CN114989079B - RIPK1 kinase target inhibitor and medical application thereof - Google Patents

RIPK1 kinase target inhibitor and medical application thereof Download PDF

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CN114989079B
CN114989079B CN202210703437.6A CN202210703437A CN114989079B CN 114989079 B CN114989079 B CN 114989079B CN 202210703437 A CN202210703437 A CN 202210703437A CN 114989079 B CN114989079 B CN 114989079B
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孔令义
王小兵
李尚�
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China Pharmaceutical University
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Abstract

The invention discloses an RIPK1 kinase target inhibitor and medical application thereof. R is R 1 Selected from substituted or unsubstituted aromatic rings; r is R 2 Selected from hydroxyl or amino; r is R 3 Selected from substituted or unsubstituted aromatic rings; r is R 4 Selected from C 1‑6 Aliphatic hydrocarbons or hydrogen. The invention provides a series of novel kinase targeted inhibitors, and the compounds disclosed by the invention can effectively target RIPK1 kinase and can be used as effective therapeutic agents for neurodegenerative diseases and inflammation related diseases.
Figure DDA0003705240350000011

Description

RIPK1 kinase target inhibitor and medical application thereof
Technical Field
The invention relates to the field of pharmaceutical chemistry, and relates to an RIPK1 kinase target inhibitor and medical application thereof.
Background
Alzheimer's Disease (AD) is a complex neurodegenerative disease that is manifested clinically by hypomnesis, cognitive dysfunction, and behavioral dysfunction. The main pathological features of AD include neuronal loss, β -amyloid (aβ) deposition and hyperphosphorylated Tau protein-induced neurofibrillary tangles (NFT), which have been demonstrated in recent years to be triggered by neuronal death and neuroinflammation.
Receptor interacting protein kinase 1 (RIPK 1) is a serine/threonine family kinase that exists at the intersection of cell death and inflammatory signaling pathways. Important studies have shown that phosphorylated RIPK1 in the brain of AD patients is aberrantly expressed, and when RIPK1 is inhibited, amyloid is reduced, the occurrence and development of neuroinflammation are delayed, and memory loss is alleviated. In addition, microglial nerve cells promote degradation of aβ, and studies have found that RIPK1 promotes inflammation and aβ aggregation in alzheimer's disease, promoting microglial cells in an inflammatory activated state and attenuating degradation of aβ. These indicate that RIPK1 is a very important therapeutic target for the treatment of AD.
The reported RIPK1 inhibitor has very limited skeleton types, and few small molecules related to the AD treatment field are more few, so that the development of a novel skeleton and high kinase selectivity compound targeting RIPK1 is an urgent and important task, and has profound significance and potential value. Here we disclose the development of novel RIPK1 inhibitors with nanomolar potency for the treatment of neurodegenerative diseases such as AD and their use in the field of alzheimer's disease treatment. Animal behavioural experiments show that the novel RIPK1 inhibitor disclosed by the invention has good AD resisting efficacy and shows good clinical application prospect.
Disclosure of Invention
The present invention aims to address the above-mentioned shortcomings of the prior art and to provide a novel RIPK1 kinase target inhibitor for use as an effective therapeutic agent for neurodegenerative diseases and other inflammation-related diseases.
It is another object of the present invention to provide the use of the novel inhibitors of RIPK1 kinase targeting.
The aim of the invention can be achieved by the following technical scheme:
a novel inhibitor targeting RIPK1 kinase, which is selected from compounds with a structure shown as a general formula (I) or a general formula (II) or pharmaceutically acceptable salts thereof:
Figure SMS_1
wherein R is 1 Selected from substituted or unsubstituted aromatic rings; r is R 2 Selected from hydroxyl or amino; r is R 3 Selected from substituted or unsubstituted aromatic rings; r is R 4 Selected from C 1-6 Aliphatic hydrocarbons or hydrogen.
As a preferred mode of the invention, the novel RIPK1 kinase targeted inhibitor is selected from compounds with a structure shown as a general formula (I), wherein R 3 Is selected from substituted or unsubstituted benzene rings;R 4 is hydrogen.
As a preferred embodiment of the present invention, R in the compound represented by the general formula (I) 4 Is hydrogen, R 3 Selected from halogen, cyano, methoxy, trifluoromethylsulfanyl, C1-5 alkyl, C1-3 alkenyl, methylenedioxy, trifluoromethyl, phenoxy mono-or polysubstituted phenyl or 5,6,7, 8-tetrahydro-2-naphthylphenyl, 4- (2-furyl) phenyl, unsubstituted phenyl. 4. The novel class of RIPK1 kinase-targeting inhibitors according to claim 3, wherein R in the compound of formula (I) 4 Is hydrogen, R 3 Selected from phenyl, 3, 4-dimethylphenyl, 4-cyanophenyl, 3,4- (methylenedioxy) -phenyl, 5,6,7, 8-tetrahydro-2-naphthylphenyl, 3, 5-dimethylphenyl, 2, 4-difluorophenyl, 4-methoxyphenyl, 4-trifluoromethylsulfanyl-phenyl, 4-vinylphenyl, 4- (2-furyl) phenyl, 4-tert-butylphenyl, 4-trifluoromethylphenyl, 2-chlorophenyl, 2-methylphenyl, 4-n-propylphenyl, 4-phenoxyphenyl, 4-methoxy-2-methylphenyl, 2-fluoro-4-methoxyphenyl.
As a preferable aspect of the present invention, the R 1 Selected from phenyl, halogen or C1-2 mono-or polysubstituted phenyl, R 2 Selected from hydroxyl or amino; preferably when R 2 R is selected from hydroxy 1 Selected from phenyl or chloro substituted phenyl; when R is 2 When selected from amino, R 1 Selected from phenyl or fluoro, chloro, methyl mono-or poly-substituted phenyl.
As a preferred aspect of the present invention, when R 2 R is selected from hydroxy 1 Selected from phenyl or 4-chlorophenyl; when R is 2 When selected from amino, R 1 Selected from 3-fluoro-5-methylphenyl, 3-fluoro-4-chlorophenyl, 2-fluoro-3-chlorophenyl.
The compound of the invention is selected from any one of the following:
Figure SMS_2
Figure SMS_3
Figure SMS_4
as a preferred aspect of the present invention, the pharmaceutically acceptable salts of the compounds of formula I-II are selected from acid addition salts of the compounds of formula I-II with acids selected from the group consisting of: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.
A pharmaceutical composition, wherein the compound of the general formula I-II or a pharmaceutically acceptable salt thereof according to the invention and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutical composition is prepared into any one of a tablet, a capsule, a powder, a syrup, a liquid, a suspension, a freeze-dried powder injection or an injection.
The preparation method of the RIPK1 kinase target inhibitor comprises the following steps:
route 1: synthesis of target Compounds 1-17 a .
Figure SMS_5
a Reagents and reaction conditions (a) substituted aromatic amine, propionic acid, 110 ℃ for 4-6h; (b) thionyl chloride, tetrahydrofuran, and refluxing for 2 hours; (c) Concentrated ammonia water, tetrahydrofuran, 0 ℃ for 30min.
Route 2: synthesis of target Compound 18-37 a .
Figure SMS_6
a Reagent and reaction conditions (a) 3-fluoro-5-methylaniline, propionic acid, 110 ℃ for 4h; (b) Substituted aromatic amine, tris (dibenzylideneacetone) dipalladium, 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl, cesium carbonate, nitrogen protection, toluene,reacting at 100 ℃ overnight; (c) Ammonia-methanol solution (7M), magnesium methoxide, methanol, 80℃for 24h.
Route 3: synthesis of target Compound 38-43 a .
Figure SMS_7
a The reagent and the reaction conditions are (a) lithium hydroxide monohydrate, tetrahydrofuran/methanol/water, and the reaction is carried out at room temperature for 2 hours; (b) Fatty amine, 2- (7-azobenzotriazole) -N, N, N ', N' -tetramethyl urea hexafluorophosphate, N, N-diisopropylethylamine and methylene chloride react at room temperature for 4 hours, 42-89%; (c) Potassium hydroxylamine solution, methanol, was reacted at room temperature for 3h, 67%.
The beneficial effects are that:
the invention provides a series of novel kinase targeted inhibitors, and the compounds disclosed by the invention can effectively target RIPK1 kinase and can be used as effective therapeutic agents for neurodegenerative diseases and inflammation related diseases.
Drawings
FIG. 1 concentration-dependent inhibition profile of RIPK1 kinase and BV2 cell necrosis by Compound 27.
Figure 2. Cognitive level improvement test on demented mice.
Positioning a navigation experiment escape latency line graph; and B, space exploration of a shuttle frequency histogram of the experimental platform.
FIG. 3 mouse brain tissue section Iba-1 immunofluorescence.
Detailed description of the preferred embodiments and methods
Example 1 preparation of 2- (phenylamino) nicotinic acid (1):
raw material a (1.010g, 5.0 mmol) and aniline (0.558 g,6.0 mmol) were dissolved in propionic acid (5 mL) and reacted at 110℃with stirring for 4-6h. Cooling to room temperature after the reaction is finished, cooling to 0 ℃, precipitating crystals, carrying out suction filtration, washing with propionic acid and water in sequence, and drying to obtain white-like crystals with the yield of 46%. 1 H NMR(600MHz, DMSO-d 6 )δ10.49(s,1H),8.54–8.10(m,2H),7.67(dd,J＝8.5,1.2Hz,2H),7.36 (dd,J＝8.5,7.3Hz,2H),7.11–7.05(m,1H),6.91(dd,J＝7.7,5.0Hz,1H),5.09(s,1H). 13 C NMR(151MHz,DMSO)δ168.68,154.90,151.00,141.73,138.88,131.58, 129.01,123.22,121.00,113.84,108.51.HRMS(ESI)calcd for C 12 H 10 N 2 O 2 [M+H] + : 215.0806；found:215.0807.
Example 2 preparation of 2- ((4-chlorophenyl) amino) nicotinic acid (2):
raw material a (1.010g, 5.0 mmol) and 4-chloroaniline (0.764 g,6.0 mmol) were dissolved in propionic acid (5 mL), and the remaining procedure was the same as in compound 1. 1 H NMR(600MHz,DMSO-d 6 )δ10.51(s,1H),8.38 (dd,J＝4.9,2.0Hz,1H),8.29(dd,J＝7.7,2.0Hz,1H),7.75(d,J＝8.9Hz,2H),7.38(d,J＝8.8Hz,2H),6.92(dd,J＝7.7,4.9Hz,1H),5.61(s,1H). 13 C NMR(151MHz, DMSO)δ168.82,154.99,151.82,141.17,138.34,128.69,126.11,121.96,114.29,108.36.HRMS(ESI)calcd for C 12 H 9 ClN 2 O 2 [M+H] + :249.0426；found:249.0426.
Example 3 preparation of 2- ((3-chlorophenyl) amino) nicotinic acid (3):
raw material a (1.010g, 5.0 mmol) and 3-chloroaniline (0.764 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as for compound 1, 1 H NMR(600MHz,DMSO-d 6 )δ10.57(s,1H),8.43 (dd,J＝4.9,2.0Hz,1H),8.30(dd,J＝7.7,2.0Hz,1H),8.07(s,1H),7.49(ddd,J＝8.2,2.1,1.0Hz,1H),7.34(t,J＝8.1Hz,1H),7.07(ddd,J＝8.0,2.1,0.9Hz,1H), 6.94(dd,J＝7.7,4.8Hz,1H),6.20(s,1H). 13 C NMR(151MHz,DMSO)δ168.85, 154.99,152.08,141.01,133.19,130.38,121.97,119.35,118.63,114.61,108.49. HRMS(ESI)calcd for C 12 H 9 ClN 2 O 2 [M+H] + :249.0426；found:249.0425.
EXAMPLE 4 preparation of 2- (phenylamino) nicotinamide (4):
raw material a (1.010g, 5.0 mmol) and aniline (0.558 g,6.0 mmol) were dissolved in propionic acid (5 mL) and reacted at 110℃with stirring for 4-6h. Cooling to room temperature after the reaction is finished, cooling to 0 ℃, precipitating crystals, filtering, and washing the crystals with propionic acid and water in sequence. The corresponding crystals (0.214 g,1.0 mmol) were dissolved in tetrahydrofuran (1 mL) and thionyl chloride (5 mL), reacted under reflux for 2h, dried by spin and then dissolved in 1mL of tetradAnd (3) dropwise adding the hydrofuran into the 0 ℃ ammonia water at a constant speed, and continuing the reaction for 30min. After completion of the reaction, the system was poured into water, extracted with ethyl acetate (3×5 ml), and the organic layer was separated with Na 2 SO 4 Drying, filtration, concentration and purification by preparative TLC (PE: ea=1:1) gave a yellow powder in 72% yield. 1 H NMR(600MHz,DMSO-d 6 )δ11.17 (s,1H),8.30(dd,J＝4.8,1.8Hz,1H),8.28(s,1H),8.14(dd,J＝7.7,1.9Hz,1H),7.70(s,1H),7.68(d,J＝7.4Hz,2H),7.29(dd,J＝8.5,7.2Hz,2H),7.02–6.92(m, 1H),6.83(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ170.06,154.95, 150.89,140.25,137.54,128.74,121.57,119.40,113.34,110.27.HRMS(ESI)calcd for C 12 H 12 N 3 O[M+H] + :214.0975；found:214.0974.
Example 5 preparation of 2- ((4-chlorophenyl) amino) nicotinamide (5):
raw material a (1.010g, 5.0 mmol) and 4-chloroaniline (0.764 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.27(s,1H),8.32 (dd,J＝4.8,1.8Hz,1H),8.30(s,1H),8.16(dd,J＝7.7,1.9Hz,1H),7.75–7.71(m,3H),7.33(dd,J＝8.9Hz,2H),6.87(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz, DMSO)δ169.93,154.63,150.80,139.20,137.60,128.52,124.84,120.78,113.76,110.52.HRMS(ESI)calcd for C 12 H 11 ClN 3 O[M+H] + :245.0587；found:245.0587.
EXAMPLE 6 preparation of 2- ((3-chlorophenyl) amino) nicotinamide (6):
raw material a (1.010g, 5.0 mmol) and 3-chloroaniline (0.764 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.36(s,1H),8.37 (dd,J＝4.8,1.8Hz,1H),8.33(s,1H),8.18(dd,J＝7.8,1.9Hz,1H),8.10(t,J＝2.1Hz,1H),7.77(s,1H),7.40(ddd,J＝8.3,2.2,0.9Hz,1H),7.30(t,J＝8.1Hz,1H), 7.00(ddd,J＝7.9,2.1,0.9Hz,1H),6.91(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151 MHz,DMSO)δ169.88,154.54,150.82,141.73,137.67,133.16,130.29,120.99,118.34,117.72,114.10,110.77.HRMS(ESI)calcd for C 12 H 11 ClN 3 O[M+H] + : 247.0586；found:247.0585.
example 7 preparation of 2- ((2, 4-difluorophenyl) amino) nicotinamide (7):
raw material a (1.010g, 5.0 mmol) and 2, 4-difluoroaniline (0.774 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.26(d,J ＝2.5Hz,1H),8.50(td,J＝9.3,6.2Hz,1H),8.31(dd,J＝4.8,1.8Hz,2H),8.18(dd, J＝7.8,1.8Hz,1H),7.72(s,1H),7.30(ddd,J＝11.7,8.9,2.9Hz,1H),7.05(ddt,J＝11.1,8.8,2.2Hz,1H),6.89(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ 169.87,157.17,157.09,155.57,155.49,154.68,153.41,153.33,151.79,151.71,150.83,137.59,125.20,125.18,125.13,125.11,122.24,122.23,122.19,122.17, 113.94,110.69,103.78,103.62,103.60,103.44.HRMS(ESI)calcd for C 12 H 11 F 2 N 3 O [M+H] + :250.0786；found:250.0787.
example 8 preparation of 2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (8):
raw material a (1.010g, 5.0 mmol) and 3-fluoro-5-methylaniline (0.751 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.34(s, 1H),8.36(dd,J＝4.8,1.8Hz,1H),8.32(s,1H),8.17(dd,J＝7.7,1.9Hz,1H),7.84–7.71(m,2H),7.00(d,J＝1.4Hz,1H),6.89(dd,J＝7.7,4.8Hz,1H),6.60(ddd,J＝ 9.6,2.6,1.3Hz,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.92,163.21, 161.63,154.64,150.84,141.70,141.62,140.11,140.05,137.61,115.38,113.90,110.66,108.42,108.28,103.03,102.85,21.05,21.04.HRMS(ESI)calcd for C 13 H 13 FN 3 O[M+H] + :246.1037；found:246.1038.
EXAMPLE 9 preparation of 2- ((3, 4-dimethylphenyl) amino) nicotinamide (9)
Raw material a (1.010g, 5.0 mmol) and 3, 4-dimethylaniline (0.727 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.02(s, 1H),8.28(dd,J＝4.8,1.8Hz,1H),8.24(s,1H),8.11(dd,J＝7.7,1.9Hz,1H),7.66(s,1H),7.48(dd,J＝8.2,2.4Hz,1H),7.35(d,J＝2.4Hz,1H),7.04(d,J＝8.1Hz, 1H),6.78(dd,J＝7.7,4.8Hz,1H),2.20(s,3H),2.16(s,3H). 13 C NMR(151MHz, DMSO)δ170.13,155.13,150.98,137.98,137.45,136.27,129.61,129.30,120.81,117.03,112.84,109.94,19.64,18.74.HRMS(ESI)calcd for C 13 H 13 FN 3 O[M+H] + : 242.1288；found:242.1288.
EXAMPLE 10 preparation of 2- ((3-bromophenyl) amino) nicotinamide (10)
Raw material a (1.010g, 5.0 mmol) and 3-bromoaniline (1.032 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.35(s,1H),8.37 (dd,J＝4.8,1.8Hz,1H),8.33(s,1H),8.22(t,J＝2.0Hz,1H),8.18(dd,J＝7.8,1.8Hz,1H),7.77(s,1H),7.46(ddd,J＝8.2,2.1,1.0Hz,1H),7.24(t,J＝8.0Hz,1H), 7.13(ddd,J＝7.9,2.0,0.9Hz,1H),6.90(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ169.88,154.51,150.82,141.87,137.66,130.62,123.90,121.74, 121.15,118.11,114.11,110.77.HRMS(ESI)calcd for C 12 H 11 BrN 3 O[M+H] + : 292.0080；found:292.0081.
EXAMPLE 11 preparation of 2- ((4- (trifluoromethyl) phenyl) amino) nicotinamide (11):
raw material a (1.010g, 5.0 mmol) and 4-trifluoromethylaniline (0.967 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.56(s, 1H),8.38(dd,J＝4.8,1.8Hz,1H),8.37(s,1H),8.21(dd,J＝7.8,1.9Hz,1H),7.92 (d,J＝8.5Hz,2H),7.82(s,1H),7.63(d,J＝8.5Hz,2H),6.96(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ169.84,154.35,150.76,143.81,137.74,126.04, 126.02,125.58,123.79,121.21,121.00,118.73,114.61,111.16.HRMS(ESI)calcd for C 13 H 11 F 3 N 3 O[M+H] + :282.0849；found:282.0850.
example 12 preparation of 2- ((4-isopropylphenyl) amino) nicotinamide (12):
raw material a (1.010g, 5.0 mmol) and 4-isopropylaniline (0.611 g,6.0 mmol) were dissolved inIn propionic acid (5 mL), the remaining procedure was as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.05(s,1H), 8.27(dd,J＝4.8,1.8Hz,1H),8.25(s,1H),8.11(dd,J＝7.7,1.9Hz,1H),7.66(s,1H),7.56(d,J＝8.5Hz,2H),7.16(d,J＝8.5Hz,2H),6.79(dd,J＝7.7,4.8Hz,1H), 2.84(p,J＝6.9Hz,1H),1.19(d,J＝6.9Hz,6H). 13 C NMR(151MHz,DMSO)δ 170.09,155.08,150.94,141.72,137.94,137.48,126.41,119.74,112.98,109.99,32.82,24.07.HRMS(ESI)calcd for C 15 H 18 N 3 O[M+H] + :256.1444；found:256.1446.
EXAMPLE 13 preparation of 2- ((4-chloro-3-fluorophenyl) amino) nicotinamide (13):
raw material a (1.010g, 5.0 mmol) and 3-fluoro-4-chloroaniline (0.873 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.45(s, 1H),8.38(dd,J＝4.8,1.8Hz,1H),8.35(s,1H),8.19(dd,J＝7.8,1.8Hz,1H),8.13 (dd,J＝12.6,2.5Hz,1H),7.79(s,1H),7.44(t,J＝8.7Hz,1H),7.32(ddd,J＝8.8,2.5,1.0Hz,1H),6.93(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ169.76,157.93,156.32,154.29,150.73,140.89,140.82,137.69,130.19,116.13, 116.11,114.38,110.95,110.53,110.41,107.03,106.85.HRMS(ESI)calcd for C 12 H 10 ClFN 3 O[M+H] + :266.0491；found:266.0492.
example 14 preparation of 2- ((3-chloro-2-fluorophenyl) amino) nicotinamide (14):
raw material a (1.010g, 5.0 mmol) and 2-fluoro-3-chloroaniline (0.873 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.57(d,J ＝2.8Hz,1H),8.65–8.50(m,1H),8.37(dd,J＝4.8,1.8Hz,2H),8.22(dd,J＝7.8, 1.8Hz,1H),7.80(s,1H),7.17(td,J＝8.2,1.3Hz,1H),7.13(td,J＝8.2,6.7,1.7Hz,1H),6.95(dd,J＝7.8,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ169.80,154.36, 150.81,148.87,147.25,137.67,130.18,130.12,124.97,124.94,121.88,119.56,119.24,119.14,114.58,111.23.HRMS(ESI)calcd for C 12 H 10 ClFN 3 O[M+H] + : 266.0491；found:266.0491.
example 15 preparation of 2- ((2, 3, 4-trifluorophenyl) amino) nicotinamide (15):
raw material a (1.010g, 5.0 mmol) and 2,3, 4-trifluoroaniline (0.883 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.41(d, J＝2.1Hz,1H),8.35(s,1H),8.33(dd,J＝4.8,1.8Hz,1H),8.32–8.27(m,1H),8.21 (dd,J＝7.8,1.8Hz,1H),7.79(s,1H),7.27(td,J＝10.4,8.6,2.2Hz,1H),6.94(dd,J＝7.7,4.8Hz,1H). 13 C NMR(151MHz,DMSO)δ169.79,154.53,150.82,145.67, 145.61,144.08,144.01,142.72,141.16,141.10,139.99,139.88,139.79,138.36,138.26,138.16,137.65,126.56,126.54,126.51,126.49,115.58,115.54,114.51, 111.49,111.46,111.37,111.35,110.93.HRMS(ESI)calcd for C 12 H 9 F 3 N 3 O[M+H] + : 268.0692；found:268.0691.
example 16 preparation of 2- ((5-chloro-2-fluorophenyl) amino) nicotinamide (16):
raw material a (1.010g, 5.0 mmol) and 5-chloro-2-fluoroaniline (0.873 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as in compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.68(d,J ＝3.2Hz,1H),8.81(dd,J＝7.3,2.7Hz,1H),8.43(dd,J＝4.8,1.8Hz,1H),8.37(s, 1H),8.23(dd,J＝7.8,1.8Hz,1H),7.80(s,1H),7.29(dd,J＝11.3,8.6Hz,1H),7.00 (dt,J＝4.6,2.3Hz,1H),6.98(dd,J＝7.8,4.8Hz,1H). 13 C NMR(151MHz,DMSO) δ169.73,154.16,151.59,150.81,149.99,137.74,129.98,129.90,128.14,128.12,120.51,120.46,119.39,119.37,116.08,115.94,114.74,111.37.HRMS(ESI)calcd for C 12 H 11 ClFN 3 O[M+H] + :266.0491；found:266.0491.
EXAMPLE 17 preparation of 2- (p-toluylamino) nicotinamide (17):
raw material a (1.010g, 5.0 mmol) and 4-methylaniline (0.643 g,6.0 mmol) were dissolved in propionic acid (5 mL), the remaining procedure was the same as for compound 4, 1 H NMR(600MHz,DMSO-d 6 )δ11.07(s,1H), 8.28(dd,J＝4.8,1.8Hz,1H),8.25(s,1H),8.12(dd,J＝7.7,1.9Hz,1H),7.67(s, 1H),7.55(d,J＝8.4Hz,2H),7.10(d,J＝8.4Hz,2H),6.79(dd,J＝7.7,4.8Hz,1H),2.25(s,3H). 13 C NMR(151MHz,DMSO)δ170.11,155.07,150.94,137.72,137.48, 130.43,129.13,119.56,112.96,109.99,20.41.HRMS(ESI)calcd for C 13 H 14 N 3 O [M+H] + :228.1131；found:228.1131.
EXAMPLE 18 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- (phenylamino) nicotinamide (18):
raw material b (2.505 g,10 mmol) and 3-fluoro-5-methylaniline (1.501 g,12 mmol) were dissolved in propionic acid, reacted at 110℃for 4h, cooled for crystallization, washed with propionic acid and water and dried to give intermediate c in 58% yield. Intermediate c (678.32 mg,2 mmol), aniline (223.51 mg,2.4 mmol), tris (dibenzylideneacetone) dipalladium (18.31 mg,0.02 mmol), 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl (19.07mg,0.04 mmol), cesium carbonate (1303.28 mg,4 mmol) were dissolved in toluene (10 mL) and reacted overnight at 100 ℃ under nitrogen, after the reaction was completed monitored by TLC, the reaction solution was filtered with celite and purified by flash chromatography column (PE: etoac=1:1) to give 18d as a yellow solid in 95% yield. Compound 18d (70.28 mg,0.2 mmol) was dissolved in methanol (3 mL), and ammonia-methanol solution (7M in MeOH,2 mmol) and magnesium methoxide (17.27 mg,0.2 mmol) were added and reacted in a closed ampoule at 80℃for 24h. The reaction solution was poured into water, extracted with ethyl acetate (3×5 ml), and the organic layer was separated with Na 2 SO 4 Drying, filtration, concentration and purification by preparative TLC (PE: ea=1:1) gave a yellow powder in 84% yield. 1 H NMR(600MHz,DMSO-d 6 )δ10.95 (s,1H),8.33(s,1H),8.20(d,J＝2.6Hz,1H),8.02(d,J＝2.7Hz,1H),7.98(s,1H),7.79–7.68(m,2H),7.19(dd,J＝8.6,7.3Hz,2H),6.95(d,J＝2.0Hz,1H),6.91(s, 1H),6.90(d,J＝1.2Hz,1H),6.74(tt,J＝7.3,1.1Hz,1H),6.54(dt,J＝9.6,1.7Hz,1H),2.28(s,3H). 13 C NMR(151MHz,DMSO)δ169.79,163.34,161.75,149.70, 145.11,143.36,142.24,142.15,140.03,139.97,130.81,130.07,129.29,118.60,114.70,114.69,114.46,111.48,107.69,107.55,102.11,101.93,21.09,21.07.HRMS (ESI)calcd for C 19 H 18 FN 4 O[M+H] + :337.1459；found:337.1460.
EXAMPLE 19 preparation of 5- ((3, 4-dimethylphenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (19):
the aniline of example 18 was replaced with 3, 4-dimethylaniline (290.84 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.86(s,1H),8.30(s, 1H),8.16(d,J＝2.7Hz,1H),7.93(d,J＝2.7Hz,1H),7.77–7.67(m,3H),6.99–6.89(m,2H),6.73(d,J＝2.4Hz,1H),6.68(dd,J＝8.1,2.4Hz,1H),6.53(d,J＝9.6 Hz,0H),2.28(s,3H),2.15(s,3H),2.12(s,3H). 13 C NMR(151MHz,DMSO)δ 169.88,163.35,161.77,149.11,142.60,142.36,142.28,142.26,140.00,139.93,136.82,131.78,130.18,129.06,126.49,116.71,114.57,114.55,112.63,111.66, 107.52,107.38,101.94,101.76,21.09,21.08,19.70,18.55.HRMS(ESI)calcd for C 21 H 22 FN 4 O[M+H] + :365.1772；found:365.1773.
EXAMPLE 20 preparation of 5- ((4-cyanophenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (20):
the aniline of example 18 was replaced with 4-cyanoaniline (283.53 mg,2.4 mmol), the remainder of the procedure was identical to that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.20(s,1H),8.74(s,1H),8.35 (s,1H),8.26(d,J＝2.5Hz,1H),8.11(d,J＝2.6Hz,1H),7.81–7.73(m,2H),7.56(d,J＝8.7Hz,2H),6.99(s,1H),6.90(d,J＝8.8Hz,2H),6.59(d,J＝9.7Hz,1H), 2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.46,163.28,161.69,151.37,149.89, 145.99,141.82,141.74,140.14,140.07,133.78,132.81,127.93,120.12,115.11,113.50,111.14,108.23,108.09,102.63,102.45,98.43,21.06.HRMS(ESI)calcd for C 20 H 17 FN 5 O[M+H] + :362.1412；found:362.1413.
example 21 preparation of 5- ((3-cyanophenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (21):
the aniline of example 18 was replaced with 3-cyanoaniline (283.53 mg,2.4 mmol), the remainder of the procedure was identical to that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.14(s,1H),8.37(s,1H),8.23 (d,J＝2.6Hz,1H),8.06(d,J＝2.6Hz,1H),7.76(dt,J＝12.0,2.3Hz,2H),7.36(dd, J＝9.2,7.4Hz,1H),7.18–7.09(m,3H),6.98(d,J＝1.9Hz,1H),6.57(ddd,J＝9.5,2.7,1.4Hz,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.58,163.30,161.71, 150.88,146.49,145.11,141.97,141.89,140.09,140.03,131.75,130.57,128.95,121.49,119.20,118.68,116.04,114.97,112.04,111.26,108.04,107.90,102.46, 102.28,21.08,21.06.HRMS(ESI)calcd for C 20 H 17 FN 5 O[M+H] + :362.1412；found: 362.1411.
EXAMPLE 22 preparation of 5- (benzo [ d ] [1,3] dioxol-5-ylamino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (22):
the aniline of example 18 was replaced with 3,4- (methylenedioxy) aniline (329.13 mg,2.4 mmol), the remainder of the procedure was identical to that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.84(s,1H),8.31(s, 1H),8.12(d,J＝2.6Hz,1H),7.89(d,J＝2.7Hz,1H),7.77(s,1H),7.71(dt,J＝12.6,2.4Hz,2H),6.93(s,1H),6.77(d,J＝8.3Hz,1H),6.60(d,J＝2.2Hz,1H),6.52(dt, J＝9.5,1.7Hz,1H),6.39(dd,J＝8.3,2.3Hz,1H),5.92(s,2H),2.27(s,3H). 13 C NMR(151MHz,DMSO)δ169.87,163.35,161.77,149.06,147.90,142.36,142.28,141.94,140.43,139.99,139.93,132.17,128.41,114.54,111.67,108.68,107.83, 101.91,101.74,100.56,98.43,21.09.HRMS(ESI)calcd for C 20 H 18 FN 4 O 3 [M+H] + : 381.1356；found:381.1358.
EXAMPLE 23 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((5, 6,7, 8-tetrahydronaphthalen-2-yl) amino) nicotinamide (23):
the aniline of example 18 was replaced with 5,6,7, 8-tetrahydro-2-naphthylaniline (353.33mg,2.4 mmol), the remainder of the procedure was identical to that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.87(s,1H), 8.30(s,1H),8.15(d,J＝2.7Hz,1H),7.93(d,J＝2.7Hz,1H),7.76–7.68(m,3H),6.93(s,1H),6.88(d,J＝8.2Hz,1H),6.69(dd,J＝8.1,2.4Hz,1H),6.61(d,J＝2.4 Hz,1H),6.53(d,J＝9.5Hz,1H),2.62(d,J＝16.5Hz,3H),2.27(s,3H),1.69(t,J＝ 3.3Hz,3H). 13 C NMR(151MHz,DMSO)δ169.87,163.35,161.77,149.14,142.30, 142.25,139.99,139.93,137.22,131.76,129.63,129.08,127.22,115.14,114.57,113.33,111.61,107.52,107.38,101.95,101.77,29.06,28.12,23.16,22.92,21.09. HRMS(ESI)calcd for C 23 H 24 FN 4 O[M+H] + :391.1929；found:391.1930.
EXAMPLE 24 preparation of 5- ((3, 5-dimethylphenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (24):
the aniline of example 18 was replaced with 3, 5-dimethylaniline (290.84 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.96(s,1H),8.33(s, 1H),8.18(d,J＝2.6Hz,1H),7.98(d,J＝2.7Hz,1H),7.79(s,1H),7.77–7.70(m,2H),6.97–6.93(m,1H),6.54(d,J＝9.6Hz,0H),6.50(s,2H),6.39(s,1H),2.28(s, 3H),2.17(s,6H). 13 C NMR(151MHz,DMSO)δ169.81,163.34,161.75,149.66, 145.17,143.46,142.25,142.16,140.02,139.96,138.20,131.04,130.54,120.55,114.69,114.68,112.43,111.48,107.67,107.53,102.11,101.93,21.19.HRMS(ESI) calcd for C 21 H 22 FN 4 O[M+H] + :365.1772；found:365.1771.
example 25 preparation of 5- ((2, 4-difluorophenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (25):
the aniline of example 18 was replaced with 2, 4-difluoroaniline (309.86 mg,2.4 mmol), the remaining procedure was as for compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.97(s,1H),8.29(s,1H), 8.15(d,J＝2.7Hz,1H),7.91(d,J＝2.7Hz,1H),7.77–7.69(m,3H),7.25(ddd,J＝11.7,8.9,2.9Hz,1H),7.06(td,J＝9.4,5.7Hz,1H),6.95(s,1H),6.93(tt,J＝8.1,1.5 Hz,1H),6.54(dt,J＝9.5,1.8Hz,1H),2.28(s,3H). 13 C NMR(151MHz,DMSO)δ 169.72,163.33,161.74,156.11,156.04,154.54,154.46,152.92,152.84,151.30, 151.22,149.80,142.96,142.22,142.14,140.03,139.97,130.86,129.51,117.57,117.54,117.51,117.48,114.71,114.70,111.35,107.71,107.57,104.53,104.38, 104.35,104.20,102.11,101.93,21.08,21.07.HRMS(ESI)calcd for C 19 H 16 F 3 N 4 O [M+H] + :373.1271；found:373.1272.
EXAMPLE 26 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4-methoxyphenyl) amino) nicotinamide (26):
substitution of the aniline in example 18 with 4-methylOxyaniline (295.57 mg,2.4 mmol), the remaining steps were identical to compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.76(s,1H),8.29(s,1H), 8.12(d,J＝2.7Hz,1H),7.87(d,J＝2.7Hz,1H),7.71(s,2H),7.69(d,J＝2.4Hz,1H),6.94(d,J＝2.3Hz,1H),6.93(s,2H),6.83(d,J＝8.9Hz,2H),6.51(dd,J＝9.6, 1.3Hz,1H),3.69(s,3H),2.27(s,3H). 13 C NMR(151MHz,DMSO)δ169.94, 163.36,161.78,153.04,148.59,142.47,142.38,140.99,139.97,139.91,137.78,132.75,127.45,117.48,114.73,114.43,111.82,107.37,107.23,101.77,101.59,55.27, 21.09.HRMS(ESI)calcd for C 20 H 19 FN 4 O 2 [M+H] + :367.1565；found:367.1567.
EXAMPLE 27 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4- ((trifluoromethyl) thio) phenyl) amino) nicotinamide (27):
the aniline of example 18 was replaced with 4-trifluoromethylthioaniline (463.65 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.16(s,1H),8.53(s, 1H),8.34(s,1H),8.24(d,J＝2.6Hz,1H),8.11(d,J＝2.6Hz,1H),7.78(s,1H),7.76(t,J＝2.3Hz,1H),7.47(d,J＝8.6Hz,2H),6.98(s,1H),6.92(d,J＝8.7Hz,2H), 6.58(dt,J＝9.5,1.7Hz,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.53, 163.29,161.70,151.05,148.89,145.60,141.91,141.83,140.11,140.04,138.20,132.43,130.73,128.61,115.02,114.38,111.16,108.56,108.11,107.97,102.51, 102.33,21.06.HRMS(ESI)calcd for C 20 H 17 F 4 N 4 OS[M+H] + :437.1054；found: 437.1053.
EXAMPLE 28 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4-vinylphenyl) amino) nicotinamide (28):
the aniline of example 18 was replaced with 4-vinylaniline (286.00 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.98(s,1H),8.33(s,1H), 8.20(d,J＝2.6Hz,1H),8.14(s,1H),8.02(d,J＝2.7Hz,1H),7.80–7.70(m,2H),7.30(d,J＝8.5Hz,2H),6.96(s,1H),6.88(d,J＝8.6Hz,2H),6.61(dd,J＝17.6, 10.9Hz,1H),6.55(d,J＝10.1Hz,1H),5.58(dd,J＝17.6,1.2Hz,1H),5.02(dd,J＝ 10.8,1.2Hz,1H),2.28(s,3H). 13 C NMR(151MHz,DMSO)δ169.74,163.33, 161.74,149.85,144.96,143.57,142.17,142.09,140.05,139.98,136.47,130.43,130.22,127.80,127.35,114.74,114.27,111.42,110.04,107.76,107.62,102.17, 101.99,21.08.HRMS(ESI)calcd for C 21 H 20 FN 4 O[M+H] + :363.1616；found: 363.1616.
EXAMPLE 29 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4- (furan-2-yl) phenyl) amino) nicotinamide (29):
the aniline of example 18 was replaced with 4- (2-furyl) aniline (382.05 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.01(s,1H),8.34(s, 1H),8.23(d,J＝2.6Hz,1H),8.19(s,1H),8.05(d,J＝2.7Hz,1H),7.76(s,1H),7.74(s,1H),7.63(dd,J＝1.8,0.8Hz,1H),7.53(d,J＝8.7Hz,2H),6.96(s,1H),6.94(d,J＝8.7Hz,2H),6.67(dd,J＝3.3,0.8Hz,1H),6.55(d,J＝9.8Hz,1H),6.52(dd,J＝ 3.4,1.8Hz,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.73,163.33,161.75, 153.76,149.97,144.66,143.78,142.16,142.08,141.52,140.06,139.99,130.44,130.26,124.87,121.19,114.77,114.43,111.86,111.41,107.80,107.65,102.91, 102.21,102.03,21.09,21.08.HRMS(ESI)calcd for C 23 H 20 FN 4 O 2 [M+H] + :403.1565； found:403.1564.
EXAMPLE 30 preparation of 5- ((4- (tert-butyl) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (30):
the aniline of example 18 was replaced with 4-tert-butylaniline (358.17 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.90(s,1H),8.31(s,1H), 8.16(d,J＝2.7Hz,1H),7.99(d,J＝2.7Hz,1H),7.89(s,1H),7.73(dt,J＝12.5,2.3Hz,1H),7.71(s,1H),7.22(d,J＝8.6Hz,2H),6.94(s,1H),6.87(d,J＝8.6Hz,2H), 6.53(d,J＝9.5Hz,1H),2.28(s,3H),1.24(s,9H). 13 C NMR(151MHz,DMSO)δ 169.84,163.35,161.76,149.25,142.49,142.33,142.27,142.25,141.09,140.00,139.93,131.46,129.04,125.89,114.58,111.52,107.55,107.41,101.97,101.79, 33.71,31.38,21.08.HRMS(ESI)calcd for C 23 H 26 FN 4 O[M+H] + :393.2085；found: 393.2085.
example 31 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4- (trifluoromethyl) phenyl) amino) nicotinamide (31):
the aniline of example 18 was replaced with 4-trifluoromethylaniline (386.70 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.16(s,1H),8.52(s,1H), 8.34(s,1H),8.25(d,J＝2.6Hz,1H),8.11(d,J＝2.6Hz,1H),7.81–7.74(m,2H),7.48(d,J＝8.5Hz,2H),6.98(s,1H),6.95(d,J＝8.5Hz,2H),6.58(d,J＝9.7Hz, 1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.53,163.29,161.71,151.00, 149.15,145.48,141.93,141.85,140.10,140.04,132.25,128.76,126.64,126.61,125.95,124.16,117.84,117.63,115.01,115.00,113.24,111.18,108.09,107.95, 102.49,102.32,21.07,21.06.HRMS(ESI)calcd for C 20 H 17 F 4 N 4 O[M+H] + :405.1333； found:405.1335.
example 32 preparation of 5- ((2-chlorophenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (32):
the aniline of example 18 was replaced with 2-chloroaniline (306.17 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ11.15(s,1H),8.29(s,1H),8.25(d, J＝2.6Hz,1H),8.08(d,J＝2.6Hz,1H),7.77(dt,J＝12.3,2.3Hz,1H),7.74(s,1H),7.53(s,1H),7.37(dd,J＝7.9,1.5Hz,1H),7.13(ddd,J＝8.5,7.3,1.5Hz,1H),6.98 (s,1H),6.87(dd,J＝8.2,1.4Hz,1H),6.80–6.75(m,1H),6.57(d,J＝9.7Hz,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ169.59,163.30,161.71,150.93,146.14, 142.27,142.00,141.92,140.09,140.03,133.06,129.75,129.34,128.03,119.76, 119.51,114.96,114.95,114.51,111.11,108.02,107.88,102.44,102.26,21.08,21.07.HRMS(ESI)calcd for C 19 H 17 ClFN 4 O[M+H] + :371.1069；found:371.1071.
example 33 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- (o-tolylamino) nicotinamide (33):
the aniline of example 18 was replaced with 2-methylaniline (257.17 mg,2.4 mmol), the remainderThe procedure is the same as for compound 18, 1 H NMR(500MHz,DMSO-d 6 )δ10.94(s,1H),8.27(s,1H),8.15 (d,J＝2.6Hz,1H),7.95(d,J＝2.7Hz,1H),7.74(d,J＝12.3Hz,1H),7.69(s,1H),7.14(s,1H),7.12(s,1H),7.03(t,J＝7.7Hz,1H),6.95(s,1H),6.85(d,J＝8.0Hz, 1H),6.75(t,J＝7.3Hz,1H),6.54(d,J＝9.5Hz,1H),2.28(s,3H),2.24(s,3H). 13 C NMR(126MHz,DMSO)δ169.79,163.48,161.58,149.60,143.50,143.35,142.30,142.20,140.00,139.92,131.64,130.68,130.61,126.67,125.53,119.58,114.64, 114.32,111.51,107.63,107.45,102.07,101.85,21.05,17.93.HRMS(ESI)calcd for C 20 H 20 FN 4 O[M+H] + :351.1616；found:351.1618.
example 34 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4-propylphenyl) amino) nicotinamide (34):
the aniline of example 18 was replaced with 4-n-propylaniline (324.50 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.88(s,1H),8.31(s,1H), 8.17(d,J＝2.7Hz,1H),7.97(d,J＝2.7Hz,1H),7.86(s,1H),7.73(d,J＝12.4Hz,1H),7.71(s,1H),7.01(d,J＝8.4Hz,2H),6.94(s,1H),6.85(d,J＝8.4Hz,2H),6.53 (d,J＝9.7Hz,1H),2.44(t,J＝7.6Hz,2H),2.28(s,3H),1.59–1.48(m,2H),0.88(t,J＝7.3Hz,3H). 13 C NMR(151MHz,DMSO)δ169.86,163.35,161.77,149.25, 142.62,142.49,142.33,142.25,140.00,139.94,132.49,131.50,129.11,114.90,114.58,111.57,107.56,107.41,101.97,101.79,36.60,24.41,21.09,21.08,13.68. HRMS(ESI)calcd for C 22 H 24 FN 4 O[M+H] + :379.1929；found:379.1931.
EXAMPLE 35 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4-phenoxyphenyl) amino) nicotinamide (35):
the aniline of example 18 was replaced with 4-phenoxyaniline (444.54 mg,2.4 mmol), the remainder of the procedure was identical to that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.92(s,1H),8.33(s,1H), 8.20(d,J＝2.7Hz,1H),7.99(d,J＝2.7Hz,1H),7.98(s,1H),7.76–7.71(m,2H),7.34(dd,J＝8.7,7.3Hz,2H),7.07–7.03(m,1H),6.99–6.90(m,7H),6.54(dt,J＝ 9.5,1.8Hz,1H),2.28(s,3H). 13 C NMR(151MHz,DMSO)δ169.80,163.34,161.76, 158.30,149.50,148.12,142.80,142.27,142.19,141.46,140.02,139.95,131.33,129.84,129.40,122.31,120.90,117.03,116.18,114.65,111.55,107.64,107.50, 102.05,101.87,21.09,21.07.HRMS(ESI)calcd for C 25 H 22 FN 4 O 2 [M+H] + :429.1721； found:429.1722.
example 36 preparation of 2- ((3-fluoro-5-methylphenyl) amino) -5- ((4-methoxy-2-methylphenyl) amino) nicotinamide (36):
the aniline of example 18 was replaced with 4-methoxy-2-methylaniline (329.24 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(500MHz,DMSO-d 6 )δ10.67(s,1H),8.23(s, 1H),7.95(d,J＝2.7Hz,1H),7.72–7.61(m,3H),7.04(s,1H),6.94(d,J＝8.7Hz,1H),6.91(s,1H),6.81(d,J＝2.9Hz,1H),6.70(dd,J＝8.7,3.0Hz,1H),6.49(d,J＝9.0Hz,1H),3.70(s,3H),2.26(s,3H),2.19(s,3H). 13 C NMR(126MHz,DMSO)δ 170.00,163.53,161.63,154.38,147.97,142.65,142.55,139.92,139.85,139.56,135.24,134.43,130.70,126.58,120.23,116.41,114.27,112.07,111.74,107.18, 107.01,101.59,101.38,55.15,21.08,21.07,18.11.HRMS(ESI)calcd for C 21 H 22 FN 4 O 2 [M+H] + :381.1721；found:381.1722.
example 37 preparation of 5- ((2-fluoro-4-methoxyphenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (37):
the aniline of example 18 was replaced with 3, 5-dimethylaniline (338.75 mg,2.4 mmol), the remaining procedure was the same as that of compound 18, 1 H NMR(600MHz,DMSO-d 6 )δ10.77(s,1H),8.26(s, 1H),8.05(d,J＝2.7Hz,1H),7.76(d,J＝2.8Hz,1H),7.72–7.63(m,2H),7.51(s,1H),7.08(t,J＝9.9Hz,1H),6.93(s,1H),6.89(dd,J＝13.0,2.8Hz,1H),6.70(dd,J ＝8.8,2.5Hz,1H),6.51(d,J＝9.6Hz,1H),3.73(s,3H),2.27(s,3H). 13 C NMR(151 MHz,DMSO)δ169.88,163.35,161.76,154.88,154.30,154.23,153.27,148.64,142.46,142.38,140.46,139.95,139.89,132.77,127.07,124.75,124.67,120.41, 120.39,114.42,111.70,110.14,110.12,107.36,107.22,102.73,102.58,101.77,101.59,55.63,21.07.HRMS(ESI)calcd for C 20 H 19 F 2 N 4 O 2 [M+H] + :385.1471；found: 385.1473.
example 38 preparation of 5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) -N-methylnicotinamide (38):
intermediate 27d (902.88 mg,2 mmol) was dissolved in a tetrahydrofuran/methanol/water (4:4:1, 10 mL) system and lithium hydroxide monohydrate (419.60 mg,10 mmol) was added and reacted at room temperature for 2h. The reaction solution was concentrated, then water was added thereto for 5mL, and the pH was adjusted to pH=4 with dilute hydrochloric acid, whereby a solid was precipitated. Filtered, dried to give intermediate 27e as a yellow solid in 95% yield. Intermediate 27e (109.35 mg,0.25 mmol) was dissolved in dichloromethane (5 mL), 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate (142.59 mg,0.375 mmol) and N, N-diisopropylethylamine (96.93 mg,0.75 mmol) were added and stirring was continued at room temperature for 15min, followed by methylamine hydrochloride (20.26 mg,0.3 mmol) and reaction was continued at room temperature for 3h. After TLC monitoring the reaction, the reaction was poured into water and extracted with ethyl acetate (3×5 ml), the organic layer was separated, and the reaction mixture was taken up in Na 2 SO 4 Dried, filtered, concentrated and purified by preparative TLC (PE: ea=2:1) to give a yellow solid in 89% yield. 1 H NMR(600 MHz,DMSO-d 6 )δ11.00(s,1H),8.81(d,J＝4.6Hz,1H),8.54(s,1H),8.23(d,J＝ 2.6Hz,1H),8.05(d,J＝2.7Hz,1H),7.76(dt,J＝12.2,2.2Hz,1H),7.48(d,J＝8.7Hz,2H),6.57(dt,J＝9.7,1.8Hz,1H),2.79(d,J＝4.5Hz,3H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ167.45,163.30,161.71,150.55,148.79,145.11,141.95,141.87,140.11,140.04,138.22,131.43,128.73,114.94,114.45,111.83,108.04, 107.90,102.36,102.18,26.33,21.07.HRMS(ESI)calcd for C 21 H 18 F 4 N 4 OS[M+H] + : 451.1210；found:451.1210.
Example 39 preparation of N-ethyl-5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (39):
the methylamine hydrochloride in example 38 was replaced with ethylamine hydrochloride (24.46 mg,0.3 mmol), the remaining procedure was as for compound 38, 1 H NMR(600MHz,DMSO-d 6 )δ10.77(s,1H),8.26(s, 1H),8.05(d,J＝2.7Hz,1H),7.76(d,J＝2.8Hz,1H),7.72–7.63(m,2H),7.51(s,1H),7.08(t,J＝9.9Hz,1H),6.93(s,1H),6.89(dd,J＝13.0,2.8Hz,1H),6.70(dd,J ＝8.8,2.5Hz,1H),6.51(d,J＝9.6Hz,1H),3.73(s,3H),2.27(s,3H). 13 C NMR(151 MHz,DMSO)δ169.88,163.35,161.76,154.88,154.30,154.23,153.27,148.64,142.46,142.38,140.46,139.95,139.89,132.77,127.07,124.75,124.67,120.41, 120.39,114.42,111.70,110.14,110.12,107.36,107.22,102.73,102.58,101.77,101.59,55.63,21.07.HRMS(ESI)calcd for C 20 H 19 F 2 N 4 O 2 [M+H] + :465.1327；found: 465.1327.HRMS(ESI)calcd for C 22 H 20 F 4 N 4 OS[M+H] + :465.1365；found:465.1367.
EXAMPLE 40 preparation of N-cyclopropyl-5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (40):
the methylamine hydrochloride in example 38 was replaced with cyclopropylamine hydrochloride (28.07 mg,0.3 mmol), the remaining procedure was as for compound 38, 1 H NMR(600MHz,DMSO-d 6 )δ10.99(s,1H),8.79(d, J＝4.1Hz,1H),8.50(s,1H),8.25(d,J＝2.6Hz,1H),8.05(d,J＝2.6Hz,1H),7.77(dd,J＝12.2,2.2Hz,1H),7.48(d,J＝8.7Hz,2H),7.03(s,1H),6.91(d,J＝8.7Hz, 2H),6.68–6.48(m,1H),3.01–2.84(m,1H),2.30(s,3H),0.73(s,1H),0.60(dd,J＝4.2,2.4Hz,2H). 13 C NMR(151MHz,DMSO)δ168.42,163.28,161.69,150.59, 148.97,145.41,138.19,132.11,128.56,114.32,111.66,108.56,108.08,107.94,102.42,102.24,23.16,21.05,5.63.HRMS(ESI)calcd for C 23 H 20 F 4 N 4 OS[M+H] + : 477.1367；found:477.1367.
EXAMPLE 41 preparation of N-cyclobutyl-5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (41):
the methylamine hydrochloride in example 38 was replaced with cyclobutylamine hydrochloride (32.27 mg,0.3 mmol), the remaining procedure was as for compound 38, 1 H NMR(600MHz,DMSO-d 6 )δ10.95(s,1H),8.95(d, J＝7.1Hz,1H),8.51(s,1H),8.26(d,J＝2.6Hz,1H),8.13(d,J＝2.6Hz,1H),7.76(dt,J＝12.1,2.3Hz,1H),7.48(d,J＝8.6Hz,2H),7.00(s,1H),6.92(d,J＝8.8Hz, 2H),6.63–6.52(m,1H),4.42(q,J＝8.0Hz,1H),2.26–2.22(m,1H),2.07(td,J＝9.5,2.7Hz,2H),1.70(dtd,J＝10.5,6.4,3.2Hz,2H). 13 C NMR(151MHz,DMSO)δ 166.08,163.27,161.68,150.80,149.06,145.45,141.87,141.79,140.09,140.02,138.21,132.30,128.56,115.00,114.29,111.69,108.55,108.09,107.95,102.44, 102.26,44.69,29.74,21.03,14.83.HRMS(ESI)calcd for C 24 H 22 F 4 N 4 OS[M+H] + : 491.1523；found:491.1523.
EXAMPLE 42 preparation of N-cyclopentyl-5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (42):
the methylamine hydrochloride in example 38 was replaced with cyclopentylamine hydrochloride (36.48 mg,0.3 mmol), the remaining procedure was as for compound 38, 1 H NMR(600MHz,DMSO-d 6 )δ10.92(s,1H),8.65(d, J＝7.0Hz,1H),8.50(s,1H),8.26(d,J＝2.6Hz,1H),8.09(d,J＝2.6Hz,1H),7.83–7.69(m,1H),7.48(d,J＝8.7Hz,2H),7.00(s,1H),6.92(d,J＝8.8Hz,2H),6.58 (ddd,J＝9.5,2.6,1.3Hz,1H),4.25(q,J＝7.0Hz,1H),2.29(s,3H),1.96–1.86(m,1H),1.68(t,J＝3.0Hz,1H),1.61–1.48(m,4H). 13 C NMR(151MHz,DMSO-d 6 )δ166.67,163.28,161.70,150.64,149.07,145.17,141.88(d,J＝12.4Hz),140.05(d,J ＝9.9Hz),138.20,132.44,130.71,128.57,114.94,114.28,112.21,108.53,107.96(d,J＝21.4Hz),102.28(d,J＝26.9Hz),51.13,31.91,23.67,21.03.HRMS(ESI)calcd for C 25 H 24 F 4 N 4 OS[M+H] + :505.1680；found:505.1680.
example 43 preparation of 5- ((2-fluoro-4- ((trifluoromethyl) thio) phenyl) amino) -2- ((3-fluoro-5-methylphenyl) amino) -N-hydroxynicotinamide (43):
intermediate 27d (225.72 mg,0.5 mmol)) was dissolved in methanol (5 mL), and a free hydroxylamine solution (165 mg,5 mmol) was added dropwise thereto, and after the completion of the reaction, ph=4 was adjusted with dilute hydrochloric acid, a solid was precipitated, filtered, washed with water and dried to give a yellow solid in 67% yield. 1 H NMR(600MHz,DMSO-d 6 )δ11.55 (s,1H),10.47(s,1H),9.47–9.21(m,1H),8.58(s,1H),8.23(d,J＝2.6Hz,1H),7.86(d,J＝2.7Hz,1H),7.76–7.67(m,1H),7.48(d,J＝8.7Hz,2H),6.99(s,3H),6.97(s, 1H),6.68–6.49(m,1H),2.29(s,3H). 13 C NMR(151MHz,DMSO)δ164.70, 163.30,161.72,149.91,148.31,144.32,141.92,141.84,140.16,140.10,138.18,130.73,130.02,128.99,114.81,114.69,110.58,108.90,108.10,107.95,102.31, 102.13,21.09.HRMS(ESI)calcd for C 20 H 16 F 4 N 4 O 2 S[M+H] + :453.1004；found: 453.1003.
EXAMPLE 44 inhibition Activity assay of RIPK1 fragment Long kinase and BV2 cell apoptosis
The RIPK1 (#VA 7591) fragment of a long human kinase was purchased from Promega. The assay components included 25mM HEPES (pH 7.2), 20mM MgCl2, 12.5mM MnCl2, 5mM EGTA, 2mM EDTA, 12.5mM beta-glycerophosphate and 2mM DTT (added immediately prior to use). mu.L of RIPK1 was incubated with 2. Mu.L of compound or DMSO at different concentrations in assay buffer for 15min at 24 ℃. Then 2. Mu.L of ATP/MBP mixture (final concentration 25. Mu.M and 5. Mu.M respectively) was added to trigger the kinase reaction and incubated for 90min at 37 ℃. mu.L of the kinase reaction solution was added with 5. Mu.L of ADP-glo TM Quenching the reagent. After a further 60min incubation, 10 μ L Kinase Detection reagent was added and incubated at 24℃for 30min. The inhibition (%) was calculated by reading using Lum module of Molecular Devices multifunctional microplate reader.
Mouse microglial cells BV2 cells (available from Wuhanplauosai Life technologies Co., ltd., china) were grown in a medium containing 4mM L-glutamine, 4500mg/L glucose, 1mM sodium pyruvate (Hyclone), 10% heat-inactivated fetal bovine serum, and 1% GlutaMAXTM (Gibco). Culture environment 37 ℃, CO 2 The content is 5%. During the experiment, BV2 cells were plated at 4X 10 3 Individual cells/well were seeded in 96-well plates for 24h. Next, the medium was changed to fresh DMEM without FBS, with 200ng/mL LPS, or without addition, and the cells were incubated for an additional 23h. BV2 cells were then incubated with the indicated concentrations of compound or DMSO for 1h, and then incubated with 25. Mu.M zVAD-fmk for 24h. By CellTiter
Figure SMS_8
Aqueous Non-Radioactive Cell Proliferation (MTS) Assay (Promega) cell viability was measured.
Calculation of IC using nonlinear regression with normalized dose response fit using Prism GraphPad 8.0 software 50 And EC (EC) 50 Values. All experiments were performed independently at least 3 times.
TABLE 1 BV2 cell apoptosis inhibition EC of Compounds 1-17 50 Value of a
Figure SMS_9
Figure SMS_10
a EC 50 Values, data are expressed as mean ± SD of three independent experiments.
TABLE 2 enzyme inhibition (1. Mu.M) of Compounds 18-37 and inhibition of BV2 cell programmed necrosis EC 50 Value of a
Figure SMS_11
Figure SMS_12
a Enzyme inhibition rate and EC 50 Values, data are expressed as mean ± SD of three independent experiments.
We evaluated the BV2 necrosis inhibitory activity of the target compounds (tables 1, 2). Table 1 shows that the compounds of formula (II) series have good inhibitory effect on the programmed necrosis of BV2 cells. We further designed and synthesized compounds of the general formula (I) series (table 2), and found that most of the compounds showed nanomolar potency on both RIPK1 kinase and BV2 cells (e.g. compounds 18, 25, 27). Further structure-activity relationship studies indicate that substitution on the amide will introduce toxicity to the cell by the compound, thus stopping further optimization of the amide moiety. FinalizingCompound 27 exhibited the best inhibitory potency, IC 50 The value reached 84nM, BV2 anti-necrotic EC 50 The value reached 40nM (FIG. 1).
Example 45 evaluation of cognitive improvement Activity of Compounds in APP/PS1 double transgenic dementia mice.
Male APP/PS1 mice and littermates (5 months old) were purchased from Jiangsu Ai Ling Fit Biotech Inc. Mice were divided into 5 groups (6 mice per group) by body weight: normal control group, SHAM group, positive drug Nec-1s group (10 mg/kg), compound 27 low dose group (2 mg/kg), compound 27 high dose group (10 mg/kg). The compound was dissolved in a mixed system consisting of 1% dmso, 9% tween 80, and 90% physiological saline. The administration was continued by intraperitoneal injection for four weeks, three to four times a week. Body weight and body temperature were monitored every 2 days. Five days after dosing, a positioning navigation experiment was performed and the platform was removed on the sixth day, and a space exploration experiment was performed. Mice were sacrificed after the experiment was completed and brain tissue was taken for further pathological section investigation. Multiple comparison tests of one-way anova and Dunnett were used to determine statistically significant differences in relevant indicators of cognitive levels between treatment and control groups and SHAM groups: p <0.05, and p <0.01.
APP/PS1 double transgenic dementia mice were purchased to evaluate the cognitive level improvement efficacy of compound 27. Five groups of mice (6 mice per group) were divided according to drug dose and type: normal control group, SHAM group, positive drug Nec-1s group (10 mg/kg), compound 27 low dose group (2 mg/kg), compound 27 high dose group (10 mg/kg). The administration was continued by intraperitoneal injection four times a week. Body weight and body temperature were monitored daily. The Morris water maze experiment after administration mainly comprises a positioning navigation experiment for five days and a space exploration experiment carried out by a sixth day removing platform. Mice were sacrificed after the experiment was completed and brain tissue was taken for further pathological section investigation.
As shown in fig. 2, there was a significant decrease in escape latency in the normal control group and the dosing group, while there was a slight decrease in escape latency in APP/PS1 mice. The optimal compound 27 was effective with only a low dose (2 mg/kg), whereas from day 2, high dose treatment (10 mg/kg) brought even better cognitive levels than the control and Nec-1s mice (fig. 2A). The number of platform shuttles was significantly increased in the group dosed with compound 27 compared to the model group (fig. 2B), demonstrating the superior cognitive improvement effect of compound 27.
Iba-1 is a calbindin specifically expressed in microglia, whose up-regulation reflects microglial activation. Our immunofluorescence results indicated that in the region of the medial prefrontal cortex (mPFC) that is highly cognitively and behavioural correlated, the expression level of Iba-1 was abnormally elevated in the mice in the model group compared to the mice in the control group, while intervention with compound 27 restored the expression of Iba-1 to normal levels. Indicating that compound 27 has excellent anti-inflammatory effect.

Claims (8)

1. A novel inhibitor targeting RIPK1 kinase, which is characterized by being selected from compounds with a structure shown as a general formula (I) or pharmaceutically acceptable salts thereof:
Figure FDA0004161646700000011
wherein R is 4 Is hydrogen, R 3 Selected from halogen, cyano, methoxy, trifluoromethylsulfanyl, C1-5 alkyl, C1-3 alkenyl, methylenedioxy, trifluoromethyl, phenoxy mono-or polysubstituted phenyl or 5,6,7, 8-tetrahydro-2-naphthylphenyl, 4- (2-furyl) phenyl, unsubstituted phenyl.
2. The novel class of RIPK1 kinase-targeted inhibitors according to claim 1, characterized in that R in the compound of formula (I) 4 Is hydrogen, R 3 Selected from phenyl, 3, 4-dimethylphenyl, 4-cyanophenyl, 3,4- (methylenedioxy) -phenyl, 5,6,7, 8-tetrahydro-2-naphthylphenyl, 3, 5-dimethylphenyl, 2, 4-difluorophenyl, 4-methoxyphenyl, 4-trifluoromethylsulfanyl-phenyl, 4-vinylphenyl, 4- (2-furyl) phenyl, 4-tert-butylphenyl, 4-trifluoromethylphenyl, 2-chlorophenyl, 2-methylphenyl, 4-n-propylphenyl, 4-phenoxyphenyl, 4-methoxy-2-methylbenzeneRadical, 2-fluoro-4-methoxyphenyl.
3. The novel inhibitor of targeted RIPK1 kinase according to any one of claims 1 to 2, wherein the pharmaceutically acceptable salt of the compound of formula (I) is selected from acid addition salts of the compound of formula (I) with an acid selected from the group consisting of: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.
4. A pharmaceutical composition comprising the novel RIPK1 kinase-targeting inhibitor according to any one of claims 1-2 and a pharmaceutically acceptable carrier.
5. Use of a novel inhibitor of a targeted RIPK1 kinase according to any one of claims 1-2 in the manufacture of a medicament for the treatment of neurodegenerative or other inflammatory-related diseases.
6. Use of a novel inhibitor of a targeted RIPK1 kinase according to any one of claims 1-2 for the manufacture of a medicament for the treatment and/or prophylaxis of a disease mediated by RIPK1 kinase.
7. Use according to claim 6, characterized in that the novel inhibitors of RIPK1 kinase targeted in any of claims 1-2 are used in the preparation of a medicament for the treatment of neurodegenerative or inflammatory related diseases mediated by RIPK1 kinase.
8. Use according to claim 7, characterized in that the novel inhibitors of RIPK1 kinase targeted in any of claims 1-2 are used for the preparation of a medicament for the treatment of RIPK1 kinase mediated alzheimer's disease.
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