An efficient and practical synthetic process for Daprodustat was developed.Starting with N,N'-dicyclohexylcarbodiimide(DCC)and malonic acid,the key intermediate 1,3-dicyclohexylpyrimidine-2,4,6(1H,3H,5H)-trione wa...An efficient and practical synthetic process for Daprodustat was developed.Starting with N,N'-dicyclohexylcarbodiimide(DCC)and malonic acid,the key intermediate 1,3-dicyclohexylpyrimidine-2,4,6(1H,3H,5H)-trione was synthesized via condensation reaction with 91%yield.Subsequent activation of this intermediate by 1,1'-carbonyldiimidazole(CDI),followed by a one-pot reaction with glycine ethyl ester hydrochloride,directly afforded Daprodustat in 92%yield with>99.8%HPLC purity.The process achieved an overall yield of 84%upon validation at 62-gram scale.Structural confirmation of the key intermediate was accomplished through nuclear magnetic resonance(NMR)spectroscopy and high-resolution mass spectrometry(HRMS).Compared with existing methods,this streamlined protocol demonstrates advantages including simplified operation,reduced reaction time,and lower production costs,offering significant potential for industrial-scale synthesis of Daprodustat.展开更多
目的:探讨石斛合剂含药血清对高糖诱导的小鼠足细胞损伤的影响及作用机制。方法:体外培养小鼠肾小球足细胞(MPC5),筛选高糖造模浓度和时间及石斛合剂含药血清最佳给药浓度;将细胞分为正常组(5.5 mmol·L^(-1)葡萄糖+10%空白血清)、...目的:探讨石斛合剂含药血清对高糖诱导的小鼠足细胞损伤的影响及作用机制。方法:体外培养小鼠肾小球足细胞(MPC5),筛选高糖造模浓度和时间及石斛合剂含药血清最佳给药浓度;将细胞分为正常组(5.5 mmol·L^(-1)葡萄糖+10%空白血清)、模型组(30 mmol·L^(-1)葡萄糖+10%空白血清)、石斛合剂含药血清组(30 mmol·L^(-1)葡萄糖+10%石斛合剂含药血清),铁死亡抑制剂(Fer-1)组(30 mmol·L^(-1)葡萄糖+10%空白血清+1μmol·L^(-1)Fer-1)。采用试剂盒检测细胞中Fe^(2+)、乳酸脱氢酶(LDH)水平;酶联免疫吸附测定法(ELISA)检测细胞谷胱甘肽(GSH)、过氧化脂质(LPO)含量;荧光探针检测活性氧(ROS)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测足细胞中肾母细胞瘤基因-1(WT-1)、结蛋白(Desmin)、长链脂酰辅酶A合成酶4(ACSL4)和谷胱甘肽过氧化物酶4(GPX4)m RNA表达水平;蛋白免疫印迹法(Western blot)检测足细胞中WT-1、Desmin、ACSL4和GPX4蛋白表达水平。结果:与空白组比较,30 mmol·L^(-1)高糖干预48 h可显著降低足细胞活力(P<0.01),10%石斛合剂含药血清改善高糖诱导的足细胞活力最为显著(P<0.01)。与正常组比较,模型组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达显著升高(P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达显著降低(P<0.01);与模型组比较,石斛合剂含药血清组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达明显降低(P<0.05,P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达明显升高(P<0.05,P<0.01)。结论:石斛合剂含药血清对高糖诱导的足细胞损伤具有保护作用,与其抑制铁死亡有关。展开更多
文摘An efficient and practical synthetic process for Daprodustat was developed.Starting with N,N'-dicyclohexylcarbodiimide(DCC)and malonic acid,the key intermediate 1,3-dicyclohexylpyrimidine-2,4,6(1H,3H,5H)-trione was synthesized via condensation reaction with 91%yield.Subsequent activation of this intermediate by 1,1'-carbonyldiimidazole(CDI),followed by a one-pot reaction with glycine ethyl ester hydrochloride,directly afforded Daprodustat in 92%yield with>99.8%HPLC purity.The process achieved an overall yield of 84%upon validation at 62-gram scale.Structural confirmation of the key intermediate was accomplished through nuclear magnetic resonance(NMR)spectroscopy and high-resolution mass spectrometry(HRMS).Compared with existing methods,this streamlined protocol demonstrates advantages including simplified operation,reduced reaction time,and lower production costs,offering significant potential for industrial-scale synthesis of Daprodustat.
文摘目的:探讨石斛合剂含药血清对高糖诱导的小鼠足细胞损伤的影响及作用机制。方法:体外培养小鼠肾小球足细胞(MPC5),筛选高糖造模浓度和时间及石斛合剂含药血清最佳给药浓度;将细胞分为正常组(5.5 mmol·L^(-1)葡萄糖+10%空白血清)、模型组(30 mmol·L^(-1)葡萄糖+10%空白血清)、石斛合剂含药血清组(30 mmol·L^(-1)葡萄糖+10%石斛合剂含药血清),铁死亡抑制剂(Fer-1)组(30 mmol·L^(-1)葡萄糖+10%空白血清+1μmol·L^(-1)Fer-1)。采用试剂盒检测细胞中Fe^(2+)、乳酸脱氢酶(LDH)水平;酶联免疫吸附测定法(ELISA)检测细胞谷胱甘肽(GSH)、过氧化脂质(LPO)含量;荧光探针检测活性氧(ROS)水平;实时荧光定量聚合酶链式反应(Real-time PCR)检测足细胞中肾母细胞瘤基因-1(WT-1)、结蛋白(Desmin)、长链脂酰辅酶A合成酶4(ACSL4)和谷胱甘肽过氧化物酶4(GPX4)m RNA表达水平;蛋白免疫印迹法(Western blot)检测足细胞中WT-1、Desmin、ACSL4和GPX4蛋白表达水平。结果:与空白组比较,30 mmol·L^(-1)高糖干预48 h可显著降低足细胞活力(P<0.01),10%石斛合剂含药血清改善高糖诱导的足细胞活力最为显著(P<0.01)。与正常组比较,模型组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达显著升高(P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达显著降低(P<0.01);与模型组比较,石斛合剂含药血清组足细胞内Fe^(2+)、LDH、LPO、ROS水平及Desmin、ACSL4 m RNA和蛋白表达明显降低(P<0.05,P<0.01),GSH水平及WT-1、GPX4 m RNA和蛋白表达明显升高(P<0.05,P<0.01)。结论:石斛合剂含药血清对高糖诱导的足细胞损伤具有保护作用,与其抑制铁死亡有关。
