CN114989079A - RIPK1 kinase target inhibitor and medical application thereof - Google Patents

RIPK1 kinase target inhibitor and medical application thereof Download PDF

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CN114989079A
CN114989079A CN202210703437.6A CN202210703437A CN114989079A CN 114989079 A CN114989079 A CN 114989079A CN 202210703437 A CN202210703437 A CN 202210703437A CN 114989079 A CN114989079 A CN 114989079A
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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 a medical application thereof. R 1 Selected from substituted or unsubstituted aromatic rings; r 2 Selected from hydroxyl or amino; r 3 Selected from substituted or unsubstituted aromatic rings; r 4 Is selected from C 1‑6 Aliphatic hydrocarbons or hydrogen. The invention provides a series of novel kinase targeted inhibitors, compounds disclosed by the inventionThe compound can effectively target RIPK1 kinase, and can be used as an effective therapeutic agent 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, clinically manifested by memory impairment, cognitive disorders and behavioral disorders. The main pathological features of AD include neuronal loss, β -amyloid (a β) deposition and hyperphosphorylated Tau-induced neurofibrillary tangles (NFTs), which have been shown in recent years to be probably triggered by neuronal death and neuroinflammation.
Receptor-interacting protein kinase 1(RIPK1) is a serine/threonine family kinase that is present at the intersection of cell death and inflammatory signaling pathways. Important research shows that phosphorylated RIPK1 in the brain of AD patients is abnormally expressed, and when RIPK1 is inhibited, amyloid is reduced, the occurrence and development of neuroinflammation are delayed, and the loss of memory is reduced. In addition, microglia promote the degradation of A beta, and research finds that in Alzheimer's disease, RIPK1 promotes inflammation and A beta aggregation, promotes the microglia to be in an inflammatory activation state, and reduces the degradation of the A beta. These indicate that RIPK1 is a very important therapeutic target for the treatment of AD.
The reported skeleton types of the RIPK1 inhibitor are very limited, and small molecules related to the field of AD treatment are fewer and fewer, so that the development of a compound which has a novel skeleton and high kinase selectivity and targets RIPK1 is a very urgent and important task and has profound significance and potential value. Here we disclose the development of a novel RIPK1 inhibitor with nanomolar potency for the treatment of neurodegenerative diseases such as AD and its application in the field of alzheimer's disease treatment. Animal behavior experiments show that the novel RIPK1 inhibitor disclosed by the inventor has good anti-AD efficacy and shows good clinical application prospects.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a novel RIPK1 kinase target inhibitor which is an effective therapeutic agent for neurodegenerative diseases and other inflammation-related diseases.
Another object of the present invention is to provide the use of the novel inhibitors targeting RIPK1 kinase.
The purpose of the invention can be realized by the following technical scheme:
a novel inhibitor targeting RIPK1 kinase is selected from compounds with the structure shown as a general formula (I) or a general formula (II) or pharmaceutically acceptable salts thereof:
Figure RE-GDA0003784052690000021
wherein R is 1 Selected from substituted or unsubstituted aromatic rings; r 2 Selected from hydroxyl or amino; r 3 Selected from substituted or unsubstituted aromatic rings; r 4 Is selected from C 1-6 Aliphatic hydrocarbons or hydrogen.
Preferably, the novel inhibitor targeting RIPK1 kinase is selected from compounds with the structure shown in the general formula (I), wherein R is 3 Is selected from substituted or unsubstituted benzene rings; r 4 Is hydrogen.
As a preferable aspect of the present invention, R in the compound represented by the general formula (I) 4 Is hydrogen, R 3 Selected from halogen, cyano, methoxy, trifluoromethyl sulfanyl, C1-5 alkyl, C1-3 alkenyl, methylenedioxy, trifluoromethyl, and phenyl mono-or polysubstituted with phenoxy, or 5,6,7, 8-tetrahydro-2-naphthylphenyl, 4- (2-furyl) phenyl, unsubstituted phenyl. 4. The novel inhibitor of RIPK1 targeted kinase according to claim 3, characterized in that R in the compound shown as the general formula (I) 4 Is hydrogen, R 3 Selected from the group consisting of 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-methoxyphenylphenyl-2-methylphenyl, 2-fluoro-4-methoxyphenyl.
As a preferable aspect of the present invention, R is 1 Selected from phenyl, halogen or C1-2 mono-or polysubstituted phenyl, R 2 Selected from hydroxyl or amino; preferably when R is 2 When selected from hydroxy, R 1 Selected from phenyl or chloro-substituted phenyl; when R is 2 When selected from amino, R 1 Is selected from phenyl or phenyl mono-or polysubstituted by fluorine, chlorine and methyl.
As a preference of the present invention, when R 2 When selected from hydroxy, R 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 RE-GDA0003784052690000022
Figure RE-GDA0003784052690000031
Figure RE-GDA0003784052690000041
preferably, the pharmaceutically acceptable salt of the compound represented by the general formula I-II is selected from an acid addition salt formed by the compound represented by the general formula I-II and an acid, wherein the acid is selected from: 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.
The invention relates to a pharmaceutical composition, wherein the compound of general formula I-II or pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
As a preferable mode of the invention, the pharmaceutical composition is prepared into any one of tablets, capsules, powder, syrup, liquid, suspending agent, freeze-dried powder injection or injection.
The invention relates to a preparation method of an RIPK1 kinase target inhibitor, which comprises the following steps:
route 1: synthesis of target Compounds 1 to 17 a .
Figure RE-GDA0003784052690000042
a The reagent and the reaction condition are (a) substituted aromatic amine and propionic acid at 110 ℃ for 4-6 h; (b) refluxing thionyl chloride and tetrahydrofuran for 2 h; (c) concentrated ammonia water, tetrahydrofuran, 0 deg.C, 30min.
Route 2: synthesis of target Compounds 18-37 a .
Figure RE-GDA0003784052690000051
a The reagent and the reaction condition are (a) 3-fluoro-5-methylaniline, propionic acid, 110 ℃ and 4 h; (b) substituted aromatic amine, tris (dibenzylideneacetone) dipalladium, 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl, cesium carbonate, protection of nitrogen and toluene are reacted at 100 ℃ overnight; (c) ammonia-methanol solution (7M), methoxy magnesium, methanol, 80 ℃,24h.
Route 3: synthesis of target Compounds 38-43 a .
Figure RE-GDA0003784052690000052
a The reagent and the reaction condition are (a) lithium hydroxide monohydrate, tetrahydrofuran/methanol/water, and the reaction is carried out for 2 hours at room temperature; (b) fatty amine, 2- (7-azobenzotriazol) -N, N, N ', N' -tetramethylurea hexafluorophosphate, N, N-diisopropylethylamine and dichloromethane are reacted at room temperature for 4h, and the reaction time is 42-89%; (c) reacting hydroxylamine potassium solution with methanol at room temperature for 3h, 67%.
Has the advantages that:
the invention provides a series of novel kinase targeted inhibitors, and the disclosed compound can effectively target RIPK1 kinase and can be used as an effective therapeutic agent for neurodegenerative diseases and inflammation related diseases.
Drawings
Figure 1 concentration-dependent inhibition curves of compound 27 against RIPK1 kinase and BV2 cell necrosis.
Figure 2 cognitive level improvement test on dementia mice.
A, positioning a flight experiment escape latency line graph; and B, searching a shuttle times histogram of the experimental platform in the space B.
FIG. 3 shows the Iba-1 immunofluorescence of mouse brain tissue sections.
Embodiments and methods of the invention
Example 1 preparation of 2- (phenylamino) nicotinic acid (1):
the starting material a (1.010g, 5.0mmol) and aniline (0.558g, 6.0mmol) were dissolved in propionic acid (5mL) and the reaction was stirred at 110 ℃ for 4-6 h. And after the reaction is finished, cooling to room temperature, cooling to 0 ℃, separating out crystals, performing 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):
the starting material a (1.010g, 5.0mmol) and 4-chloroaniline (0.764g, 6.0mmol) were dissolved in propionic acid (5mL) and the remainder of the procedure was taken with 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):
the starting material a (1.010g, 5.0mmol) and 3-chloroaniline (0.764g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
the starting material a (1.010g, 5.0mmol) and aniline (0.558g, 6.0mmol) were dissolved in propionic acid (5mL) and the reaction was stirred at 110 ℃ for 4-6 h. And after the reaction is finished, cooling to room temperature, cooling to 0 ℃, separating out crystals, carrying out suction filtration, and washing the crystals with propionic acid and water in sequence. Dissolving the corresponding crystal (0.214g, 1.0mmol) in tetrahydrofuran (1mL) and thionyl chloride (5mL), refluxing for 2h, spinning to dry, dissolving in 1mL tetrahydrofuran, slowly dropping into 0 ℃ ammonia water at a constant speed, and continuing the reaction for 30min. After the reaction was completed, the system was poured into water and extracted with ethyl acetate (3 × 5mL), and the organic layer was separated and Na was added 2 SO 4 Dry, filter, concentrate and purify by preparative TLC (PE: EA ═ 1:1) to give 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):
starting material a (1.010g, 5.0mmol) and 4-chloroaniline (0.764g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
starting material a (1.010g, 5.0mmol) and 3-chloroaniline (0.764g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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＝2.1 Hz,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):
starting material a (1.010g, 5.0mmol) and 2, 4-difluoroaniline (0.774g, 6.0mmol) were dissolved in propionic acid (5mL), compound 4 was used for the remaining procedures, 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):
starting material a (1.010g, 5.0mmol) and 3-fluoro-5-methylaniline (0.751g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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)
The starting material a (1.010g, 5.0mmol) and 3, 4-dimethylaniline (0.727g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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)
Starting material a (1.010g, 5.0mmol) and 3-bromoaniline (1.032g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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＝7.8,1.8 Hz,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(151 MHz,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):
starting material a (1.010g, 5.0mmol) and 4-trifluoromethylaniline (0.967g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
the starting material a (1.010g, 5.0mmol) and 4-isopropylaniline (0.811g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
starting material a (1.010g, 5.0mmol) and 3-fluoro-4-chloroaniline (0.873g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
starting material a (1.010g, 5.0mmol) and 2-fluoro-3-chloroaniline (0.873g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
starting material a (1.010g, 5.0mmol) and 2,3, 4-trifluoroaniline (0.883g, 6.0mmol) were dissolved in propionic acid (5mL), the remaining procedure was followed with 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):
starting material a (1.010g, 5.0mmol) and 5-chloro-2-fluoroaniline (0.873g, 6.0mmol) were dissolved in propionic acid (5mL), the remaining procedure was followed with 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):
the starting material a (1.010g, 5.0mmol) and 4-methylaniline (0.643g, 6.0mmol) were dissolved in propionic acid (5mL) and the remaining procedure was followed with 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):
dissolving a raw material b (2.505g,10mmol) and 3-fluoro-5-methylaniline (1.501g,12mmol) in propionic acid, reacting for 4h at 110 ℃, cooling for crystallization, washing with propionic acid and water, and drying to obtain an intermediate c with the yield of 58%. Intermediate c (678.32mg,2mmol), aniline (223.51mg,2.4mmol), tris (dibenzylideneacetone) dipalladium (18.31mg,0.02mmol), 2-dicyclohexylphosphine-2 ',4',6' -triisopropylbiphenyl (19.07mg,0.04 mmol), cesium carbonate (1303.28mg,4mmol) were dissolved in toluene (10mL) under nitrogen protection 10After the reaction was monitored by TLC at 0 ℃ overnight, the reaction was filtered through celite and purified by flash chromatography (PE: EtOAc 1:1) to give 18d as a yellow solid in 95% yield. Compound 18d (70.28mg,0.2mmol) was dissolved in methanol (3mL), and ammonia-methanol solution (7M in MeOH,2mmol) and magnesium methoxide (17.27mg,0.2 mmol) were added and reacted in a closed ampoule at 80 ℃ for 24h. The reaction was poured into water and extracted with ethyl acetate (3 x 5mL), the organic layer was separated and Na was added 2 SO 4 Dry, filter, concentrate and purify by preparative TLC (PE: EA ═ 1:1) to give 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 from example 18 was replaced with 3, 4-dimethylaniline (290.84mg,2.4mmol), the remaining procedure was followed for 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 from example 18 was replaced with 4-cyanoaniline (283.53mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 3-cyanoaniline (283.53mg,2.4mmol), the remaining procedure was followed with 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] Dioxolan-5-ylamino) -2- ((3-fluoro-5-methylphenyl) amino) nicotinamide (22):
the aniline in example 18 was replaced with 3,4- (methylenedioxy) aniline (329.13mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 5,6,7, 8-tetrahydro-2-naphthylaniline (353.33mg,2.4 mmol), the remaining procedure was followed for 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 from example 18 was replaced with 3, 5-dimethylaniline (290.84mg,2.4mmol), the remaining procedure was followed for 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 from example 18 was replaced with 2, 4-difluoroaniline (309.86mg,2.4mmol), the remaining procedure was followed 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):
the aniline from example 18 was replaced with 4-methoxyaniline (295.57mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 4-trifluoromethylsulfanylaniline (463.65mg,2.4mmol), the remaining procedure was followed for 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 from example 18 was replaced with 4-vinylaniline (286.00mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 4- (2-furyl) aniline (382.05mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 4-tert-butylaniline (358.17mg,2.4mmol), the remaining procedure was followed with 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＝12.5,2.3 Hz,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 from example 18 was replaced with 4-trifluoromethylaniline (386.70mg,2.4mmol), and the remaining procedure was followed for 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 from example 18 was replaced with 2-chloroaniline (306.17mg,2.4mmol), the remaining procedure was followed with 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-toluylamino) nicotinamide (33):
the aniline from example 18 was replaced with 2-methylaniline (257.17mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 4-n-propylaniline (324.50mg,2.4mmol), the remaining procedure was followed with 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 from example 18 was replaced with 4-phenoxyaniline (444.54mg,2.4mmol), the remaining procedure was followed with 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 in example 18 was replaced with 4-methoxy-2-methylaniline (329.24mg,2.4mmol), the remaining procedure was followed with 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 in example 18 was replaced with 3, 5-dimethylaniline (338.75mg,2.4mmol), the remaining procedure was followed with 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 (902.88mg,2mmol) was dissolved in a tetrahydrofuran/methanol/water (4:4:1,10mL) system, and lithium hydroxide monohydrate (419.60mg,10mmol) was added and reacted at room temperature for 2 h. After the reaction mixture was concentrated, 5mL of water was added thereto, and the pH was adjusted to 4 with dilute hydrochloric acid to precipitate a solid. Filtration and drying gave intermediate 27e as a yellow solid in 95% yield. Intermediate 27e (109.35mg,0.25mmol) was dissolved in dichloromethane (5mL) and 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium hexafluorophosphate (142.59mg,0.375mmol) and N, N-diisopropyl urea were addedEthylamine (96.93mg,0.75mmol) was stirred at room temperature for 15min, followed by the addition of methylamine hydrochloride (20.26mg,0.3mmol) and reaction continued at room temperature for 3 h. After the completion of the reaction monitored by TLC, the reaction was poured into water and extracted with ethyl acetate (3X 5mL), and the organic layer was separated and Na was added 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＝8.7 Hz,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 (24.46mg,0.3mmol), the remaining procedure was followed with 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 (28.07mg,0.3mmol), the remaining procedures were performed with 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 (32.27mg,0.3mmol), the remaining procedure was followed 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 salt in example 38The acid salt was replaced with cyclopentylamine hydrochloride (36.48mg,0.3mmol), the remaining procedure was followed with 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 (225.72mg,0.5mmol)) was dissolved in methanol (5mL) and the free hydroxylamine solution (165mg,5mmol) was added dropwise thereto, after the reaction was completed, the pH was adjusted to 4 with dilute hydrochloric acid, a solid 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 assay of inhibitory Activity of RIPK1 fragment Long kinase and BV2 apoptosis
The RIPK1(# VA7591) fragment long human kinase was purchased from Promega. The components of the assay solution included 25mM HEPES (pH 7.2), 20mM MgCl2, 12.5mM MnCl2,5mM EGTA, 2mM EDTA, 12.5mM β -glycerophosphate and 2mM DTT (added just prior to use). mu.L of RIPK1 was incubated with 2. mu.L of different concentrations of compound or DMSO in assay buffer at 24 ℃ for 15 min. 2 μ L of ATP/MBP mix (final concentrations 25 μ M and 5 μ M respectively) was then added to trigger the kinase reaction and incubated for 90min at 37 ℃. mu.L of kinase reaction mixture 5. mu.L of ADP-glo was added TM Quenching the reagent. After a further 60min incubation, 10. mu.L of Kinase Detection reagent was added and incubated at 24 ℃ for 30min. The inhibition (%) was calculated by reading using the Lum module of a Molecular Devices multifunctional microplate reader.
Mouse microglia BV2 cells (purchased from Wuhan Protech, Inc., China) were grown in medium containing 4mM L-glutamine, 4500mg/L glucose, 1mM sodium pyruvate (Hyclone), 10% heat-inactivated fetal bovine serum, and 1% GlutaMAXTM (Gibco). The culture environment is 37 ℃ and CO 2 The content is 5 percent. During the experiment, BV2 cells were cultured at 4X 10 3 Individual cells/well were seeded in 96-well plates for 24h. Next, the medium was replaced with fresh DMEM without FBS, with 200ng/mL LPS, or without addition, and the cells were incubated for an additional 23 h. BV2 cells were then incubated with the indicated concentrations of compound or DMSO for 1h, followed by 25. mu.M zVAD-fmk for 24h. By CellTiter
Figure RE-GDA0003784052690000223
Aqueou Non-Radioactive Cell Proliferation (MTS) assay (Promega) for Cell viability.
IC was calculated using Prism GraphPad 8.0 software using non-linear regression with normalized dose-response fit 50 And EC 50 The value is obtained. All experiments were performed at least 3 times independently.
TABLE 1 BV2 apoptosis inhibition EC of Compounds 1-17 50 Value of a
Figure RE-GDA0003784052690000221
Figure RE-GDA0003784052690000222
a EC 50 Values, data are expressed as mean ± SD of three independent experiments.
TABLE 2 enzyme inhibition (1. mu.M) and BV2 apoptosis inhibition EC for compounds 18-37 50 Value of a
Figure RE-GDA0003784052690000231
Figure RE-GDA0003784052690000232
a Enzyme inhibition and EC 50 Values, data are expressed as mean ± SD of three independent experiments.
We evaluated BV2 necrosis-inhibiting activity of the compound of interest (tables 1, 2). Table 1 shows that the compounds of the general formula (II) series have good inhibitory effect on the programmed necrosis of BV2 cells. We further designed and synthesized a series of compounds of general formula (I) (table 2) and found that most of the compounds exhibited nanomolar potency on both RIPK1 kinase and BV2 cells (e.g., compounds 18, 25, 27). Further structure-activity relationship studies have shown that substitution on the amide can induce toxicity of the compound to cells, thus stopping further optimization of the amide moiety. Final Compound 27 exhibits optimal inhibitory potency, IC 50 The value reaches 84nM, BV2 anti-necrosis EC 50 Values reached 40nM (FIG. 1).
Example 45 evaluation of cognitive improvement Activity of Compounds in APP/PS1 Dual transgenic dementia mice.
Male APP/PS1 mice and their littermate controls (5 months old) were purchased from Jiangsu Elaeagni Biotech, Inc. Mice were divided into 5 groups by weight (6 mice per group): normal control group, SHAM group, positive drug Nec-1s group (10mg/kg), compound 27 low dose group (2mg/kg), compound 27 high dose group (10 mg/kg). The compounds were dissolved in a mixed system consisting of 1% DMSO, 9% Tween 80 and 90% 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. The location navigation experiment was performed five days after dosing and the platform was removed on the sixth day for space exploration experiments. After completion of the experiment, mice were sacrificed and brain tissue was taken for further pathological section examination. One-way analysis of variance and multiple comparative tests of Dunnett were used to determine statistically significant differences in relevant indicators such as cognitive levels between the treated and control groups and the SHAM group: (x) p <0.05, and (x) p < 0.01.
APP/PS1 double transgenic dementia mice were purchased to evaluate the cognitive level improving 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 (10mg/kg), compound 27 low dose group (2mg/kg), compound 27 high dose group (10 mg/kg). The administration was continued by intraperitoneal injection four weeks, 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 removing a platform on the sixth day. After completion of the experiment, mice were sacrificed and brain tissue was taken for further pathological section investigation.
As shown in FIG. 2, the escape latency of the normal control group and the administration group is significantly reduced, while the escape latency of APP/PS1 mice is slightly reduced. Optimized compound 27 was effective with only low dose (2mg/kg) administration, whereas high dose treatment (10mg/kg) resulted in even better cognitive levels from day 2 than control and Nec-1s mice (fig. 2A). The number of platform shuttling was significantly increased in the compound 27 administration group compared to the model group (fig. 2B), demonstrating the excellent cognitive improvement effect of compound 27.
Iba-1 is a calcium binding protein specifically expressed in microglia, the upregulation of which reflects microglial activation. Our immunofluorescence results (FIG. 3) show that, in the region of the medial prefrontal cortex (mPF) which is highly correlated with cognition and behavior, Iba-1 expression levels are abnormally elevated in model mice compared to control mice, while interventional treatment with Compound 27 restores Iba-1 expression to normal levels. The compound 27 is shown to have excellent anti-inflammatory effect.

Claims (10)

1. A novel inhibitor of targeted RIPK1 kinase is characterized by being selected from compounds with the structure shown as a general formula (I) or a general formula (II) or pharmaceutically acceptable salts thereof:
Figure FDA0003705240320000011
wherein R is 1 Selected from substituted or unsubstituted aromatic rings; r 2 Selected from hydroxyl or amino; r is 3 Selected from substituted or unsubstituted aromatic rings; r 4 Is selected from C 1-6 Aliphatic hydrocarbons or hydrogen.
2. The novel inhibitor of the RIPK1 targeted kinase according to claim 1, wherein the novel inhibitor of the RIPK1 targeted kinase is selected from compounds represented by the general formula (I), wherein R is 3 Is selected from substituted or unsubstituted benzene rings; r 4 Is hydrogen.
3. The novel inhibitor of RIPK1 targeted kinase according to claim 2, characterized in that R in the compound shown as the general formula (I) 4 Is hydrogen, R 3 Selected from halogen, cyano, methoxy, trifluoromethyl sulfanyl, C1-5 alkyl, C1-3 alkenyl, methylenedioxy, trifluoromethyl, and phenyl mono-or polysubstituted with phenoxy, or 5,6,7, 8-tetrahydro-2-naphthylphenyl, 4- (2-furyl) phenyl, unsubstituted phenyl.
4. The novel inhibitor of RIPK1 targeted kinase according to claim 3, characterized in that R in the compound shown as the general formula (I) 4 Is hydrogen, R 3 Selected from the group consisting of 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-butylphenylA group, 4-trifluoromethylphenyl group, 2-chlorophenyl group, 2-methylphenyl group, 4-n-propylphenyl group, 4-phenoxyphenyl group, 4-methoxy-2-methylphenyl group, 2-fluoro-4-methoxyphenyl group.
5. The novel class of inhibitors targeting RIPK1 kinase according to claim 1, wherein R is 1 Selected from phenyl, halogen or C1-2 mono-or polysubstituted phenyl, R 2 Selected from hydroxyl or amino; preferably when R is 2 When selected from hydroxy, R 1 Selected from phenyl or chloro-substituted phenyl; when R is 2 When selected from amino, R 1 Is selected from phenyl or phenyl mono-or polysubstituted by fluorine, chlorine and methyl.
6. The novel class of inhibitors targeting RIPK1 kinase according to claim 1, wherein R is when R 2 When selected from hydroxy, R 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.
7. The novel inhibitor of RIPK1 targeted kinase according to any of claims 1 to 6, wherein the compound of formula (I) or formula (II) and the pharmaceutically acceptable salt thereof is selected from the group consisting of acid addition salts of compounds of formula (I) or (II) 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.
8. A pharmaceutical composition comprising the novel inhibitor of the targeted RIPK1 kinase of any one of claims 1-6 and a pharmaceutically acceptable carrier.
9. Use of the novel inhibitors of the targeted RIPK1 kinase of any one of claims 1-6 in a medicament for the treatment of neurodegenerative or other inflammatory related diseases; preferably during the preparation.
10. Use of the novel inhibitor of RIPK 1-targeted kinase according to any one of claims 1 to 6 for the preparation of a medicament for the treatment and/or prevention of diseases mediated by RIPK1 kinase, preferably for the preparation of a medicament for the treatment of neurodegenerative or inflammatory-related diseases mediated by RIPK1 kinase, further preferably for the preparation of a medicament for the treatment of alzheimer's disease mediated by RIPK1 kinase.
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