Neuroprotective agents 개발을 위한 imidazoisoquinolinone과 ferulic acid 유도체 합성 및 활성 연구

Abstract

Calpain is Ca²^(+)-dependent cysteine protease that plays an important role in cell differentiation and in apoptosis/necrosis. Activated calpain induces the conversion of p35 into p25 in neurons, and then p25 modulates cdk5 activity. Consequently, p25/cdk5 kinase hyperphosphorylates tau and promotes apoptosis of primary neurons. Intracellular neurofibrillary tangle is composed by hyperphosphorylated tau and it is one of the Alzheimer's disease characteristic hallmarks with extra cellular plaques of amyloid β-peptide aggregates. Overactivated calpain induces cathepsin release. Cathepsin is also a member of the papain superfamily of cysteine proteases and it mediates caspase-activation. Activated caspase 1, 3 can induce calpastatin degradation leading to the sustained calpain activation. At the same time, caspase 3 mediatied proteolysis plays a role in the neurons, membranes, cytoskeleton integry and nuclear functions. Therefore, the use of calpain and cathepsin inhibitors is of potential therapeutic utility in ischemic injuries. Especially, the best strategy for neuroprotection after ictus seems to be indentifying and targeting more downstream cellular events that are triggered by Ca²^(+). The known calpain and cathepsin inhibitors have not been successful for any clinical usage for neurodegenerative diseases duing to poor selectivity to other cystein proteases and insufficient stability. Therefore, this research has been focused on the discovery of non peptide calpain and cathepsin inhibitors. Derivatives of calpain and cathepsin inhibitor based on ferulic acid with various activities were designed and synthesized. Synthesized compounds were evaluated by calpain and cathepsin inhibition test and MDL28170 and CA-074 were used as a positive control. All compounds didn't show good biological activities as the calpain inhibitor, but showed 50% inhibition activities at 10 nM as the cathepsin inhibitor. Among the compounds, F20 showed as good activity as CA-074, so it is possible to synthesize non peptide cathepsin inhibitors with modification of F20.;세포 내에 풍부하게 존재하는 단백질 중 하나인 Poly (ADP-ribose) polymerase-1 (PARP-1)은 DNA가 손상되었을 때 활성이 나타난다. DNA-damaging chemotherapeutics에 의해 손상된 DNA는 그 정도에 따라 PARP-1에 의해 수선되거나 사멸되는데 PARP-1 저해제를 DNA-damaging chemotherapeutics와 병용 투여하면 종양세포에 수선을 막아 apoptotic cell death를 일어나게 하고, 과도한 PARP-1의 활성을 억제하여 necrosis가 아닌 apoptosis를 유도하게 된다. 본 연구에서는 Inoteck사에서 isoquinolinone을 기본구조로 하여 개발한 INO-1001을 바탕으로 Pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5-(6H)-one 유도체를 합성하여 PARP-1 저해 능력을 평가하는 것을 목표로 하였다. 기존에 알려진 pyrido[2',1':2,3]imidazo[4,5-c]isoquinolin-5-(6H)-one 합성법을 이용하여 14종의 유도체를 합성할 수 있었다. 합성된 14종의 유도체는 세포 수준에서의 PARP-1 생리활성 테스트를 실시 하였으며 세포 독성을 확인하기 위하여 MTT assay를 실시 하였다. 테스트 결과 P3가 PARP-1 저해 능력이 가장 우수하고 세포 독성이 없는 것으로 확인되었다. 따라서 현재의 치환기인 methyl기 대신 INO-1001과 같은 긴 치환기를 도입한다면 지금 보다 뛰어난 PARP-1 저해제를 개발 할 수 있을 것이다.