhibitory ability on the expression level of TNF-α in U937 cells.

[0309] Test Example 4: Pharmacokinetics in SD rats

[0310] 1. Experimental purpose:

[0311] SD rats were used as test animals to study the pharmacokinetic behavior of the compound of the present invention in rats (plasma) at a dose of 5 mg / kg or 2 mg / kg orally and a dose of 1 mg / kg intravenously.

[0312] 2. Experimental Plan

[0313] 2.1 Trial Drugs:

[0314] The compound of the present invention is homemade.

[0315] 2.2 Experimental Animals:

[0316] There were 3 male SD rats in each group, from Shanghai Jiesijie Experimental Animal Co., Ltd., with animal production license number (SCXK (Shanghai) 2013-0006N0.311620400001794).

[0317] 2.3 Drug preparation:

[0318] PO, 10% solutol HS15, dissolve by ultrasound, prepare as a clear solution or homogeneous suspension.

[0319] IV, 5% DMSO + 10% Solutol HS15 + 85% PBS, sonicated to prepare a clear solution.

[0320] 2.4 Dosage regimen:

[0321] 3 male SD rats were administered in each group after overnight fasting, with a dose of 5 mg / kg or 2 mg / kg or 10 mL / kg, respectively; and an iv dose of 1 mg / kg or 5 mL / kg, respectively.

[0322] 2.5 Sample collection:

[0323] After oral administration to rats, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours were measured; after intravenous administration to rats, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours were measured; 0.2 mL of blood was collected from the jugular vein, placed in an EDTA-K2 test tube, centrifuged at 6000 rpm at 4°C for 6 min to separate plasma, and stored at -80°C.

[0324] 2.6 Sample processing:

[0325] 1) Add 100uL acetonitrile to 20uL of plasma sample for precipitation, mix and centrifuge at 3500×g for 5 to 20 minutes.

[0326] 2) The supernatant solution after treatment is subjected to LC / MS / MS analysis to determine the concentration of the test compound. The LC / MS / MS analysis instrument is AB Sciex API 4000Qtrap.

[0327] 2.7 Liquid phase analysis:

[0328] Liquid phase conditions: Shimadzu LC-20AD pump

[0329] ·Chromatographic column: Waters Xbridge C18 5μm, 4.6X 50mm Mobile phase: Liquid A is 0.1% formic acid in water, Liquid B is methanol

[0330] Flow rate: 1.0mL / min

[0331] Elution time: 0-4.0 minutes, eluent is as follows:

[0332]

[0333] 3. Test results and data processing:

[0334] The main pharmacokinetic parameters were calculated using WinNonlin 6.1.

[0335] 4. Experimental conclusion

[0336] The results of pharmacokinetic assay in SD rats showed that the compound of the present invention exhibited good PK properties.

[0337] Test Example 5: Metabolism study of the compounds of the present invention in human liver S9 incubation system

[0338] 1. Experimental purpose: The purpose of this test example is to test the metabolism of the compound of the present invention in the human liver S9 incubation system.

[0339] 2. Experimental Procedure

[0340] 2.1 Incubation system

[0341] Human liver S9 incubation concentration: 1.0mg / mL Test compound: Compounds of the present invention Incubation concentration of test compound: 10μM Reducing coenzyme (β-NADPH): 1.0mM Incubation medium: <![CDATA[50 mM K2HPO4 buffer (pH 7.4)]]> Incubation conditions: 37℃ Incubation time: 0,60min Incubation volume: 200μL

[0342] 2.2 Preparation of test substance working solution

[0343] Test compound stock solution: 20 mM test compound stock solution was prepared in DMSO and stored at 2-8°C until use.

[0344] Working solution of test compound: dilute the compound stock solution to 1.00 mM with 50% ACN / H2O (v / v) to use as working solution.

[0345] 2.3 Preparation of human liver S9 working solution

[0346] The human liver S9 suspension (20 mg / mL) was diluted to 1.27 mg / mL with 50 mM phosphate buffer for later use.

[0347] 2.4 Preparation of reducing coenzyme solution

[0348] Dissolve the reducing coenzyme (β-NADPH) in phosphate buffered saline to 5.0 mM for later use.

[0349] 2.5 Preparation of enzyme reaction termination solution

[0350] An acetonitrile solution containing 0.1% FA was used as the reaction termination solution and stored at 2-8°C until use.

[0351] 2.6 Liver S9 incubation and sample processing

[0352] For samples with T=60 minutes, Group 1 (with NADPH): Pipette 158 μL of human liver S9 working solution (1.27 mg / mL) into a centrifuge tube, add 2 μL of compound working solution (1000 μM), and add 40 μL of reduced coenzyme to start the reaction. Group 2 (without NADPH): Pipette 158 μL of human liver S9 working solution (1.27 mg / mL) into a centrifuge tube, add 2 μL of compound working solution (1000 μM), and add 40 μL of phosphate buffered saline.

[0353] For the sample at T=0 minutes, 158 μL of human liver S9 working solution (1.27 mg / mL) was transferred to a centrifuge tube, and 40 μL of reduced coenzyme was added to start the reaction, and the reaction was incubated in parallel without the compound.

[0354] After incubation at 37°C for 60 minutes, the samples were removed from the incubation water bath and 400 μL of stop solution was added to terminate the reaction.

[0355] For negative control, after stopping the reaction, vortex thoroughly to denature the microsomes, and then add 2 μL of 1000 μM compound working solution.

[0356] The sample will be centrifuged at 5000 rpm for 15 minutes. Take 60 μL of the centrifuged sample and 120 μL of water to the test plate for mass spectrometry and high performance liquid chromatography analysis.

[0357] 3. Sample Analysis and Data Processing

[0358] 3.1 Sample Analysis

[0359] 3.1.1 Mass spectrometry analysis of samples An analytical method was established on QTRAP 5500 and LC-30AD liquid spectrometers, and the pMRM-IDA-EPI scanning mode was used for sample analysis.

[0360] 3.1.2 High Performance Liquid Chromatography Analysis of Samples The analysis method was established on Vanquish HPLC and the Vanquish HPLC series was used for sample analysis:

[0361] Thermo Scientific Vanquish series high performance liquid chromatography (HPLC) was used for analysis:

[0362] Mobile phase preparation:

[0363] a. Take 0.5 ml of formic acid, add it to 1000 ml of pure water, mix well, and mark it as mobile phase C.

[0364] b. Take 0.5 ml of formic acid, add it to 1000 ml of acetonitrile, mix well, and mark it as mobile phase D.

[0365] Chromatographic conditions / HPLC method parameters

[0366]

[0367] Injection volume 5.00ul

[0368] 3.2 Data processing

[0369] 3.2.1 Mass spectrometry data processing: Set appropriate parameters and methods according to the compound structure, using System Integration Metabolite identification software The software processes the data. After data processing, potential metabolites are analyzed and interpreted.

[0370] 3.2.2 HPLC data processing: Appropriate parameters and methods were set according to the compound structure, and the data were processed using Chromeleon 7 software. After data processing, potential metabolites were analyzed and resolved.

[0371] 4. Experimental results and conclusions

[0372] The compound of the present invention shows excellent stability and produces less major metabolites or even no metabolites in a human liver S9 experiment, so that the compound can produce good drug effects even at a low dose.

[0373] Test Example 6: Study on the metabolic stability of the compounds of the present invention in human hepatocytes

[0374] 1. Experimental purpose: The purpose of this test example is to test the metabolic stability of the compounds of the present invention in human hepatocytes.

[0375] 2. Experimental reagents:

[0376] The compound of the present invention is self-made.

[0377] 3. Experimental methods:

[0378] 3.1. Preparation of working solution

[0379] 1) Prepare 10 mM stock solutions of test compounds in an appropriate solvent (DMSO).

[0380] 2) In a separate conical tube, dilute the 10 mM test compound to 100 μM by mixing 198 μL of acetonitrile and 2 μL of the 10 mM stock solution.

[0381] 3.2. Preparation of hepatocytes

[0382] 1) Place the culture medium (L-15 medium) and hepatocyte thawing medium in a 37°C water bath and warm for at least 15 minutes before use.

[0383] 2) Transfer a vial of cryopreserved hepatocytes from the warehouse, making sure the vial remains at low temperature until the thawing process is complete. Place the vial in a 37°C water bath and gently shake the vial for 2 minutes to thaw the cells. Once thawing is complete, spray the vial with 70% ethanol and transfer the vial to a biosafety cabinet.

[0384] 3) Use a wide-bore pipette tip to transfer the hepatocytes to a 50 mL conical tube containing thawing medium. Place the 50 mL conical tube in a centrifuge and centrifuge at 100 g for 10 minutes. After the spin is complete, aspirate the thawing medium and resuspend the hepatocytes in enough medium to produce approximately 1.5 × 106 cells / mL.

[0385] 4) Count the cells and determine the viable cell density using AO / PI staining. Dilute the cells to a working cell density of 1×106 viable cells / mL using culture medium.

[0386] 3.3. Stability determination procedure

[0387] 1) Transfer 247.5 μL of the hepatocyte solution to each well of a pre-warmed culture plate (1 mL 96-well deep-well plate). Pre-warm in an Eppendorf Thermomixer Comfort plate shaker at 600 rpm and 50°C to 37°C.

[0388] 2) Add 2.5 μL of 100 μmol / L test compound and control compound to the corresponding wells of the culture plate containing cells to start the reaction. The incubation concentration of the test compound is 1 μmol / L.

[0389] 3) Incubate the plate at 37°C (using a thermometer to measure the temperature) and 900 rpm on an Eppendorf Thermomixer Comfort plate shaker. At 0.5, 5, 15, 30, 45, 60, 80, 100 and 120 minutes, transfer 20 μL of the incubated mixture to a separate quenching plate. Then mix the aliquot with 12 volumes (300 μL) of acetonitrile containing the internal standard IS (100 nM alprazolam, 200 nM caffeine and 100 nM toluene butyramide) to terminate the reaction. Vortex for 5 minutes. Centrifuge the sample at 3220 g for 45 minutes. Dilute an aliquot of 100 μL of the supernatant with 100 μL of ultrapure water, and use the mixture for LC / MS / MS analysis.

[0390] 4. Experimental data processing method:

[0391] All calculations were performed using Microsoft Excel. Peak areas were determined from the extracted ion chromatograms. In vitro half-lives (t1 / 2) of the parent compounds were determined by regression analysis of the parent disappearance percentage versus time curves.

[0392] The in vitro t1 / 2 (in minutes) was converted to in vitro intrinsic clearance (in vitro CLint, in μL / min / 1×106 cells) using the following equation: in vitro CLint=kV / N (V=culture volume (0.25 mL); N=number of hepatocytes per well (0.25×106 cells))

[0393] 5. Experimental results:

[0394]

[0395] 6. Experimental conclusion: The compounds of the present invention have stronger stability in human hepatocytes.

[0396] Test Example 7: PD test in Wistar rat acute inflammation model

[0397] 1. Experimental purpose:

[0398] Wistar rats aged 6-8 weeks were used as test animals to study the pharmacodynamic behavior of the compound of the present invention in rats (serum) after oral administration at different doses and time points.

[0399] 2. Experimental Plan

[0400] 2.1 Test samples:

[0401] The compound of the present invention is self-made.

[0402] Wistar rats, Vital River Company.

[0403] 2.2 Drug preparation:

[0404] An appropriate amount of compound was weighed, and the corresponding solvent 10% Solutol HS15 was added, and a suspension with a final concentration of 0.2 mg / mL was obtained by ultrasound, vortexing and stirring.

[0405] 2.3 Dosage regimen:

[0406] Eight male Wistar rats were administered orally to each group after overnight fasting at a dose of 0.1 mg / kg in a dosing volume of 5 mL / kg.

[0407] The experiment was started when the rats weighed 200-230 g. Vehicle or drug was administered 1 h, 5 h, 11 h, 17 h, and 23 h before the administration of the modeling agent LPS. Samples were collected 1 h after the tail vein injection of the modeling agent LPS or PBS.

[0408] 2.4 Sample collection:

[0409] Blood was collected from the jugular vein of rats, added to a centrifuge tube, placed at room temperature for 60 minutes, and centrifuged at 8000 rpm for 5 minutes. 50 μL of the serum after centrifugation was transferred to a new labeled centrifuge tube, and 2 portions were taken, quickly frozen with dry ice, and stored in a -80°C refrigerator for detection.

[0410] 2.5 Sample processing:

[0411] Serum samples: After thawing, the normal group and the 6-hour administration group were diluted 1.5 times, that is, 80.0 μL of sample was added to 40.0 μL 1x reagent diluent buffer (prepared by R&D kit); the modeling group and the 18-hour administration group were diluted 3 times, that is, 40.0 μL of sample was added to 80.0 μL 1x reagent diluent buffer.

[0412] 2.6 Experimental Procedure

[0413] TNF-α test (Kit DY510):

[0414] 1) Coat the test plate one day in advance: Take appropriate coating antibody and add it to PBS to obtain a coating antibody solution with a concentration of about 4.00 μg / mL, add it to the test plate, 100.0 μL per well, and place it in a 24°C incubator for overnight incubation.

[0415] 2) Washing: Discard the coating solution in the test plate, add 300.0ul of wash buffer to each well, and repeat washing 3 times, removing all residual liquid in the test plate each time.

[0416] 3) Blocking: Add 300.0 μL of 1× blocking buffer to each well for blocking and incubate at 24°C for at least 1 h.

[0417] 4) Preparation of standard products: First, dilute the Rat TNFa standard product to 1000 pg / ml, take 4.17 μL of the standard product and add it to 495.83 μL of 1xreagent dilute, and then dilute it twice with 1xRegent dilute to prepare standard products with 8 concentrations.

[0418] 5) Take out the sealed test plate and wash the plate.

[0419] 6) Sample incubation: Take 100.0 μL of the diluted sample or standard into the corresponding test well of the ELISA test plate, seal it with adhesive tape and place it in a 24°C incubator for 2 hours.

[0420] 7) Wash the plate.

[0421] 8) Detection antibody incubation: Prepare the detection antibody working solution: Add appropriate detection antibody to 1xRegent Dilute to obtain the detection antibody solution. Add 100.0 μL of detection antibody working solution to each well, seal with new adhesive tape, and incubate in a 24°C incubator for 2 hours.

[0422] 9) Wash the plate.

[0423] 10) Secondary antibody HRP incubation in dark: Take appropriate amount of 40xStreptavidin-HRP and dilute it to 1x in 1xRegent Dilute. Add 100.0μL to each well and incubate in 24℃ incubator for about 20min.

[0424] 11) Wash the plate and repeat step 2.

[0425] 12) Color development and light protection: Mix equal volumes of color reagent A and B to prepare substrate solution, add 100.0ul to each well, and incubate at room temperature for about 20 minutes.

[0426] 13) Stop: When the color development is appropriate, add 50.0 μL STOP solution to each well.

[0427] IL-6 and KC / GRO detection (Kit K15059D-2):

[0428] 1) Reagent preparation: Preparation: Take out the reagent kit from the refrigerator and equilibrate it at room temperature for 30 minutes.

[0429] 2) Prepare the standard: add 1 ml of diluent 42 to the standard, shake thoroughly, and leave at room temperature for 15-20 minutes. As the highest concentration point, make 4-fold dilutions in sequence, 7 standards + 1 Blank.

[0430] 3) Prepare samples: dilute rat serum appropriately

[0431] 4) Prepare the detection antibody diluent: aspirate the detection antibody and make up the volume with diluent 40.

[0432] 5) Prepare the cleaning solution: prepare 1*PBS (0.05% Tween-20) for later use.

[0433] 6) Prepare plate reading solution: Prepare 2* plate reading solution. (Prepare 10 minutes before the end of antibody incubation)

[0434] 7) Take out the rewarmed MSD plate and Blocker H, add 150ul Block H to each well and incubate at room temperature with shaking at 800rpm for 1h.

[0435] 8) Wash the MSD plate 3 times, each time with 150ul of washing solution.

[0436] 9) Add 50ul of diluted samples and standards to each well, seal with sealing film, and shake at room temperature for 2h.

[0437] 10) Wash the MSD plate 3 times, each time with 150ul of washing solution.

[0438] 11) Add 25ul of the prepared detection antibody to each well, seal with sealing film, and shake at room temperature for 2 hours.

[0439] 12) Wash the MSD plate 3 times, each time with 150ul of washing solution.

[0440] 13) Add 150ul of prepared plate reader solution to each well and test on the machine.

[0441] 3 Experimental results and conclusions:

[0442] Data processing was performed using Graph pad.

[0443] The results of the PD experiment on the Wistar rat acute inflammation model showed that the compounds of the present invention exhibited a more excellent PD effect and had a good inhibition rate on TNF-α; 6 hours after administration, the inhibition rate of TNF-α of the compounds of the present invention was higher than 60.0%, and the inhibition rate of TNF-α of the superior compounds was higher than 90.0%.

Claims

1. A compound represented by general formula (I) or (II), a stereoisomer thereof or a pharmaceutically acceptable salt thereof: in: M1, M2 and M3 are each independently selected from carbon or oxygen, and are not oxygen at the same time; M4, M5 and M6 are each independently selected from carbon or oxygen, and are not oxygen at the same time; R1 is selected from deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 The alkynyl group may be further substituted with deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl and C 2-6 substituted by one or more substituents in the alkynyl group; R2 is selected from deuterium, halogen, nitro, hydroxyl, thiol, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl or C 2-6 The alkynyl group may be further substituted with deuterium, halogen, nitro, hydroxyl, mercapto, cyano, amino, oxo, thio, carboxyl, C 1-6 Alkyl, C 1-6 Deuterated alkyl, C 1-6 Haloalkyl, C 1-6 Hydroxyalkyl, C 1-6 Alkoxy, C 1-6 Deuterated alkoxy, C 1-6 Halogenated alkoxy, C 2-6 Alkenyl and C 2-6 substituted by one or more substituents in the alkynyl group; n is selected from 0, 1, 2 or 3; and m is selected from 0, 1, 2 or 3.

2. The compound according to claim 1, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: M1 is selected from carbon or oxygen; M2 and M3 are selected from carbon; M4 is selected from carbon or oxygen; M5 and M6 are selected from carbon; R1 is selected from deuterium, halogen, oxo or C 1-3 alkyl; R2 is selected from deuterium, halogen, oxo or C 1-3 alkyl; and n is 2; and m is 2.

3. A compound, a stereoisomer thereof or a pharmaceutically acceptable salt thereof, characterized in that: The structure of the compound is shown below:

4. A compound represented by the general formula (M-1), its stereoisomer or a pharmaceutically acceptable salt thereof: in: M1, M2, M3, R1 and n are as described in claim 1 or 2.

5. The compound represented by the general formula (M-1) as shown in claim 4, its stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: Selected from the following compounds:

6. A method for preparing the compound of general formula (I) or its stereoisomers and pharmaceutically acceptable salts thereof according to claim 1, characterized in that: The following steps are included: The compound represented by the general formula (M-1) is reacted to obtain the target compound represented by the general formula (I); in, M1, M2, M3, R1 and n are as described in claim 1 or 2.

7. A pharmaceutical composition comprising a therapeutically effective dose of the compound according to any one of claims 1 to 3, its stereoisomers or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable carriers, diluents or excipients.

8. Use of the compound according to any one of claims 1 to 3, its stereoisomer or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 7 in the preparation of a drug for treating a disease mediated by p38 kinase.

9. Use of the compound according to any one of claims 1 to 3, its stereoisomers or pharmaceutically acceptable salts thereof, or the pharmaceutical composition according to claim 7 in the preparation of a medicament for treating autoimmune diseases, chronic inflammatory diseases, acute inflammatory conditions, autoinflammatory diseases, atherosclerosis, diabetes, fibrotic diseases, metabolic diseases, cancers, tumors, leukemia and lymphoma.

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