Nitrogen-containing fused ring compound, pharmaceutical composition containing the compound and application thereof

Abstract

The present invention provides a nitrogen-containing fused ring compound, a pharmaceutical composition containing the compound and its application, and relates to the field of pharmaceutical technology. The nitrogen-containing fused ring compound of the present invention has a structure as shown in formula (I), #imgabs0# wherein Y, W, V, Z, X, L1, L2, R1, R3, and R4 are as defined herein. The nitrogen-containing fused ring compound of the present invention has a strong inhibitory effect on SIKs enzyme activity, and the IC value of the inhibition of SIKs enzyme activity in vitro is 50 In the experiment, the compounds of the present invention have good inhibitory activity against SIK2 and SIK3 kinases, and have a certain selectivity for SIK1 kinase; the compounds of the present invention have an inhibitory effect on the expression of TNF-α in the serum of acutely inflammatory mice, and have a promoting effect on the secretion of the anti-inflammatory factor IL-10; in pharmacokinetics, the compounds of the present invention have a good oral exposure, and the exposure of the representative compounds is significantly improved compared with compound 1.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to a nitrogen-containing fused ring compound, a pharmaceutical composition containing the compound, and applications thereof. Background Art

[0002] Salt-inducible kinase (SIK) belongs to the AMP-activated protein kinase (AMPK) family and includes three isoforms, namely SIK1, SIK2 and SIK3. The activity of SIK can be affected by a variety of different forms of post-translational modifications, including LKB1, CaMK, PKA-mediated phosphorylation, p300-mediated acetylation, etc. The SIK family regulates multiple aspects of physiology, including metabolism, neurophysiology, immune function and development. Recent studies have shown that SIK activity is at the center of the kinase cascade that regulates sleep and circadian rhythms, and these processes in turn are the main regulators of all physiology and behavior (including metabolism, immune function and neurophysiology). Based on the very diverse biological functions of SIK, the potential application space of SIKs targeted drugs may include tumors, inflammatory diseases, obesity-induced diabetes and other diseases related to kinase activity disorders.

[0003] Salt-inducible kinase 2 (SIK2) is a serine / threonine protein kinase and a member of the adenosine monophosphate-activated protein kinase (AMPK) subfamily. SIK2 gene expression level is highest in adipose tissue. It negatively regulates the activity of cAMP-responsive element-binding protein (CREB) by phosphorylation to regulate transcription co-activator (CRTC / TORC) 2 and 3. It also affects the expression of genes related to gluconeogenesis and lipid synthesis by phosphorylating p300 / CBP, playing an important role in fat and glucose metabolism. Knockdown of SIK2 in ovarian cancer cells leads to a significant decrease in the phosphorylation of PKB at amino acid position 473 and a significant increase in the expression level of LKB1. The degree of reduction in the phosphorylation level of PKB and the degree of increase in the level of LKB1 are significantly correlated with the degree of inhibition of ovarian cancer cell proliferation. The expression of SIKs, especially SIK2, is significantly upregulated during the differentiation of monocytes into macrophages. Therefore, SIK2 has a more precise correlation with inflammatory diseases among all subtypes of SIKs. Related studies have shown that SIK3 is overexpressed in ovarian cancer cells and is a new ovarian cancer-related antigen that promotes the G1 / S process and cell proliferation of cancer cells, and may become a new target for the treatment of ovarian cancer. Activation of the cAMP signaling pathway can inhibit the activity of SIK2 / 3, and PKA mediates the phosphorylation of SIK2 / 3, inhibits its kinase activity, promotes the nuclear entry of CRTC, induces the secretion of anti-inflammatory factor IL-10, and inhibits the expression of pro-inflammatory factors IL-6 and TNF-α. Inhibition of SIK2 / SIK3 can stimulate anti-inflammatory cytokines and inhibit pro-inflammatory cytokines, achieve two-way regulation of inflammation, and rebuild immune balance.

[0004] At present, inhibitors targeting SIKs targets can be divided into non-selective inhibitors and selective inhibitors. Researchers have screened out some marketed drugs, such as pan-protein tyrosine kinase inhibitors Dasatinib and Bosutinib, which have certain activity against SIKs targets. HG-9-91-01, YKL-05-099, and MRT-199665 are all first-generation pan-SIKs inhibitors with poor selectivity for members of each subfamily, and most drugs have defects such as off-target and poor pharmacokinetic properties. In addition, subtype-selective SIK2 / 3 inhibitors represented by ARN-3261 and GLPG3970 are still in early clinical or preclinical studies. In summary, it is of great significance to further develop new SIK inhibitors and fully study their in vivo mechanisms of action.

[0005] RIPK2 kinase is an emerging drug target for the treatment of autoimmune and inflammatory diseases. RIPK2 is mainly involved in regulating the signal transduction of NOD1 and NOD2 pathways and the activation of their downstream nuclear factor NF-κB and mitogen-activated protein kinase MAPK pathways, thereby regulating a series of inflammatory responses in the body. The inhibitory effect of compounds on RIPK2 kinase can be examined at the cellular level by detecting the downstream gene NF-κB or cytokine TNF-α (Nachbur U., Stafford C.A., Bankovacki A., et al. A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production. Nat Commun. 2015, 6: 6442.). Currently, there are no RIPK2 inhibitors on the market for disease treatment. Compounds such as WEHI-345 and GSK583 have been shown to have good RIPK2 kinase inhibitory activity and inflammatory treatment potential. The development of RIPK2 inhibitors has good application prospects for autoimmune and inflammatory disease drugs. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a nitrogen-containing fused ring compound, a pharmaceutical composition comprising the compound and its application. The compound obtained by the present invention can inhibit the activity of SIKs and can be used to treat cancer, immune inflammation, bone formation, diabetes, and central nervous system diseases (depression, insomnia, epilepsy).

[0007] The present invention provides a nitrogen-containing fused ring compound, wherein the compound has a structure shown in formula (I):

[0008]

[0009] or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein:

[0010] Y, W, V, Z, X are independently selected from C or N;

[0011] W, Y, and Z are not all N; when W, Y, and Z are all C, V is N;

[0012] L1 is selected from O, NH, CH2 or none;

[0013] L2 is selected from C 1-3 Alkanes or none;

[0014] R1 is selected from R csubstituted 5-6 membered cycloalkyl, 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl, -NR a R b , -OH, unsubstituted or R e Substituted 5-6 membered aromatic heterocyclic group, unsubstituted or R e substituted 9-10 membered fused heteroaromatic ring group, 4-6 membered heterocyclic group; the 4-6 membered heterocyclic group contains 1 or 2 N atoms, the 4-6 membered heterocyclic group can be methyl, fluorine, -(CH2) m OH; the 7-12-membered spirocycloalkyl, 7-12-membered spirocycloheteroalkyl, 7-12-membered bridged cycloalkyl, 7-12-membered bridged cycloheteroalkyl, 8-12-membered cycloalkyl and 8-12-membered cycloheteroalkyl may be C 1-3 Alkyl, -OH, -NH2, -NHCH3, -N(CH3)2, halogen, halogen-substituted C 1-3 Alkyl or C 1-3 Alkoxy substitution;

[0015] R a , R b Independently selected from H or C 1-3 Alkanes;

[0016] R c Selected from -OH, -NH2, -NHCH3 or -N(CH3)2;

[0017] m is selected from 1, 2 or 3;

[0018] R3 is selected from H, cyano, C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 1-3 Alkyl, F, C 3-6 Cycloalkyl, one or more fluorine-substituted C 1-3 Alkoxy or

[0019] R4 is selected from F, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy or -NHS(O)2R d ; preferably F, methoxy, difluoromethoxy, trifluoromethoxy or -NHS(O)2R d ;

[0020] R d Selected from C 1-3 Alkyl or cyclopropyl; preferably methyl, ethyl or cyclopropyl;

[0021] R eSelected from H, tetrahydropyranyl, C 1-3 Alkyl, cyclopropyl, halogen-substituted C 1-3 Alkyl, Preferred are tetrahydropyranyl, methyl, ethyl, cyclopropyl, trifluoromethyl, trifluoroethyl,

[0022] A preferred embodiment of the present invention is to provide a nitrogen-containing fused ring compound represented by formula (II) or formula (III), the structure of which is as follows:

[0023]

[0024] or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein:

[0025] Y, W, V, Z, X are independently selected from C or N;

[0026] W, Y, and Z are not all N; when W, Y, and Z are all C, V is N;

[0027] L1 is selected from O or NH or CH2; preferably O or NH;

[0028] L2 is selected from C 1-3 Alkane; preferably methyl, ethyl;

[0029] R1 is selected from R c substituted 5-6 membered cycloalkyl, 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl, -NR a R b , -OH, unsubstituted or R e Substituted 5-6 membered aromatic heterocyclic group, unsubstituted or R e A substituted 9-10 membered fused heteroaromatic ring group, a 4, 5 or 6 membered heterocyclic ring group, wherein the 4, 5 or 6 membered heterocyclic ring group contains 1 or 2 N atoms, and the 4, 5 or 6 membered heterocyclic ring group may be substituted by methyl, fluorine, -(CH2) m OH substituted; 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl may be C 1-3 Alkyl, -OH, -NH2, -NHCH3, -N(CH3)2, halogen, halogen-substituted C 1-3 Alkyl, C 1-3 Alkoxy substituted; preferably -NR a R b , no substitution or R eA substituted 5-6 membered aromatic heterocyclic group, a 4, 5 or 6 membered heterocyclic group, wherein the 4, 5 or 6 membered heterocyclic group contains 1 or 2 N atoms, and the 4, 5 or 6 membered heterocyclic group may be substituted with methyl, fluorine, -(CH2) m OH substitution;

[0030] R a , R b Each independently selected from H, C 1-3 Alkane; preferably H, methyl, ethyl;

[0031] R c Selected from -OH, -NH2, -NHCH3, -N(CH3)2;

[0032] m is an integer selected from 1 to 3;

[0033] R3 is selected from H, cyano, C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 11-3 Alkyl, F, C 3-6 Cycloalkyl, one or more fluorine-substituted C 1-3 Alkoxy, Preferred are cyano, methyl, ethyl, methoxy, trifluoromethyl, difluoromethyl, F, cyclopropyl, cyclobutyl, cyclopentyl, trifluoromethoxy, difluoromethoxy,

[0034] R4 is selected from F, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy, -NHS(O)2R d ; preferably F, methoxy, difluoromethoxy, trifluoromethoxy, -NHS(O)2R d ;

[0035] R d Selected from C 1-3 Alkyl, cyclopropyl; preferably methyl, ethyl, cyclopropyl;

[0036] R e Selected from H, tetrahydropyranyl, C 1-3 Alkyl, cyclopropyl, halogen-substituted C 1-3 Alkyl, Preferred are tetrahydropyranyl, methyl, ethyl, cyclopropyl, trifluoromethyl, trifluoroethyl,

[0037] A preferred embodiment of the present invention is to provide a nitrogen-containing fused ring compound represented by formula (IV) or formula (V) or formula (VI) or formula (VII):

[0038]

[0039] or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein:

[0040] Y, W, V, Z, X are independently selected from C or N;

[0041] W, Y, and Z are not all N; when W, Y, and Z are all C, V is N;

[0042] L1 is selected from O or NH or CH2; preferably O or NH;

[0043] L2 is selected from C 1-3 Alkane; preferably methyl or ethyl;

[0044] R1 is selected from R c substituted 5-6 membered cycloalkyl, 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl, -NR a R b , -OH, unsubstituted or R e Substituted 5-6 membered aromatic heterocyclic group, unsubstituted or R e A substituted 9-10 membered fused heteroaromatic ring group, a 4, 5 or 6 membered heterocyclic ring group, wherein the 4, 5 or 6 membered heterocyclic ring group contains 1 or 2 N atoms, and the 4, 5 or 6 membered heterocyclic ring group may be substituted by methyl, fluorine, -(CH2) m OH substituted; 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl may be C 1-3 Alkyl, -OH, -NH2, -NHCH3, -N(CH3)2, halogen, halogen-substituted C 1-3 Alkyl, C 1-3 Alkoxy substituted. Preferably -NR a R b , no substitution or R e A substituted 5-6 membered aromatic heterocyclic group, a 4, 5 or 6 membered heterocyclic group, wherein the 4, 5 or 6 membered heterocyclic group contains 1 or 2 N atoms, and the 4, 5 or 6 membered heterocyclic group may be substituted with methyl, fluorine, -(CH2) m OH substitution;

[0045] R a , R b Each independently selected from H, C 1-3 Alkane; preferably H, methyl, ethyl;

[0046] R cSelected from -OH, -NH2, -NHCH3, -N(CH3)2;

[0047] m is an integer selected from 1 to 3;

[0048] R3 is selected from H, cyano, C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 1-3 Alkyl, F, C 3-6 Cycloalkyl, one or more fluorine-substituted C 1-3 Alkoxy, Preferred are cyano, methyl, ethyl, methoxy, trifluoromethyl, difluoromethyl, F, cyclopropyl, cyclobutyl, cyclopentyl, trifluoromethoxy, difluoromethoxy,

[0049] R4 is selected from F, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy; preferably F, methoxy, difluoromethoxy, trifluoromethoxy;

[0050] R5 is selected from C 1-3 Alkyl, cyclopropyl; preferably methyl, ethyl, cyclopropyl;

[0051] R e Selected from H, tetrahydropyranyl, C 1-3 Alkyl, cyclopropyl, halogen-substituted C 1-3 Alkyl, Preferred are tetrahydropyranyl, methyl, ethyl, cyclopropyl, trifluoromethyl, trifluoroethyl,

[0052] A preferred embodiment of the present invention is to provide a nitrogen-containing fused ring compound represented by Formula I, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein Formula I Unit selection from

[0053] L1 is selected from O or NH or CH2; preferably O or NH;

[0054] L2 is selected from C 1-3 Alkane; preferably methyl, ethyl.

[0055] Preferably, when R1 of the nitrogen-containing fused ring compound represented by the above formula (I) is a 5-6 membered aromatic heterocyclic group substituted by Re, the 5-6 membered aromatic heterocyclic group is preferably pyrazolyl, triazolyl, imidazolyl, pyrrolyl, thiazolyl, pyridyl, pyrimidinyl or pyridazinyl.

[0056] Preferably, in the nitrogen-containing fused ring compound represented by the above formula (III) or (VI) or (VII), the R1 is selected from a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains 1 or 2 N atoms, and the 4-6 membered heterocyclic group can be methyl, fluorine, -(CH2) m OH substitution.

[0057] Preferably, in the nitrogen-containing fused ring compounds represented by the above formula (I) to (VII), Y, V, and Z are C, and W is N.

[0058] Preferably, in the nitrogen-containing fused ring compounds represented by the above formula (I) to (VII), the R3 is selected from C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 1-3 Alkyl, one or more fluorine-substituted C 1-3 Alkoxy; R4 is selected from C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy or -NHS(O)2R d .

[0059] A preferred embodiment of the present invention is to provide a nitrogen-containing fused ring compound of formula I, or a stereoisomer or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the compounds numbered A1-A31:

[0060]

[0061]

[0062]

[0063]

[0064] The present invention also relates to a pharmaceutical composition, characterized in that it comprises the above-mentioned nitrogen-containing fused ring compound, or a stereoisomer or a pharmaceutically acceptable salt thereof.

[0065] The above-mentioned pharmaceutical composition may also include other chemotherapeutic drugs.

[0066] The chemotherapy drug in the above-mentioned pharmaceutical composition is at least one selected from paclitaxel, carboplatin, cisplatin, capecitabine, irinotecan and azacitidine.

[0067] The present invention also relates to the use of the compound represented by the above general formula, its stereoisomer, its pharmaceutically acceptable salt or a pharmaceutical composition comprising the compound represented by the above general formula, its stereoisomer, or its pharmaceutically acceptable salt in the preparation of drugs for treating diseases caused by abnormal activation of SIK kinase.

[0068] In some embodiments, the disease is selected from one or more of cancer, immune inflammation, diabetes, and central nervous system diseases (depression, insomnia, epilepsy).

[0069] In some embodiments, the cancer is selected from one or more of ovarian cancer, breast cancer, prostate cancer, glioma, and diffuse large B-cell lymphoma.

[0070] In some embodiments, the immune inflammation is selected from one or more of arthritis, colitis, psoriasis, atopic dermatitis, psoriatic arthritis, systemic lupus erythematosus, osteoarthritis, complications caused by organ transplantation, autoimmune thyroid disease, Sjögren's syndrome, vitiligo, autoimmune kidney damage, autoimmune liver damage, chronic obstructive pulmonary disease, multiple sclerosis, and amyotrophic lateral sclerosis.

[0071] Definition and Description

[0072] The structural formula of the present invention Here, N refers to the nitrogen atom. The fragment must comply with the carbon bond substitution rules of carbon atoms or nitrogen atoms and belong to a 6-membered and 5-membered aromatic condensed ring.

[0073] The term "isomer" means that the compounds of the present invention may exist in specific geometric or stereoisomer forms. In the present invention, isomers mainly refer to the (R)- and (S)-enantiomers, racemates that appear in the connection of R1 and L2 in R1-L2, and (R)- and (S)-enantiomers, racemates that appear in the substituents in the R1 group. All these isomers and their mixtures are included in the scope of the present invention.

[0074] When L1 and L2 are selected from none, R1 is directly connected to the condensed ring.

[0075] Unless otherwise specified, C 1-3 Alkanes represent straight or branched saturated hydrocarbon groups, which may be monosubstituted (e.g., -CH2CH3, -CH2F) or polysubstituted (e.g., -CH(CH2), -CF3,), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), and propyl (e.g., n-propyl and isopropyl).

[0076] Unless otherwise specified, a numerical range represents all integers including the numbers at both ends of the range. Unless otherwise specified, an integer from 0 to 3 represents 0, 1, 2, 3; an integer from 7 to 12 represents 7, 8, 9, 10, 11, 12; 1 to 2 represents 1, 2; 5 to 6 represents 5, 6; and so on.

[0077] Beneficial technical effects: The nitrogen-containing fused ring compounds of the present invention can achieve strong inhibition of SIKs enzyme activity, and the IC of SIKs enzyme activity inhibition in vitro is 50 In the experiment, it has good inhibitory activity against SIK2 and SIK3 kinases, and the inhibitory effect on SIK2 is better than or equivalent to the positive drug GLPG3970, and has a certain selectivity for SIK1 kinase; some compounds of the present invention have an inhibitory effect on TNFα in the serum of acute inflammatory mice and a promoting effect on the secretion of IL-10; in pharmacokinetics, the compounds of the present invention have a good oral exposure, and the exposure of representative compounds is significantly improved compared with compound 1; some compounds of the present invention have a significantly better inhibitory effect on RIPK2 kinase than positive drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 The inhibitory effect of compounds A18 and A26 on TNF-α secretion of BMDM cells under MDP stimulation;

[0079] Figure 2 The inhibitory effect of compounds A18 and A26 on NF-κB gene expression in THP-1 cells under MDP stimulation. DETAILED DESCRIPTION

[0080] Example 1 N-(3-(difluoromethoxy)-5-(7-((piperidin-4-methylene)amino)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A1)

[0081]

[0082] Synthesis step 1 Synthesis of intermediate 1-2

[0083] 2-Amino-4-bromopyridine (2.275 g) and sodium bicarbonate (2.18 g) were placed in a round-bottom flask, followed by the addition of 24 mL of anhydrous ethanol to fully dissolve the raw materials, followed by the addition of 5.3 mL of chloroacetaldehyde solution (40% wt aqueous solution), and stirred and refluxed at 82°C overnight. After the reaction was completed, the reaction solution was filtered through a sand core funnel / diatomaceous earth and extracted three times with ethyl acetate / water. After the solvent was removed in vacuo, the product was purified by column chromatography (PE:EA=1:2) to obtain 1.27 g (1-2) of a brown solid. ESI-MS: m / z=198 [M+H] + .

[0084] Synthesis step 2 Synthesis of intermediate 1-3

[0085] Add intermediate 1-2 (394 mg), 1-tert-butyloxycarbonyl-4-aminomethylpiperidine (129 mg), 36 mg Pd2(dba)3, 69 mg Xantphos, 288 mg t-BuONa, and 3 mL Toluene to a two-necked bottle, seal, replace N2, and react overnight at 100°C. After the reaction is completed, quench the reaction with EA. Add 50 mL of water and extract three times, and the organic phase is dried over anhydrous Na2SO4. The organic phase is spin-dried and purified by column chromatography (PE:EA=1:2~1:3) to obtain the product as a brown-yellow solid (1-3). ESI-MS: m / z=331[M+H] + .

[0086] Synthesis step 3 Synthesis of intermediate 1-5

[0087] 3-Bromo-5-nitroanisole 1-4 (5 g) was placed in a round-bottom flask, and then 20 mL of dichloromethane was added to dissolve the raw material, and then 32 mL of boron tribromide solution (2.0 mol / L in Dichloromethane) was added dropwise under ice bath conditions, and stirred at room temperature overnight. After the reaction was completed, the reaction solution was extracted three times with dichloromethane / water. After the solvent was removed in vacuo, the product was purified by column chromatography (PE:EA=15:1) to obtain 4.4 g of the product as a yellow solid (1-5).

[0088] Synthesis Step 4 Synthesis of Intermediate 1-6

[0089] Potassium hydroxide (11.38 g) was dissolved in 50 mL of water under ice bath conditions, followed by addition of intermediate 1-5 (4.4 g) and 50 mL of acetonitrile and stirring for 30 minutes, followed by slow dropwise addition of diethyl bromofluoromethylphosphonate (7.21 mL) and stirring at room temperature for 2 hours. After the reaction was completed, the reaction solution was extracted three times with ethyl acetate / water, and the organic phase was dried over anhydrous Na2SO4. After the solvent was removed in vacuo, the product was purified by column chromatography (PE:EA=1:0) to obtain 4.9 g of the product (1-6) as a yellow solid.

[0090] Synthesis Step 5 Synthesis of Intermediate 1-7

[0091] The intermediate 1-6 (4.9 g) was dissolved in 20 mL of anhydrous ethanol, and stannous chloride (17.4 g) was added under ice bath, and stirred and refluxed at 90°C for 4 hours. After the reaction was completed, the pH was adjusted to weak alkalinity with sodium carbonate, filtered, and the filtrate was extracted with ethyl acetate three times, and the organic phases were combined and the solvent was removed by vacuum distillation to obtain the desired compound, which was directly used in the next step. ESI-MS: m / z=238[M+H] + .

[0092] Synthesis Step 6 Synthesis of Intermediate 1-8

[0093] The intermediate 1-7 (4.11 g) was dissolved in 15 mL of pyridine, followed by the addition of 4-dimethylaminopyridine (847 mg) and cyclopropanesulfonyl chloride (2.68 g), and the mixture was stirred and refluxed at 80°C overnight. After the reaction, the pH was adjusted to a weak alkaline state with dilute hydrochloric acid, and then extracted three times with ethyl acetate / water. The solvent was removed by distillation under reduced pressure, and column chromatography (PE:EA) = 25:1 was used to obtain 5.13 g (1-8) of a white solid. ESI-MS: m / z = 342 [M+H] + .

[0094] Synthesis Step 7 Synthesis of Intermediate 1-9

[0095] The intermediate 1-8 (2 mmol) was dissolved in 5 mL DCM, and (Boc)2O (2.4 mmol), 4-dimethylaminopyridine (0.4 mmol), and triethylamine (3 mmol) were added under ice bath. After the addition, the mixture was stirred under ice bath for 10 min and then transferred to room temperature for reaction for 6 h. After the reaction was completed, the organic phases were combined after extraction with EA three times, and the solvent was removed by vacuum distillation. The mixture was separated by column chromatography (PE:EA) = 4:1 to obtain a light yellow solid (1-9). ESI-MS: m / z = 442 [M+H] + .

[0096] Synthesis Step 8 Synthesis of N-(3-(difluoromethoxy)-5-(7-((piperidin-4-methylene)amino)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A1)

[0097] Intermediate 1-3 (330 mg, 1 mmol) and intermediate 1-9 (442 mg, 1 mmol) were dissolved in DMF (4 mL), and Pd(dppf)Cl2 (36 mg, 0.05 mmol) and KOAc (196 mg, 2 mmol) were added. The mixture was protected by nitrogen and reacted at 110°C for 12 h. After the reaction, the mixture was washed with water and extracted with EA three times. The organic phases were combined and the solvent was removed by distillation under reduced pressure. The intermediate was purified by column chromatography (DCM: MeOH = 30: 1). The protective group was removed by hydrochloric acid / 1,4-dioxane solution (4 M) at 25°C. The reaction was terminated after 1 h. The compound A1 was obtained after purification by column chromatography (DCM: MeOH = 15: 1). 1HNMR(400MHz,Methanol-d4)δ8.16(d,J=7.6Hz,1H),7.35(s,1H),7.24(t,J=1.7Hz,1H),7. 09–6.67(m,3H),6.51(dd,J=7.6,2.3Hz,1H),6.32(d,J=2.3Hz,1H),3.15(d,J=12.5Hz,2H) ,3.05(d,J=6.3Hz,2H),2.70(td,J=12.6,2.5Hz,2H),2.61(tt,J=8.0,4.9Hz,1H),1.93–1. 81(m,3H),1.37–1.24(m,3H),1.09–1.02(m,2H),0.98–0.89(m,2H).ESI-MS: m / z=492[M+H] + .

[0098] Example 2 N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A2)

[0099]

[0100] Synthesis step 1 Synthesis of intermediate 2-3

[0101] 4-Methoxypyridine (2-1, 10.9 g, 10 mmol) and 2,4-dinitrophenylhydroxy (2-2, 20.9 g, 11.5 mmol) were added to DCM (30 mL), stirred at room temperature, and after the reaction was completed as monitored by TLC, the solvent was removed by distillation under reduced pressure and used directly in the next step.

[0102] Synthesis Step 2 Synthesis of Intermediate 2-4

[0103] Intermediate 2-3 and methyl propiolate (10 g, 12 mmol) were dissolved in DMF (30 mL), potassium carbonate (16.6 g, 12 mmol) was added, and the mixture was reacted at 80°C. After the reaction was completed, water was added, and the mixture was extracted three times with EA. The organic phases were combined and the solvent was removed by vacuum distillation. The mixture was purified by column chromatography (PE:EA=1:1) to obtain intermediate 2-4. ESI-MS: m / z=207[M+1] + .

[0104] Synthesis step 3 Synthesis of intermediate 2-5

[0105] Add 125 mL of 50% H2SO4 aqueous solution to the intermediate 2-4 (14 g), stir at 120°C for 3-6 h, adjust the pH to neutral with NaOH after the reaction is completed, extract with ether 3-5 times, combine the organic phases, remove the solvent by vacuum distillation, and purify by column chromatography (DCM: MeOH = 20: 1) to obtain the intermediate 2-5, ESI-MS: m / z = 135 [M+1] + .

[0106] Synthesis Step 4 Synthesis of Intermediate 2-7

[0107] 4-Boc-2-hydroxymethylmorpholine (2-1, 1.09 g, 5 mmol) was dissolved in DCM (10 mL), and triethylamine TEA (1.4 mL, 10 mmol) and methanesulfonyl chloride (0.6 mL, 7.5 mmol) were added under ice bath conditions. After the addition, the mixture was reacted at room temperature for 6 h. After washing with water, the mixture was extracted with EA, and the solvent was removed by distillation under reduced pressure. The obtained liquid was directly used in the next reaction.

[0108] Synthesis Step 5 Synthesis of Intermediate 2-8

[0109] Intermediate 2-5 (2 mmol) was dissolved in DMF solvent (5 mL), and intermediate 2-7 (3 mmol) and cesium carbonate (6 mmol) were added, and the mixture was heated to 80°C and reacted overnight. The solvent was removed by distillation under reduced pressure, and intermediate 2-8 was obtained after purification by column chromatography (DCM: MeOH = 20: 1) with a yield of 51%; ESI-MS: m / z = 334 [M+1] + .

[0110] Synthesis Step 6 Synthesis of N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A2)

[0111] Synthesis steps: Referring to step 8 of Example 1, intermediate 2-8 was used instead of intermediate 1-3 to obtain compound A2. 1H NMR(400MHz,Chloroform-d)δ8.32(dd,J=7.6,0.6Hz,1H),8.06(s,1H),7.38(t,J=1.7Hz,1 H),7.09(td,J=1.5,0.8Hz,1H),7.06(d,J=2.7Hz,1H),6.87(t,J=2.1Hz,1H),6.58(d,J=2. 4Hz,1H),4.12–3.91(m,4H),3.71(td,J=11.2,2.9Hz,1H),3.06(d,J=12.1Hz,1H),3.00–2. 76(m,3H),2.60–2.52(m,1H),1.05–0.99(m,2H),0.91–0.82(m,2H).ESI-MS:m / z=495[M+H] + .

[0112] Example 3 N-(3-(difluoromethoxy)-5-(5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A3)

[0113]

[0114] Synthesis step 1 Synthesis of intermediate 3-2

[0115] Synthesis steps: Refer to step 5 of Example 2, replace intermediate 2-7 with N,N-dimethylaminoethyl bromide hydrobromide to obtain intermediate 3-2, ESI-MS: m / z=206 [M+H] + .

[0116] Synthesis Step 2 Synthesis of N-(3-(difluoromethoxy)-5-(5-(2-(dimethylamino)ethoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A3)

[0117] Synthesis steps: Refer to step 8 of Example 1, replace intermediate 1-3 with intermediate 3-2 to obtain compound A3, 1H NMR(400MHz,Chloroform-d)δ8.28(d,J=7.6Hz,1H),8.03(s,1H),7.40(t,J=1.8Hz,1H),7.12(d,J=2.7Hz,1H),7.01(t,J=1.8Hz,1H),6.90(t,J=2.1Hz ,1H),6.76–6.37(m,2H),4.33(t,J=5.5Hz,2H),2.99(t,J=5.5Hz,2H),2.57 –2.51(m,7H),1.02–0.93(m,3H),0.91–0.77(m,3H).ESI-MS:m / z=467[M+H] + .

[0118] Example 4 (R)-N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A4)

[0119]

[0120] Synthesis step 1 Synthesis of intermediate 4-3

[0121] Synthesis steps Referring to steps 4 and 5 of Example 2, (R)-N-Boc-2-hydroxymethylmorpholine was used instead of 4-Boc-2-hydroxymethylmorpholine to obtain intermediate 4-3, ESI-MS: m / z=334 [M+H] + .

[0122] Synthesis Step 2 Synthesis of (R)-N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A4)

[0123] Synthesis steps: Refer to step 8 of Example 1, replace intermediate 1-3 with intermediate 4-3 to obtain compound A4, 1H NMR(400MHz, DMSO-d6)δ8.63(d,J=7.6Hz,1H),8.32(s,1H),7.42(t,J=1.7Hz,1H),7.19–7.1 1(m,2H),6.89(t,J=2.1Hz,1H),6.72(dd,J=7.6,2.6Hz,1H),4.07(d,J=5.1Hz,2H),3.79(dt d,J=11.0,4.3,3.5,1.9Hz,2H),3.56–3.35(m,2H),2.92(dd,J=12.1,2.4Hz,1H),2.84–2.73 (m,1H),2.73–2.62(m,2H),1.24(d,J=7.4Hz,1H),1.02–0.92(m,4H).ESI-MS:m / z=495[M+H] + .

[0124] Example 5 (S)-N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A5)

[0125]

[0126] Synthesis Step 1 Synthesis of Intermediate 5-3

[0127] Synthesis steps: Referring to steps 4 and 5 of Example 2, (S)-N-Boc-2-hydroxymethylmorpholine was used instead of 4-Boc-2-hydroxymethylmorpholine to obtain intermediate 5-3, ESI-MS: m / z=334 [M+H] + .

[0128] Synthesis Step 2 Synthesis of (S)-N-(3-(difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A5)

[0129] Synthesis steps: Refer to step 8 of Example 1, replace intermediate 1-3 with intermediate 5-3 to obtain compound A5, 1H NMR(400MHz, DMSO-d6)δ8.63(d,J=7.6Hz,1H),8.32(s,1H),7.44(t,J=1.7Hz,1H),7.22–7 .11(m,2H),6.90(t,J=2.1Hz,1H),6.72(dd,J=7.6,2.6Hz,1H),4.09(d,J=5.0Hz,2H),3.83 (dddd,J=13.2,8.5,4.2,2.0Hz,2H),3.55(td,J=11.1,3.0Hz,1H),2.97(dd,J=12.3,2.4H z,1H),2.81–2.73(m,2H),1.23(s,2H),0.99(dt,J=7.7,2.2Hz,4H).ESI-MS:m / z=495[M+H] + .

[0130] Example 6 N-(3-(difluoromethoxy)-5-(6-(2-morpholinylethoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A6)

[0131]

[0132] Synthesis Step 1 Synthesis of Intermediate 6-2

[0133] Dissolve 6-bromopyrazolo[1,5-A]pyridine 6-1 (91 mg, 0.46 mmol) in DMSO solution (2 mL), add diboric acid pinacol ester (142 mg, 0.56 mmol), potassium acetate (147 mg, 1.5 mmol), and react overnight at 80°C under nitrogen protection. After the reaction, extract with EA three times and combine the organic phases, dry with anhydrous sodium sulfate, and distill under reduced pressure to remove the solvent, and purify with column chromatography (PE:EA=2:1) ​​to obtain compound 6-2. ESI-MS: m / z=197[M+H] + .

[0134] Synthesis step 2 Synthesis of intermediate 6-3

[0135] Intermediate 6-2 (732 mg, 3 mmol) was dissolved in THF (10 mL), and H2O2 (1.02 g, 9 mmol) and NaOH (360 mg, 4.5 mL, 9 mmol) were added under ice bath conditions. The reaction was completed after 30 min. The solvent was removed by distillation under reduced pressure and the product was directly used for the next step reaction.

[0136] Synthesis step 3 Synthesis of intermediate 6-4

[0137] Synthesis steps Referring to step 2 of Example 5, 4-(2-bromoethyl)morpholine hydrobromide was used to replace intermediate 5-2, and intermediate 2-5 was replaced by intermediate 6-3 to obtain intermediate 6-4, yield: 42%; ESI-MS: m / z=248[M+1] + .

[0138] Synthesis Step 3 Synthesis of N-(3-(difluoromethoxy)-5-(6-(2-morpholinylethoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A6)

[0139] Synthesis steps: Referring to step 8 of Example 1, intermediate 6-4 was used instead of intermediate 1-3 to obtain compound A6, ESI-MS: m / z=509 [M+H] + .

[0140] Example 7 (S)-N-(3-cyano-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A7)

[0141]

[0142] Synthesis Step 1 Synthesis of Intermediate 7-2

[0143] Synthesis steps: Refer to step 6 of Example 1, and use 5-amino-3-bromobenzonitrile 7-1 to replace intermediate 1-7 to obtain intermediate 7-2. ESI-MS: m / z=301 [M+H] + .

[0144] Synthesis Step 2 Synthesis of Intermediate 7-3

[0145] Synthesis steps: Refer to step 7 of Example 1, and use intermediate 7-2 instead of intermediate 1-8 to obtain intermediate 7-3. ESI-MS: m / z=401[M+H] + .

[0146] Synthesis Step 3 Synthesis of (S)-N-(3-cyano-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A7)

[0147] Synthesis steps: Referring to step 8 of Example 1, intermediate 5-3 was used to replace intermediate 1-3, and intermediate 7-3 was used to replace intermediate 1-9 to obtain compound A7, ESI-MS: m / z=454 [M+H] + .

[0148] Example 8 N-(3-(difluoromethoxy)-5-(7-(2-morpholinylethoxy)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A8)

[0149]

[0150] Synthesis Step 1 Synthesis of Intermediate 8-2

[0151] Synthesis steps: Referring to step 1 of Example 1, 2-amino-4-fluoropyridine was used instead of 2-amino-4-bromopyridine to obtain intermediate 8-2, ESI-MS: m / z=137 [M+H] + .

[0152] Synthesis step 2 Synthesis of intermediate 8-3

[0153] Synthesis steps: Refer to step 5 of Example 2, replace intermediate 2-7 with 2-morpholinoethanol to obtain intermediate 8-3, ESI-MS: m / z=248[M+H] + .

[0154] Synthesis Step 3 Synthesis of N-(3-(difluoromethoxy)-5-(7-(2-morpholinylethoxy)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A8)

[0155] Synthesis steps: Referring to step 8 of Example 1, intermediate 8-3 was used instead of intermediate 1-3 to obtain compound A8; 1 H NMR(400MHz,DMSO-d6)δ10.15(s,1H),8.40(d,J=7.5Hz,1H),7.65(s,1H),7.3 1(d,J=13.4Hz,2H),7.20–7.11(m,1H),7.10–6.99(m,2H),6.73(dd,J=7.6,2. 6Hz,1H),4.19(t,J=5.6Hz,2H),3.58(t,J=4.5Hz,4H),3.38(s,4H),2.85–2.7 7(m,1H),2.73(t,J=5.6Hz,2H),0.99(d,J=6.4Hz,4H).ESI-MS:m / z=509[M+H] + .

[0156] Example 9 (S)-N-(3-difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)methanesulfonamide (A9)

[0157]

[0158] Synthesis Step 1 Synthesis of Intermediate 9-1

[0159] Synthesis steps: Referring to step 6 of Example 1, methanesulfonyl chloride was used instead of cyclopropanesulfonyl chloride to obtain intermediate 9-1, ESI-MS: m / z=316 [M+H] + .

[0160] Synthesis Step 2 Synthesis of Intermediate 9-2

[0161] Synthesis steps: Refer to step 7 of Example 1, and use intermediate 9-1 instead of intermediate 1-8 to obtain intermediate 9-2. ESI-MS: m / z=416 [M+H] + .

[0162] Synthesis Step 3 Synthesis of (S)-N-(3-difluoromethoxy)-5-(5-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)methanesulfonamide (A9)

[0163] Synthesis steps: Referring to step 8 of Example 1, intermediate 9-2 was used to replace intermediate 1-9, and intermediate 5-3 was used to replace intermediate 1-3 to obtain compound A9. ESI-MS: m / z=469 [M+H] + .

[0164] Example 10 (S)-N-(3-(difluoromethoxy)-5-(6-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)methanesulfonamide (A10)

[0165]

[0166] Synthesis Step 1 Synthesis of Intermediate 10-1

[0167] Synthesis steps: Referring to step 5 of Example 2, intermediate 2-7 is replaced by (S)-N-Boc-2-hydroxymethylmorpholine, and intermediate 2-5 is replaced by intermediate 6-3 to obtain intermediate 10-1, ESI-MS: m / z=334 [M+H] + .

[0168] Synthesis Step 3 Synthesis of (S)-N-(3-(difluoromethoxy)-5-(6-(morpholine-2-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)methanesulfonamide (A10)

[0169] Synthesis steps: Referring to step 8 of Example 1, intermediate 9-2 was used to replace intermediate 1-9, and intermediate 10-1 was used to replace intermediate 1-3 to obtain compound A9, ESI-MS: m / z=469 [M+H] + .

[0170] Example 11 (S)-N-(3-(difluoromethoxy)-5-(5-(pyrrole-3-methoxy)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A11)

[0171]

[0172] Synthesis steps: Referring to Example 4 of Compound A4, (S)-1-BOC-3-hydroxymethylpyrrolidine was used instead of (R)-N-Boc-2-hydroxymethylmorpholine to obtain Compound A11, ESI-MS: m / z=479 [M+H] + .

[0173] Example 12 N-(3-(difluoromethoxy)-5-(6-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A12)

[0174]

[0175] Synthesis Step 1 Synthesis of Intermediate 12-3

[0176] 4-Pyrazoleboronic acid pinacol ester (388 mg, 2.0 mmol), methyl oxirane (173 mg, 2.4 mmol), cesium carbonate (1.3 g, 4.0 mmol) were dissolved in DMF and heated to 80 ° C for 16 h. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography to obtain intermediate 12-3 (160 mg). ESI-MS: m / z = 267 [M + H] +

[0177] Synthesis step 2 Synthesis of intermediate 12-4

[0178] Intermediate 12-3 (160 mg, 0.6 mmol), intermediate 6-1 (118 mg, 0.6 mmol), Pd(dppf)2Cl2 (22 mg, 0.03 mmol), sodium carbonate (127 mg, 1.2 mmol) were dissolved in a mixed solution of 1,4-dioxane and water (1,4-dioxane: water = 4:1), replaced with nitrogen protection, and heated to 80 ° C for 18 hours. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography to obtain product 12-4 (90 mg). ESI-MS: m / z = 257 [M+H] + .

[0179] Synthesis Step 3 Synthesis of N-(3-(difluoromethoxy)-5-(6-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A12)

[0180] Synthesis steps: Referring to step 8 of Example 1, intermediate 12-4 was used instead of intermediate 1-3 to obtain compound A12, ESI-MS: m / z=518 [M+H] +

[0181] Example 13 N-(3-(difluoromethoxy)-5-(5-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A13)

[0182]

[0183] Synthesis Step 1 Synthesis of Intermediate 13-3

[0184] Synthesis steps: Refer to step 2 of Example 12, replace compound 6-1 with compound 5-bromopyrazolo[1,5-A]pyridine 13-2, and replace intermediate 12-3 with intermediate 13-1 to obtain intermediate 13-3, ESI-MS: m / z=199[M+H] +

[0185] Synthesis Step 2 Synthesis of N-(3-(difluoromethoxy)-5-(5-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A13)

[0186] Synthesis steps: Referring to step 8 of Example 1, intermediate 13-3 was used instead of intermediate 1-3 to obtain compound A13, ESI-MS: m / z=460 [M+H] +

[0187] Example 14 N-(3-(difluoromethoxy)-5-(7-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A14)

[0188]

[0189] Synthesis Step 1 Synthesis of Intermediate 14-1

[0190] Synthesis steps: Refer to step 2 of Example 12, replace compound 6-1 with intermediate 1-2 to obtain intermediate 14-1, ESI-MS: m / z=257 [M+H] +

[0191] Synthesis Step 2 Synthesis of N-(3-(difluoromethoxy)-5-(7-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)imidazo[1,2-a]pyridin-3-yl)phenyl)cyclopropanesulfonamide (A14)

[0192] Synthesis steps: Referring to step 8 of Example 1, intermediate 14-1 was used instead of intermediate 1-3 to obtain compound A14, ESI-MS: m / z=518 [M+H] +

[0193] Example 15 N-(3-(difluoromethoxy)-5-(6-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)phenyl)cyclopropanesulfonamide (A15)

[0194]

[0195] Synthesis Step 1 Synthesis of Intermediate 15-2

[0196] Synthesis steps: Refer to step 2 of Example 12, replace compound 6-1 with 6-bromopyrazolo[1,5-A]pyrimidine 15-1 to obtain intermediate 15-2, ESI-MS: m / z=258[M+H] +

[0197] Synthesis Step 2 Synthesis of N-(3-(difluoromethoxy)-5-(6-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrimidin-3-yl)phenyl)cyclopropanesulfonamide (A15)

[0198] Synthesis steps: Referring to step 8 of Example 1, intermediate 15-2 was used instead of intermediate 1-3 to obtain compound A15, ESI-MS: m / z=519 [M+H] +

[0199] Example 16 N-(3-(Difluoromethoxy)-5-(5-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)phenyl)methanesulfonamide (A16)

[0200]

[0201] Synthesis Step 1 Synthesis of Intermediate 16-2

[0202] Synthesis steps: Refer to step 4 of Example 1, use 3-hydroxy-5-nitrobenzoic acid 16-1 instead of intermediate 1-5 to obtain intermediate 16-2.

[0203] Synthesis Step 2 Synthesis of Intermediate 16-3

[0204] Synthesis steps: Refer to step 5 of Example 1, and use intermediate 16-2 instead of intermediate 1-6 to obtain intermediate 16-3. ESI-MS: m / z=204 [M+H] + .

[0205] Synthesis step 3 Synthesis of intermediate 16-4

[0206] Synthesis steps: Refer to step 6 of Example 1, and use intermediate 16-3 instead of intermediate 1-7 to obtain intermediate 16-4. ESI-MS: m / z=282[M+H] + .

[0207] Synthesis Step 4 Synthesis of Intermediate 16-5

[0208] Intermediate 16-4 (1128 mg, 4.0 mmol), DPPA (1.3 g, 4.8 mmol) and DIEA (1.5 g, 12.0 mmol) were dissolved in 10 mL 1,4-dioxane and stirred at room temperature for 0.5 h; then 10 mL water was added and the temperature was raised to 90 °C for 6 h. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 4: 1) to obtain intermediate 16-5. ESI-MS: m / z = 253 [M + H] +

[0209] Synthesis Step 5 Synthesis of Intermediate 16-7

[0210] The intermediate 16-5 (329 mg, 1.3 mmol), 4-bromo-1-iodo-2-nitrobenzene 16-6 (511 mg, 1.6 mmol), Pd2(dba)3 (59 mg, 0.065 mmol), BINAP (80.9 mg, 0.13 mmol), 18-crown-6 (17 mg, 0.065 mmol) and potassium carbonate (358 mg, 2.6 mmol) were dissolved in toluene and heated to 100 ° C under nitrogen protection to react overnight. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 10: 1) to obtain 16-7. ESI-MS: m / z = 452 [M + H] +

[0211] Synthesis Step 6 Synthesis of Intermediate 16-8

[0212] Synthesis steps: Refer to step 5 of Example 1, and use intermediate 16-7 to replace intermediate 1-6 to obtain intermediate 16-8. ESI-MS: m / z=422 [M+H] + .

[0213] Synthesis Step 7 Synthesis of Intermediate 16-9

[0214] Intermediate 16-8 (287 mg, 0.62 mmol), trimethyl orthoformate (197 mg, 1.86 mmol) and p-toluenesulfonic acid (117 mg, 0.68 mmol) were dissolved in tetrahydrofuran and heated to 70°C for 1 h. After the reaction was completed, the mixture was directly concentrated under reduced pressure and purified by column chromatography (PE:EA=4:1) to obtain intermediate 16-9. ESI-MS: m / z=432[M+H] +

[0215] Synthesis Step 8 Synthesis of N-(3-(difluoromethoxy)-5-(5-(1-(2-hydroxy-2-methylpropyl)-1H-pyrazol-4-yl)-1H-benzo[d]imidazol-1-yl)phenyl)methanesulfonamide (A16)

[0216] Synthesis steps: Referring to step 8 of Example 1, intermediate 16-9 was used to replace intermediate 1-3, and intermediate 12-3 was used to replace intermediate 1-9 to obtain compound A16, ESI-MS: m / z=492 [M+H] +

[0217] Example 17 3-(2-(Difluoromethoxy)-6-methoxypyridin-4-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A17)

[0218]

[0219] Synthesis Step 1 Synthesis of Intermediate 17-2

[0220] 4-Bromo-2-methoxypyridine (1.88 g, 10.0 mmol) and m-chloroperbenzoic acid (2.0 g, 11.6 mmol) were dissolved in dichloromethane and stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain product 17-2 (1.0 g). ESI-MS: m / z=204 [M+H] +

[0221] Synthesis Step 2 Synthesis of Intermediate 17-3

[0222] Intermediate 17-2 (406 mg, 2.0 mmol) and triethylamine (1.0 g, 10.0 mmol) were dissolved in 1,4-dioxane, trifluoroacetic anhydride (630 mg, 6.0 mmol) was added under ice bath, and stirred at room temperature overnight. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain intermediate 17-3 (200 mg). ESI-MS: m / z=204[M+H] +

[0223] Synthesis Step 3 Synthesis of Intermediate 17-4

[0224] The intermediate 17-3 (305 mg, 1.5 mmol) was dissolved in acetonitrile, and ethyl bromodifluoroacetate (609 mg, 3.0 mmol), DBU (342 mg, 2.3 mmol) and tert-butyl hydroperoxide (386 mg, 3.0 mmol) were added in sequence under a nitrogen atmosphere, and heated to 80°C and stirred overnight. After the reaction was completed, the mixture was concentrated under reduced pressure and purified by column chromatography to obtain the product 17-4 (300 mg). ESI-MS: m / z=254 [M+H] +

[0225] Synthesis Step 4 Synthesis of Intermediate 17-5

[0226] Synthesis steps: Refer to step 2 of Example 12, replace compound 12-3 with intermediate 13-3 to obtain intermediate 17-5, ESI-MS: m / z=199 [M+H] +

[0227] Synthesis Step 5 Synthesis of 3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)-6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A17)

[0228] Synthesis steps: Referring to step 8 of Example 1, intermediate 17-5 was used to replace intermediate 1-3, and intermediate 17-4 was used to replace intermediate 1-9 to obtain compound A17, ESI-MS: m / z=372 [M+H] + ; 1 H NMR(400MHz,Chloroform-d)δ8.65–8.58(m,1H),8.18(s,1H),7.84(d,J=9.2Hz,1H),7.77(s,1H),7.65(d,J=3.0Hz,1H),7 .47(t,J=73.6Hz,1H),7.40(dd,J=9.2,1.6Hz,1H),6.73(d,J=1.1Hz,1H),6.69(d,J=1.2Hz,1H),3.98(s,3H),3.93(s,3H).

[0229] Example 18 1-(4-(3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A18)

[0230]

[0231] Synthesis steps: Referring to step 8 of Example 1, intermediate 12-4 was used to replace intermediate 1-3, and intermediate 17-4 was used to replace intermediate 1-9 to obtain compound A18, ESI-MS: m / z=430 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.58–8.52(m,1H),8.12(s,1H),7.81–7.69(m,3H),7.45(t,J=73.6Hz,1H),7.69(m,3H ),7.33(dd,J=9.2,1.6Hz,1H),6.66(d,J=1.2Hz,1H),6.62(d,J=1.2Hz,1H),4.11(s,2H),3.90(s,3H),1.22(s,6H).

[0232] Example 19 4-(2-((3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)oxy)ethyl)morpholine (A19)

[0233]

[0234] Synthesis steps: Referring to step 8 of Example 1, intermediate 6-4 was used to replace intermediate 1-3, and intermediate 17-4 was used to replace intermediate 1-9 to obtain compound A19, ESI-MS: m / z=421 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.11(d,J=2.9Hz,2H),7.72(d,J=9.7Hz,1H),7.68–7.20(m,1H),7.46(t,J=73.6Hz,1H),7.11(dd,J=9.7,2.1Hz,1H),6 .70(d,J=1.0Hz,1H),6.66(d,J=1.1Hz,1H),4.12(t,J=5.5Hz,2H),3.92(s ,3H),3.75(t,J=4.7Hz,4H),2.84(t,J=5.5Hz,2H),2.59(t,J=4.7Hz,4H).

[0235] Example 20 (R)-2-(((3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-5-yl)oxy)methyl)morpholine (A20)

[0236]

[0237] Synthesis steps: Referring to step 8 of Example 1, intermediate 4-3 was used instead of intermediate 1-3, and intermediate 17-4 was used instead of intermediate 1-9 to obtain compound A20, ESI-MS: m / z=407 [M+H] + . 1 H NMR (400MHz, Methanol-d4) δ8.21(d,J=7.5Hz,1H),8.03(s,1H),7.50(t,J=73.4Hz,1H),6.84(d,J=2.6Hz,1H),6.51(td,J=6.6,6.0,1.9Hz, 3H),3.98–3.84(m,7H),3.67(ddd,J=11.5,10.1,4.0Hz,1H),2.97(dd,J=12.5,2.4Hz,1H),2.89–2.79(m,2H),2.72(dd,J=12.5,10.6Hz,1H).

[0238] Example 21 (S)-2-(((3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)oxy)methyl)morpholine (A21)

[0239]

[0240] Synthesis steps: Referring to step 8 of Example 1, intermediate 10-1 was used to replace intermediate 1-3, and intermediate 17-4 was used to replace intermediate 1-9 to obtain compound A21, ESI-MS: m / z=407 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.49(d,J=2.0Hz,2H),7.99(d,J=9.8Hz,1H),7.73(t,J=73.4Hz,1H),7.21(dd,J=9.7,2.2Hz,1H),6.91(s,2H),4.00 (d,J=5.0Hz,2H),3.86(s,3H),3.75(d,J=3.2Hz,1H),3.50(td,J=11.1,2.9Hz,2H),2.96–2.89(m,1H),2.78–2.64(m,2H),2.62–2.52(m,1H).

[0241] Example 22 2-((3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)imidazo[1,2-a]pyridin-7-yl)oxy)-N,N-dimethylethylamine (A22)

[0242]

[0243] Synthesis Step 1 Synthesis of Intermediate 22-2

[0244] 2-Amino-4-hydroxypyridine (1.0 mmol) and chloroacetaldehyde were placed in a round-bottom flask, and then acetonitrile (3 mL) was added to fully dissolve the raw materials, and stirred at 90°C for 10 h. After the reaction was completed, the solvent was removed in vacuo, and column chromatography was used for purification (DCM: MeOH = 15: 1) to obtain a light yellow solid product (1-2). Yield: 90.6%; ESI-MS: m / z = 135 [M+H] + .

[0245] Synthesis Step 1 Synthesis of Intermediate 22-3

[0246] Intermediate 22-2 (1.0 mmol), N,N-dimethylaminoethyl bromide hydrobromide (1.1 mmol), K2CO3 (3.0 mmol) were placed in a round-bottom flask, and dioxane: water = 20:1 (4 mL) was added, and stirred at 80°C for 6 h. After the reaction was completed, it was extracted three times with dichloromethane / water, the organic phases were combined and dried over anhydrous Na2SO4, and the solvent was removed in vacuo to obtain a yellow oily product intermediate 22-3. Yield: 27.5%; ESI-MS: m / z = 206 [M+H] + .

[0247] Synthesis Step 3 Synthesis of 2-((3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)imidazo[1,2-a]pyridin-7-yl)oxy)-N,N-dimethylethylamine (A22)

[0248] Synthesis steps: Referring to step 8 of Example 1, intermediate 22-3 was used to replace intermediate 1-3, and intermediate 17-4 was used to replace intermediate 1-9 to obtain compound A22, ESI-MS: m / z=379 [M+H] + . 1 H NMR (400MHz, Methanol-d4) δ8.48(d,J=7.6Hz,1H),7.74(s,1H),7.59(t,J=73.1Hz,1H),7.06(d,J=2.6Hz,1H),6.87(dd,J=7.6, 2.5Hz,1H),6.77(d,J=1.4Hz,1H),6.72(d,J=1.4Hz,1H),4.48(t,J=4.9Hz,2H),3.95(s,3H),3.52(t,J=4.9Hz,2H),2.90(s,6H).

[0249] Example 23 1-(4-(3-(2-(difluoromethoxy)-6-methoxypyridin-4-yl)imidazo[1,2-a]pyridin-7-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A23)

[0250]

[0251] Synthesis steps: Referring to step 8 of Example 1, intermediate 14-1 was used instead of intermediate 1-3, and intermediate 17-4 was used instead of intermediate 1-9 to obtain compound A23, ESI-MS: m / z=430 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ8.68(d,J=7.2Hz,1H),8.29(s,1H),8.07(s,1H),8.01(s,1H),7.91(d,J=1.8Hz,1H),7.77(t ,J=73.2Hz,1H),7.27(dd,J=7.3,1.9Hz,1H),6.98–6.92(m,2H),4.76(s,1H),4.02(s,2H),3.89(s,3H),1.07(s,6H).

[0252] Example 24 3-(2,6-difluoromethoxy-4-)-6-(1-(tetrahydropyran-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A24)

[0253]

[0254] Synthesis Step 1 Synthesis of Intermediate 24-2

[0255] 4-Bromo-2,6-difluoropyridine (24-1, 582 mg, 3 mmol) was placed in a round-bottom flask, 15 mL of methanol was added to fully dissolve, and sodium hydroxide (240 mg, 6 mmol) was added to fully dissolve, and stirred in an oil bath at 70°C for 12 h. After the solvent was removed in vacuo, column chromatography was used for purification (pure PE) to obtain 660 mg of the product as a white solid (24-2). ESI-MS: m / z = 219 [M+H] + .

[0256] Synthesis Step 2 Synthesis of Intermediate 24-4

[0257] Synthesis steps: Referring to step 2 of Example 12, 1-(tetrahydropyran-4-yl)-1H-pyrazole-4-boronic acid pinacol ester (24-3) was used instead of compound 12-3 to obtain intermediate 24-4, ESI-MS: m / z=269 [M+H] +

[0258] Synthesis Step 3 Synthesis of 3-(2,6-difluoromethoxy-4-)-6-(1-(tetrahydropyran-4-yl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A24)

[0259] Synthesis steps: Referring to step 8 of Example 1, intermediate 24-4 was used to replace intermediate 1-3, and intermediate 24-2 was used to replace intermediate 1-9 to obtain compound A24, ESI-MS: m / z=406 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.62(s,1H),8.17(s,1H),7.85(d,J=9.2Hz,1H),7.80(s,1H),7.71(s,1H),7.36(dd,J=9.2,1.5Hz,1H), 6.55(s,2H),4.40(dd,J=10.4,5.0Hz,1H),4.14(dt,J=11.6,3.4Hz,2H),3.97(s,6H),3.61–3.50(m,2H),2.15(dt,J=6.6,3.8Hz,4H).

[0260] Example 25 2-(4-(3-(2,6-dimethoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-N,N-dimethylethylamine (A25)

[0261]

[0262] Synthesis Step 1 Synthesis of Intermediate 25-1

[0263] 4-Pyrazoleboronic acid pinacol ester (1.0 g, 5.15 mmol) and dimethylaminochloroethane hydrochloride (1.11 g, 7.73 mmol) were added to 30 mL of acetonitrile, cesium carbonate (3.36 g, 10.31 mmol) was added to the reaction solution, and the temperature was raised to 90°C and stirred for 16 h. After the reaction was completed, water (60 mL) was added to the reaction solution, and it was extracted with EtOAc (3×60 ml), and the organic phase was washed with saturated brine (3×30 ml), dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation. The product was directly used in the next step reaction.

[0264] Synthesis Step 2 Synthesis of Intermediate 25-2

[0265] Compound 25-1 (800 mg, 3.02 mmol), 6-bromopyrazolo[1,5-a]pyridine (594 mg, 3.02 mmol), Pd(dppf)Cl2 (221 mg, 0.302 mmol) and sodium carbonate (640 mg, 6.03 mmol) were added to a mixed solvent of 10 mL 1,4-dioxane and 1 mL H2O, and the mixture was heated to 100 °C and stirred for 7 h under N2 atmosphere. After the reaction was completed, the solid was removed by filtration, and the filtrate was dried by spin drying and purified with DCM:MeOH=30:1 to obtain 530 mg of a black oil. ESI-MS: m / z=256[M+H] +

[0266] Synthesis Step 3 Synthesis of 2-(4-(3-(2,6-dimethoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-N,N-dimethylethylamine (A25)

[0267] Synthesis steps: Referring to step 8 of Example 1, intermediate 25-2 is used to replace intermediate 1-3, and intermediate 24-2 is used to replace intermediate 1-9 to obtain compound A25. 1 H NMR (400MHz, Chloroform-d) δ8.62(s,1H),8.17(s,1H),7.85(d,J=9.2Hz,1H),7.79(d,J=4.9Hz,2H),7.37(d,J=9.3 Hz,1H),6.55(s,2H),4.34(t,J=6.4Hz,2H),3.97(s,6H),2.89(t,J=6.4Hz,2H),2.35(s,6H).ESI-MS:m / z=393[M+H] +

[0268] Example 26 1-(4-(3-(2,6-dimethoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A26)

[0269]

[0270] Synthesis steps: Referring to step 8 of Example 1, intermediate 12-4 was used to replace intermediate 1-3, and intermediate 24-2 was used to replace intermediate 1-9 to obtain compound A26, ESI-MS: m / z=394 [M+H] + . 1H NMR (400MHz, Chloroform-d) δ8.63(dd,J=1.6,0.9Hz,1H),8.19(s,1H),7.87(dd,J=9.3,1.0Hz,1H),7.84(d,J=0. 8Hz,1H),7.73(d,J=0.9Hz,1H),7.37(dd,J=9.3,1.6Hz,1H),6.55(s,2H),4.14(s,2H),3.98(s,6H),1.23(s,6H).

[0271] Example 27 3-(2,6-dimethoxypyridin-4-yl)-6-(1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A27)

[0272]

[0273] Synthesis Step 1 Synthesis of Intermediate 27-1

[0274] 4-Pyrazoleboronic acid pinacol ester (1.5g, 7.73mmol) and 2,2,2-trifluoroiodoethane (2.4g, 11.60mmol) were added to 30mL of acetonitrile, cesium carbonate (3.8g, 11.60mmol) was added to the reaction solution, and the temperature was raised to 90°C and stirred for 16h. After the reaction was completed, water (60mL) was added to the reaction solution, and it was extracted with EtOAc (3×60ml), and the organic phase was washed with saturated brine (3×30ml) and dried over anhydrous sodium sulfate. Column chromatography purification (PE:EtOAc=2:1) ​​gave 570mg of a light yellow oil.

[0275] Synthesis Step 2 Synthesis of Intermediate 27-2

[0276] Synthesis steps: Refer to step 2 of Example 12, replace compound 12-3 with intermediate 27-1 to obtain intermediate 27-2, ESI-MS: m / z=267 [M+H] +

[0277] Synthesis Step 3 Synthesis of 3-(2,6-dimethoxypyridin-4-yl)-6-(1-(2,2,2-trifluoroethyl)-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridine (A27)

[0278] Synthesis steps: Referring to step 8 of Example 1, intermediate 27-2 is used to replace intermediate 1-3, and intermediate 24-2 is used to replace intermediate 1-9 to obtain compound A27. 1H NMR(400MHz,Chloroform-d)δ8.64(s,1H),8.19(s,1H),7.88(t,J=4.6Hz,2H),7.79(s,1H),7.3 6(dd,J=9.2,1.6Hz,1H),6.55(s,2H),4.78(q,J=8.3Hz,2H),3.97(s,6H).ESI-MS:m / z=404[M+H] +

[0279] Example 28 1-(4-(3-(2,6-dimethoxypyridin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A28)

[0280]

[0281] Synthesis steps: Referring to step 8 of Example 1, intermediate 15-2 was used to replace intermediate 1-3, and intermediate 24-2 was used to replace intermediate 1-9 to obtain compound A28, ESI-MS: m / z=395 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.46(dd,J=2.2,1.0Hz,1H),8.34(d,J=2.2Hz,1H),8.06(d,J=2.4Hz,1H),7 .82(s,1H),6.65(dd,J=2.4,1.0Hz,1H),6.20(s,2H),4.67(s,1H),3.99(s,2H),3.94(s,6H),1.08(s,6H).

[0282] Example 29 1-(4-(1-(2,6-dimethoxypyridin-4-yl)-1H-benzo[d]imidazol-5-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A29)

[0283]

[0284] Synthesis Step 1 Synthesis of Intermediate 29-2

[0285] 2,6-dimethoxypyridine-4-carboxylic acid 29-1 (732 mg, 4.0 mmol), DPPA (1.3 g, 4.8 mmol) and DIEA (1.5 g, 12.0 mmol) were dissolved in 10 mL 1,4-dioxane and stirred at room temperature for 0.5 h; then 10 mL water was added and the temperature was raised to 90 ° C for 6 h. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 4: 1) to obtain the product 29-2 (420 mg). ESI-MS: m / z = 155 [M + H] +

[0286] Synthesis Step 2 Synthesis of Intermediate 29-4

[0287] Intermediate 29-2 (200 mg, 1.3 mmol), 4-bromo-1-iodo-2-nitrobenzene 29-3 (511 mg, 1.6 mmol), Pd2(dba)3 (59 mg, 0.065 mmol), BINAP (80.9 mg, 0.13 mmol), 18-crown-6 (17 mg, 0.065 mmol) and potassium carbonate (358 mg, 2.6 mmol) were dissolved in toluene and heated to 100 ° C under nitrogen protection to react overnight. After the reaction was completed, it was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and purified by column chromatography (PE: EA = 10: 1) to obtain 29-4 (270 mg). ESI-MS: m / z = 354 [M + H] +

[0288] Synthesis Step 3 Synthesis of Intermediate 29-5

[0289] Intermediate 29-4 (270 mg, 0.76 mmol) and zinc powder (149 mg, 2.29 mmol) were dissolved in 2 mL of a mixed solution of acetic acid and methanol (AcOH: MeOH = 1:1) and reacted at room temperature for 4 h. After the reaction was completed, the reaction solution was adjusted to a weak alkaline state with a saturated sodium bicarbonate aqueous solution, and then extracted three times with ethyl acetate. The organic phases were combined and concentrated under reduced pressure to obtain a crude product 29-5 (210 mg), which was used directly in the next step without further purification. ESI-MS: m / z = 324 [M+H] +

[0290] Synthesis Step 4 Synthesis of Intermediate 29-6

[0291] Intermediate 29-5 (200 mg, 0.62 mmol), trimethyl orthoformate (197 mg, 1.86 mmol) and p-toluenesulfonic acid (117 mg, 0.68 mmol) were dissolved in tetrahydrofuran and heated to 70°C for 1 h. After the reaction was completed, the mixture was directly concentrated under reduced pressure and purified by column chromatography (PE:EA=4:1) to obtain 29-6 (130 mg). ESI-MS: m / z=334 [M+H] +

[0292] Synthesis Step 5 Synthesis of 1-(4-(1-(2,6-dimethoxypyridin-4-yl)-1H-benzo[d]imidazol-5-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A29)

[0293] Intermediate 29-6 (120 mg, 0.36 mmol), intermediate 12-3 (94 mg, 0.36 mmol), Pd(PPh3)4 (42 mg, 0.036 mmol) and potassium carbonate (149 mg, 1.08 mmol) were dissolved in 1,4-dioxane and heated to 90°C under nitrogen protection for overnight reaction. After the reaction was completed, the mixture was extracted three times with ethyl acetate and saturated brine, the organic phases were combined, concentrated under reduced pressure, and semi-preparative purification was performed to obtain A29. 1 H NMR(400MHz,Chloroform-d)δ8.15(s,1H),7.93(d,J=1.7Hz,1H),7.86(s,1H),7.71(s,1H),7.64(d,J=8.4Hz,1H),7 .48(dd,J=8.5,1.7Hz,1H),6.49(s,2H),4.11(s,2H),4.00(s,6H),3.84(s,1H),1.22(s,6H).ESI-MS:m / z=394[M+H] +

[0294] Example 30 1-(4-(3-(2,6-dimethoxypyridin-4-yl)pyrazolo[1,5-a]pyridin-6-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A30)

[0295]

[0296] Synthesis steps: Referring to step 8 of Example 1, intermediate 12-4 was used to replace intermediate 1-3, and compound 24-1 was used to replace intermediate 1-9 to obtain compound A30, ESI-MS: m / z=370 [M+H] + . 1H NMR(400MHz,DMSO-d6)δ9.14(s,1H),8.71(s,1H),8.29(s,1H),8.22(d,J=9.3Hz,1H),8 .07(s,1H),7.75(d,J=9.3Hz,1H),7.48(s,2H),4.78(s,1H),4.04(s,2H),1.11(s,6H).

[0297] Example 31 1-(4-(3-(2,6-dimethoxypyridin-4-yl)imidazo[1,2-b]pyridazin-7-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A31)

[0298]

[0299] Synthesis Step 1 Synthesis of Intermediate 24-4

[0300] Synthesis steps: Refer to step 2 of Example 12, replace intermediate 6-1 with 7-chloroimidazole [1,2-B] pyridazine (31-1) to obtain intermediate 31-2, ESI-MS: m / z = 258 [M + H] +

[0301] Synthesis Step 2 Synthesis of 1-(4-(3-(2,6-dimethoxypyridin-4-yl)imidazo[1,2-b]pyridazine-7-yl)-1H-pyrazol-1-yl)-2-methyl-2-propanol (A31)

[0302] Synthesis steps: Referring to step 8 of Example 1, intermediate 31-2 was used to replace intermediate 1-3, and compound 24-2 was used to replace intermediate 1-9 to obtain compound A31, ESI-MS: m / z=395 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ9.01(d,J=2.2Hz,1H),8.39(s,2H),8.36(d,J=2.2Hz,1H ),8.16(s,1H),7.16(s,2H),4.78(s,1H),4.02(s,2H),3.84(s,6H),1.07(s,6H).

[0303] Comparative Example 1: Synthesis of Compound 1

[0304]

[0305] Synthesis step 1 Synthesis of intermediate I-2

[0306] Add 4-bromo-2,6-difluorobenzoic acid (5.25 g) and tert-butylamine (1.62 g) to an eggplant-shaped bottle, then add 120 mL of acetonitrile to fully dissolve the raw materials. Then add HATU (9.13 g) and DIEA (8.52 g) at 0°C, stir for 10 min, and then react at room temperature for 4 h. After the reaction is completed, extract with DCM (50 mL) / water 5 times and combine the organic phases, and dry the organic phases with anhydrous sodium sulfate. Spin dry the organic phase to obtain 5.25 g of the product as a white solid (I-2). ESI-MS: m / z=292[M+H] + .

[0307] Synthesis step 2 Synthesis of intermediate I-3

[0308] Add intermediate I-2 (5.25 g) and sodium methoxide (7.87 g) to an eggplant-shaped bottle, then add 100 mL of methanol to fully dissolve the raw materials, and react at 85 ° C overnight. After the reaction is completed, adjust the pH to 6-8 with 6N hydrochloric acid aqueous solution, then extract with DCM (60 mL) / water 3 times, combine the organic phases and dry with anhydrous sodium sulfate. The organic phase is spin-dried and purified by column chromatography (PE: EA = 10: 1-8: 1-5: 1) to obtain 4.95 g of the product as a white solid (I-3). ESI-MS: m / z = 317 [M+H] + .

[0309] Synthesis step 3 Synthesis of intermediate I-4

[0310] The intermediate I-3 (4.95 g) was dissolved in 40 mL of DCM, and 20 mL of boron trichloride (1 M DCM solution) was added dropwise at -20 ° C, and then the reaction was continued at -20 ° C for 2.5 h. After the reaction was completed, 50 mL of water was added to quench the reaction, and the pH was adjusted to 7-8 with a saturated NaHCO3 solution. The organic phases were combined and dried over anhydrous sodium sulfate after extraction with DCM (50 mL) / water for 3 times. The organic phase was spin-dried and purified by column chromatography (PE: EA = 8: 1) to obtain 2.50 g of the product as a white solid (I-4). ESI-MS: m / z = 303 [M + H] + .

[0311] Synthesis Step 4 Synthesis of Intermediate I-5

[0312] The intermediate I-4 (2.50 g) was dissolved in a solution of acetonitrile: water = 1: 1 (V: V), KOH (4.65 g) was slowly added at 0°C, and after stirring for 15 minutes, diethyl bromofluoromethylphosphonate was added dropwise, and then reacted at 40°C for 7 hours. After the reaction was completed, the pH was adjusted to 7 with 6N hydrochloric acid aqueous solution, and then extracted with DCM (50 mL) / water three times, and the organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was spin-dried and purified by column chromatography (PE: EA = 10: 1 ~ 8: 1 ~ 5: 1) to obtain the product as a white solid 2.85 g (I-5). ESI-MS: m / z = 353 [M + H] + .

[0313] Synthesis Step 5 Synthesis of Intermediate I-6

[0314] Dissolve the intermediate I-5 (2.85 g) in 25 mL of phosphorus oxychloride and react at 110 ° C overnight. After the reaction is completed, the reaction solution is cooled to room temperature and slowly added dropwise into 100 mL of ice water. Then, the organic phases are combined and dried over anhydrous sodium sulfate after extraction with DCM (50 mL) / water for 3 times. The organic phase is spin-dried and purified by column chromatography (PE: EA = 30: 1 to 20: 1) to obtain 1.7 g of the product as a white solid (I-6). ESI-MS: m / z = 278 [M+H] + .

[0315] Synthesis Step 6 Synthesis of 2-(difluoromethoxy)-6-methoxy-4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyridin-3-yl)benzonitrile (Compound 1)

[0316] Synthesis steps: Refer to step 8 of Example 1, replace intermediate 1-3 with intermediate 17-5, and replace intermediate 1-9 with intermediate 1-6 to obtain compound 1, ESI-MS: m / z=396 [M+H] + . 1 H NMR(400MHz,Chloroform-d)δ8.40(d,J=1.5Hz,1H),8.10(s,1H),7.93–7.74(m, 2H),6.70(s,1H),5.59(t,J=6.0Hz,1H),3.51(t,J=5.1Hz,4H),3.44(t,J=6.4Hz ,2H),3.25(d,J=5.9Hz,2H),3.21(q,J=6.2Hz,2H),2.58(t,J=5.1Hz,4H),2.47( q,J=7.2Hz,2H),2.41(d,J=6.4Hz,2H),1.89–1.70(m,6H),1.13(t,J=7.2Hz,3H).

[0317] Example 32 In vitro SIKs kinase activity inhibition experiment

[0318] ADP-Glo TM Kinase assay is a luminescent technique analysis of the ADP formed by the kinase reaction. In this particular study, the kinase reaction is composed of the phosphorylation of SIK1 (NCBI Reference Sequence: NP_775490.2), SIK2 (NCBI Reference Sequence: NP_001353615.1) or SIK3 (NCBI Reference Sequence: NP_001353615.1) by AMARA peptide substrate (manufacturer: Sangon Biotech, catalog number: P23855). In the second step, the kinase reaction is terminated, and all remaining ATP is exhausted. In the final step, ADP is converted into ATP and the newly synthesized ATP is measured by using luciferase / luciferin reaction. The light generated is measured using the Envision plate reader, and the luminescent signal obtained is positively correlated with the kinase activity.

[0319] Detection Kit: ADP-Glo TM Kinase Assay (Manufacturer: Promega, catalog number: V9102)

[0320] Reaction system:

[0321] 1. Prepare kinase reaction buffer: 25mM Tris-HCl (pH 7.5), 0.01% TritonX-100, 0.5mM EGTA, 2.5mM DTT, 5mM MgCl2

[0322] 2. Compound preparation: All compounds were dissolved and diluted in 100% DMSO.

[0323] 3. Take 5ul enzyme and 39.5ul kinase reaction buffer in a 96-well plate, add 0.5ul DMSO / positive drug / compound, and use them as DMSO control group, positive drug control group, and compound group respectively.

[0324] 4. Enzyme inactivation treatment: Take SIK1 / 2 / 3 enzyme and heat it in a 95℃ metal bath for 15 minutes. Take 5ul of the inactivated enzyme and 39.5ul of kinase reaction buffer in a 96-well plate, and add 0.5ul of DMSO as the enzyme inactivation group.

[0325] 5. Take 4ul of substrate AMARA (final concentration of 45uM) and 1ul of ATP (final concentration of 5uM), mix them, and add them to the 96-well plates of each group mentioned above.

[0326] 6. Centrifuge the 96-well plate at 1000 rpm for 10 seconds and incubate at room temperature for 1.5 hours.

[0327] 7. Take 5ul of the reaction solution into a 384-well white plate, add 5ul of ADP-Glo ​​reagent to stop the reaction and consume the unreacted ATP, centrifuge the 384-well plate at 1000rpm for 10 seconds, and incubate at room temperature for 40 minutes.

[0328] 8. Add 10ul of kinase assay reagent to the 384-well plate to convert ADP into ATP, centrifuge the 384-well plate at 1000rpm for 10 seconds, and incubate at room temperature for 30 minutes.

[0329] 9. Detection of luminescence on Tecan (SPARK) microplate reader

[0330] Data processing:

[0331] The inhibition rate was calculated as follows:

[0332] Inhibition rate (%) = [1-(RLU sample-RLU enzyme inactivation group) / (RLU DMSO group-RLU enzyme inactivation group)] × 100%

[0333] Data Analysis:

[0334] The log value of the concentration was used as the X-axis and the percentage inhibition rate was used as the Y-axis. The [Inhibitor] vs. normalized response--Variable slope of the analysis software GraphPad Prism9 was used to fit the dose-effect curve to obtain the IC50 value of each compound on the enzyme activity.

[0335] Table 1 Inhibition rate of SIKs kinase activity in vitro when treated with 500 nM compounds

[0336]

[0337] As can be seen from Table 1, the compounds of the present invention have a strong inhibitory effect on SIKs enzyme activity, especially in the SIK2 subtype that is more relevant to inflammatory diseases, and generally have an inhibitory activity comparable to or stronger than that of positive drugs.

[0338] The IC values ​​of the compounds of the present invention for inhibiting SIKs enzyme activity in vitro were further determined. 50 value.

[0339] Table 2 IC values ​​of compounds for inhibition of SIKs enzyme activity in vitro 50 value

[0340] Compound No. <![CDATA[SIK1(IC 50 ,nM)]]> <![CDATA[SIK2(IC 50 ,nM)]]> <![CDATA[SIK3(IC 50 ,nM)]]> GLPG3970 596.00 44.44 2.91 A10 1243.00 10.60 9.10 A17 50.07 3.98 7.26 A18 40.38 4.51 7.75 A21 >1000 38.07 28.54 A24 15.17 3.13 5.52 A26 58.83 8.15 9.47

[0341] As can be seen from Table 2, the compounds of the present invention have better inhibitory activity against SIK2 kinase than positive drugs, and have certain selectivity against SIK1 kinase.

[0342] Example 33 Endotoxin-induced acute inflammation in mice

[0343] Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria. The administration of LPS can induce systemic inflammation and cause a strong immune response in animals, which is mainly manifested as uncontrolled activation of inflammatory cells such as neutrophils and monocytes / macrophages, and the release of a large number of inflammatory factors, such as TNF-α.

[0344] Experimental materials: Lipopolysaccharide from Escherichia coli 055:B5 (LPS, manufacturer: Sigma-Aldrich, catalog number: L2880), Mouse TNF alpha Uncoated ELISA Kit (manufacturer: ThermoFishe, catalog number: 88-7324-88), Mouse IL-10 Uncoated ELISA Kit (manufacturer: ThermoFishe, catalog number: 88-7105-88).

[0345] Experimental animals: C57BL / 6N mice (male, SPF grade, 6-8 weeks old, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.)

[0346] Experimental operation:

[0347] The experimental group mice were orally gavaged with each test compound, and the control group and the modeling group were gavaged with an equal amount of solvent; at the Tmax of the test drug, LPS (20 mg / kg, 5 mL / kg) was injected intraperitoneally into the C57BL / 6N mice in the modeling group and the experimental group, and the control group mice were injected with sterile PBS.

[0348] Mice were sacrificed 90 minutes after LPS injection, and blood was collected in 1.5 mL EP tubes and centrifuged at low temperature to obtain serum, which was then subjected to cytokine analysis.

[0349] Cytokine detection:

[0350] The steps for IL-10 and TNF-α are the same and are carried out synchronously.

[0351] 1. Prepare the coating reagent with diluted 1× PBS, that is, add the capture antibody to PBS, add 100 μL of coating reagent to each well of the ELISA plate, and incubate at 4°C overnight.

[0352] 2. Prepare the ELISA plate in advance and bring it to room temperature. Pour out the coating reagent, add washing solution to each well, incubate for 1 minute, discard the washing solution, repeat 3 times, and completely pour out the residual liquid for the last time.

[0353] 3. Add 200 μL 1×ELISA Diluent to each well and block at room temperature for 1 hour.

[0354] 4. Wash at least once with washing solution, prepare standards and samples in advance, and dilute the standards in gradients according to the requirements of the kit; add 100μL 1×ELISA Diluent to the blank wells, and perform duplicate detection on both the standards and blank wells; dilute the samples with 1×ELISA Diluent, the dilution ratio of IL-10 is 1:4, and the dilution ratio of TNF is 1:2, and add 100μL to each well. After sealing with sealing film, incubate at room temperature for 2 hours.

[0355] 5. Dilute the detection antibody in advance at a dilution ratio of 1:250. Repeat step 2 for 3-5 times, add 100 μL of detection antibody to each well, seal the plate and incubate at room temperature for 1 hour.

[0356] 6. Dilute horseradish peroxidase in advance at a dilution ratio of 1:100. Repeat step 2 for 3-5 times, add 100 μL horseradish peroxidase to each well, seal the plate and incubate at room temperature for 30 minutes.

[0357] 7. Repeat the washing in step 2 5-7 times, add 100 μL 1× TMB to each well, and incubate at room temperature for 15 minutes.

[0358] 8. After color development, add 100 μL of stop solution to each well, and immediately read the OD 450nm on a microplate reader and process the data.

[0359] The calibration data were performed in the same experimental method.

[0360] Data processing:

[0361] The standard curve fitting formula was calculated using ELISA Calc to obtain the cytokine concentration (CONC) in each well.

[0362] The calculation is as follows:

[0363] Relative expression of TNFα=[(CONC experimental group×dilution factor) / (CONC modeling group×dilution factor)]×100%

[0364] Relative expression of IL-10 = [(CONC experimental group × dilution factor) / (CONC modeling group × dilution factor)] × 100%

[0365] Table 3 Effect of some compounds of the present invention on the inhibition of TNFα expression in the serum of acute inflammation mice

[0366]

[0367] Note: ND means not detected.

[0368] According to Table 3, the representative compounds of the present invention have comparable or better effects on inhibiting TNFα expression than the positive drugs.

[0369] Table 4 Promoting effect of some compounds of the present invention on IL-10 secretion in serum of acute inflammatory mice

[0370]

[0371] Note: ND means not detected.

[0372] According to Table 4, the representative compounds of the present invention have comparable or better effects on promoting IL-10 secretion than the positive drugs.

[0373] Example 34 Pharmacokinetic Test

[0374] The experiment was completed at Hangzhou Lead Pharmaceutical Technology Co., Ltd.

[0375] Experimental materials: SD rats (male, 7-9 weeks old, Shanghai Slake)

[0376] Experimental procedure: The clear solution obtained after dissolving the test compound was administered orally to male SD rats (overnight fasting, 7-9 weeks old). After administration of the test compound or control compound, blood was collected from the eye sockets at 0.25, 0.50, 1.00, 2.00, 4.00, 6.00, 8.00 and 24.00 hours, and plasma was obtained after centrifugation. The blood drug concentration was determined by LC-MS / MS, and the relevant pharmacokinetic parameters were calculated by the non-compartmental linear logarithmic trapezoidal method using WinNonLin 8.0 pharmacokinetic statistical software.

[0377] Preparation of standard curve and quality control samples: The mixed stock solution of the compound was diluted with 50% methanol water to prepare standard working solutions containing 20, 40, 100, 200, 400, 1000, 2000, 4000, and 10000 ng / mL of each compound, and quality control working solutions of 60, 600, and 8000 ng / mL. 2.50 μL of standard curve working solution and quality control working solution were added to 47.5 μL of blank rat plasma to prepare standard curves containing 1.00, 2.00, 5.00, 10.00, 20.00, 50.00, 100.00, 200.00, 500.00 ng / mL of each compound and quality control samples with concentrations of 3.00, 30.00 and 400.00 ng / mL, and 200 μL of acetonitrile (containing internal standard verapamil 2 ng / mL) was added respectively. After vortexing for 3 min, the mixture was centrifuged at 20000 rcf at 4 °C for 10 min, and the supernatant was taken for LC-MS / MS analysis.

[0378] Preparation and processing of unknown samples: Take 50 μL of plasma sample, add 200 μL of acetonitrile (containing internal standard verapamil 2 ng / mL), vortex for 3 minutes, centrifuge at 20000 rcf, 4°C for 10 minutes, and take the supernatant for LC-MS / MS analysis.

[0379] The unknown samples were diluted 10 times and prepared: 45 μL of blank plasma was taken, 5 μL of plasma sample was added, and 200 μL of acetonitrile (containing internal standard verapamil 2 ng / mL) was added. After vortexing for 3 min, centrifuged at 20000 rcf and 4 °C for 10 min, and the supernatant was taken for LC-MS / MS analysis.

[0380] The results are shown in Table 5.

[0381] Table 5 Pharmacokinetic properties of the compounds of the present invention in rats

[0382]

[0383] As can be seen from Table 5, the compounds of the present invention have good oral exposure, and the exposure of representative compounds A17, A18, and A26 is significantly improved compared with compound 1. The structural difference between compound A17 and comparative example compound 1 is only that the six-membered ring connected to the condensed ring in A17 is a pyridine ring, while the comparative example compound 1 is a benzene ring. Other molecules A18 and A26, whose six-membered ring connected to the condensed ring is a pyridine ring, have changes in the substituents, but their oral exposure is significantly improved compared with comparative example compound 1. The technical effect brought about by the above structural changes is unexpected.

[0384] Example 35 Compounds Inhibitory Activity against RIPK2 Kinase

[0385] Experimental materials: ADP-Glo ​​Kinase Assay (Promega, V9101); RIPK2 (ICE, S2303T-H17G); WEHI-345 (MCE, HY-18937).

[0386] Experimental procedure: Starting from the highest concentration of 10000nM, prepare the test compound into a 10-point dose-corresponding serial dilution with DMSO in a dilution step of 1 / 4 (the highest concentration of WEHI-345 is 1000nM, and the dilution multiple is 1 / 3), and transfer 0.1μL of the dilution to the 384-well assay plate. Each concentration of the test compound has two replicate wells. Add 5μL of enzyme working solution to each well of the 384-well assay plate, centrifuge at 1000rpm for 1min, and then incubate at 25℃ for 10min. Add 5μL of ATP working solution to each well to initiate the reaction and incubate at 25℃ for 180min. Add 5μL of ADP-Glo ​​reagent, centrifuge at 1000rpm for 1min, and incubate at 25℃ for 40min. Add 10μL of detection reagent, centrifuge at 1000rpm for 1min, and incubate at 25℃ for 40min. Detect the luminescent signal in an ELISA reader.

[0387] Data processing: Inhibition rate (%) = 100*(ave High control-cpd well) / (ave High control-ave low control), where High control group: no compound added, low control group: no compound and enzyme added. The log value of the concentration was used as the X-axis, and the percentage inhibition rate was used as the Y-axis. The IC value of the test compound of the nonlinear regression equation was calculated using XLfit 5.5.0. 50 Fit the line to obtain the IC value of each compound on the enzyme activity 50 value.

[0388] The experimental results are shown in the table below.

[0389] Table 6 Inhibitory activity of compounds on RIPK2 kinase

[0390]

[0391]

[0392] According to the results in Table 6, the inhibitory activity of the representative compounds of the present invention on RIPK2 kinase is significantly better than that of the RIPK2 kinase-positive drug WEHI-345, and better than that of the SIK kinase-positive drug GLPG3970.

[0393] Example 36 Inhibition experiment of compound on TNF-α secretion of BMDM cells under MDP stimulation

[0394] Experimental materials: MDP (InvivoGen, tlrl-mdp); Mouse TNF-α Precoated ELISA Kit (Dakoway Biotechnology Co., Ltd., 1217202); IFN-γ (PepreTech, 315-05-20UG); WEHI-345 (MCE, HY-18937).

[0395] Experimental operation: Bone marrow cells of 6-8 week old C57BL / 6 mice were isolated and seeded in 24-well plates (culture conditions: DMEM+50ng / mL M-CSF+10% FBS). The cells were cultured to the 6th day, and WEHI-345, GLPG3940, A18 and A26 were treated with concentration gradients of 2000nM, 500nM, 125nM, 31.25nM, 7.81nM and 1.95nM for 30 minutes. Except for the control group, the other groups were treated with IFN-γ (5ng / mL) for 2h, and then stimulated with MDP (10ng / mL). After 24h, the cell supernatant was collected and centrifuged at 2000-3000rpm for 20min. After that, the supernatant was aspirated into a new EP tube for standby use, and the TNF-α content in the supernatant was detected by ELISA kit.

[0396] Data processing: Inhibition rate (%) = 100*(model-cpd) / (model-control), where model group: no compound added, control group: no compound added and MDP. With the concentration value as the X-axis and the percentage inhibition rate as the Y-axis, GraphPad Prism9 was used to fit the dose-effect curve to obtain the IC value of each compound on the inhibitory activity of TNF-α secretion by BMDM cells under MDP stimulation. 50 value.

[0397] The experimental results are shown in Table 7 and Figure 1 .

[0398] Table 7 Inhibitory activity of representative compounds of the present invention on TNF-α secretion of BMDM cells

[0399] Compound No. <![CDATA[TNF-α inhibition IC 50 (nM)]]> GLPG3970 119.2 WEHI-345 70.85 A18 0.6701 A26 7.570

[0400] It can be seen from the above table that the representative compounds of the present invention have significantly better inhibitory activity on TNF-α secretion of BMDM cells than WEHI-345 and GLPG3970 at the cellular level.

[0401] Example 37 Inhibition experiment of compound on NF-κB gene expression in THP-1 cells under MDP stimulation

[0402] Experimental materials: MDP (InvivoGen, tlrl-mdp); THP-1-Dual TM Cells (InvivoGen, thpd-nfis); QUANTI-Blue (InvivoGen, rep-qbs); WEHI-345 (MCE, HY-18937).

[0403] Experimental steps: 1×10 6 Resuspend THP-1-Dual TM Cells were seeded in 96-well plates (culture conditions: RPMI-1640 + 10% FBS + 2mM L-Glu + 25mM HEPES + 5μg / mL Blasticidin + 50μg / mL Zeocin), 100μL of cell solution per well, cultured for 12h. After that, 100μL of culture medium containing gradient concentrations (8000nM, 2666nM, 888nM, 296nM, 98.7nM, 32.9nM, 10.9nM, 3.65nM, 1.21nM, 0.4nM) of WEHI-345, GLPG3970, A18 and A26 was added to each well for 30min. Except for the control group, 10μg / mL of MDP was added to each well and cultured for 24h. Thereafter, 20 μL of the culture supernatant was aspirated and mixed with 180 μL of QUANTi-Blue reagent, incubated at 37° C. for 4 h, and the absorbance was measured at 620 nm.

[0404] Data processing: Inhibition rate (%) = 100*(model-cpd) / (model-control), where model group: no compound added, control group: no compound added and MDP. With concentration value as X-axis and percentage inhibition rate as Y-axis, GraphPad Prism9 was used to fit the dose-effect curve to obtain the IC value of each compound's inhibitory activity on NF-κB gene expression in THP-1 cells stimulated by MDP. 50 value.

[0405] The experimental results are shown in Table 8 and Figure 2

[0406] Table 8 Inhibitory activity of compounds on NF-κB gene expression in THP-1 cells

[0407] Compound No. <![CDATA[NF-κB gene inhibitory IC 50 (nM)]]> WEHI-345 85.69 A18 6.507 A26 4.08

[0408] According to the results in Table 8, the representative compounds of the present invention have significantly better inhibitory activity on NF-κB gene expression in THP-1 cells than WEHI-345.

[0409] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A nitrogen-containing fused ring compound or a stereoisomer or a pharmaceutically acceptable salt thereof, characterized in that: The compound has a structure shown in formula (I): in, Y, W, V, Z, X are independently selected from C or N; W, Y, and Z are not all N; when W, Y, and Z are all C, V is N; L1 is selected from O, NH, CH2 or none; L2 is selected from C 1-3 Alkanes or none; R1 is selected from R c substituted 5-6 membered cycloalkyl, 7-12 membered spirocycloalkyl, 7-12 membered spirocycloheteroalkyl, 7-12 membered bridged cycloalkyl, 7-12 membered bridged cycloheteroalkyl, 8-12 membered paracycloalkyl, 8-12 membered paracycloheteroalkyl, -NR a R b , -OH, unsubstituted or R e Substituted 5-6 membered aromatic heterocyclic group, unsubstituted or R e substituted 9-10 membered fused heteroaromatic ring group, 4-6 membered heterocyclic group; the 4-6 membered heterocyclic group contains 1 or 2 N atoms, the 4-6 membered heterocyclic group can be methyl, fluorine, -(CH2) m OH; the 7-12-membered spirocycloalkyl, 7-12-membered spirocycloheteroalkyl, 7-12-membered bridged cycloalkyl, 7-12-membered bridged cycloheteroalkyl, 8-12-membered cycloalkyl and 8-12-membered cycloheteroalkyl may be C 1-3 Alkyl, -OH, -NH2, -NHCH3, -N(CH3)2, halogen, halogen-substituted C 1-3 Alkyl or C 1-3 Alkoxy substitution; R a , R b Independently selected from H or C 1-3 Alkanes; R c Selected from -OH, -NH2, -NHCH3 or -N(CH3)2; m is selected from 1, 2 or 3; R3 is selected from H, cyano, C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 1-3 Alkyl, F, C 3-6 Cycloalkyl, one or more fluorine-substituted C 1-3 Alkoxy or R4 is selected from F, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy or -NHS(O)2R d ; R d Selected from C 1-3 Alkyl or cyclopropyl; R e Selected from H, tetrahydropyranyl, C 1-3 Alkyl, cyclopropyl, halogen-substituted C 1-3 Alkyl, 2. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: The compound has a structure shown in formula (II) or formula (III): in, L1 is selected from O, NH or CH2; L2 is selected from C 1-3 Alkane.

3. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: The compound has a structure shown in formula (IV), formula (V), formula (VI), or formula (VII): in, L1 is selected from O, NH or CH2; L2 is selected from C 1-3 Alkanes; R4 is selected from F, C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy; R5 is selected from C 1-3 Alkyl, cyclopropyl.

4. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to any one of claims 1 to 3, characterized in that: Said Unit selected from in, L1 is selected from O or NH or CH2; L2 is selected from C 1-3 Alkane.

5. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: R1 is R e A substituted 5-6 membered aromatic heterocycle, wherein the 5-6 membered aromatic heterocycle is selected from pyrazolyl, triazolyl, imidazolyl, pyrrolyl, thiazolyl, pyridinyl, pyrimidinyl or pyridazinyl.

6. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: L1 is selected from O or NH or CH2, and L2 is selected from C 1-3 Alkane.

7. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: The L1 and L2 are both null; the R1 is selected from a 4-6 membered heterocyclic group, the 4-6 membered heterocyclic group contains 1 or 2 N atoms, the 4-6 membered heterocyclic group can be methyl, fluorine, -(CH2) m OH substitution.

8. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 7, characterized in that: The Y, V, and Z are C, and W and X are N.

9. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 8, characterized in that: The R3 is selected from C 1-3 Alkyl, C 1-3 Alkoxy, one or more fluorine-substituted C 1-3 Alkyl, one or more fluorine-substituted C 1-3 Alkoxy; R4 is selected from C 1-3 Alkoxy, halogen substituted C 1-3 Alkoxy or -NHS(O)2R d .

10. The nitrogen-containing fused ring compound or its stereoisomers or pharmaceutically acceptable salts according to claim 1, characterized in that: The compound is selected from the compounds numbered A1-A31:

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nitrogen-containing fused ring compound or its stereoisomers and pharmaceutically acceptable salts according to any one of claims 1 to 10.

12. The pharmaceutical composition according to claim 11, characterized in that The pharmaceutical composition further comprises other pharmaceutical components, which are chemotherapeutic drugs.

13. The pharmaceutical composition according to claim 12, characterized in that The chemotherapy drug is selected from at least one of paclitaxel, carboplatin, cisplatin, capecitabine, irinotecan and azacitidine.

14. Use of the nitrogen-containing fused ring compound according to any one of claims 1 to 10 or its stereoisomer, pharmaceutically acceptable salt or the pharmaceutical composition according to any one of claims 11 to 13 in the preparation of a drug for treating a disease caused by abnormal activation of SIK kinase.

15. The use according to claim 14, characterized in that: The disease is selected from at least one of cancer, immune inflammation, diabetes, and central nervous system diseases.

16. The use according to claim 15, characterized in that The cancer is selected from at least one of ovarian cancer, breast cancer, prostate cancer, glioma, and diffuse large B-cell lymphoma; the immune inflammation is selected from at least one of arthritis, colitis, psoriasis, atopic dermatitis, psoriatic arthritis, systemic lupus erythematosus, osteoarthritis, complications caused by organ transplantation, autoimmune thyroid disease, Sjögren's syndrome, vitiligo, autoimmune kidney damage, autoimmune liver damage, chronic obstructive pulmonary disease, multiple sclerosis, and amyotrophic lateral sclerosis; the central nervous system disease is selected from at least one of depression, insomnia, and epilepsy.

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