Design,Synthesis,Evaluation And Mechanism Of Novel Tetramethylpyrazine Derivatives As Potential Neuroprotective Agents

Aims Neurodegenerative diseases,including Alzheimer's,Parkinson's,Huntington's diseases and amyotrophic lateral sclerosis,are characterized by the progressive loss of structure or function of neurons in human brain or spinal cord,leading to functional loss(ataxia)or sensory dysfunction(dementia).Although progresses of research have been made and drugs for treatment of neurodegenerative diseases like Alzheimer's disease(AD)or Parkinson's disease(PD)have been developed,most clinical trials have yielded disappointing results for those drugs which aimed to reverse the degenerative progression in these diseases.Increasing evidence suggests that many causally related factors,including oxidative stress,mitochondrial dysfunction,neurotoxicity,Ca2+ influx,neuro-inflammation,autophagy and metal ion are accepted to contribute to the pathogenesis of neurodegenerative diseases.These findings lay the foundation for the design of multi-function and multi-targeted drugs for treatment of neurodegenerative diseases.In this project,we have designed and synthesized a series of tetramethylpyrazine derivatives and we found that compounds CT-011 and CT-006,with potent neuroprotection and multi-functional activities,would open new avenues for the treatment of neurological diseases including AD and PD.Methods Chemical synthesis;spectral analysis(ESI-MS,1H NMR,14 C NMR and EA);screening free radical scavenging abilities in free cell system;screening the protection of IAA-induced cells death;MTT assay to test cell viability;LDH kit to test cell toxicity and Hoechst/TUNEL kit to test cellular apoptosis;fluorescent probe to test cellular reactive oxygen species and reactive nitrogen species(ROS/RNS),mitochondrial membrane potential(??m)and Ca2+ influx;report assay;western blot,Immunofluorescence;PCR;Hu APPswe/PS1?E9 transgenic mice;MPTP mice.Results In this project,we have designed and synthesized 14 compounds and investigated their abilities for scavenging free radicals and their neuroprotection against oxidative stress in vitro.Most new compounds showed good free radicals-scavenging activities and neuroprotection in a dose-dependent manner.Compounds CT-011 and CT-006 are the most potent.Therefore,CT-011 and CT-006 were selected for further detailed research to understand their mechanism of action in neuroprotection.Here are the summary of the results.1)CT-011 prevents neurotoxin-induced(e.g.IAA/t-BHP/glutamate)neurotoxicity because of the multi-functional mechanisms:(1)both directly scavenging free radicals and inhibiting the production of ROS and RNS induced by the neurotoxins;(2)preventing glutamate-induced neurotoxicity by activating the PI3K-Akt and inhibiting GSK3? pathways;(3)preventing glutamate induced neurotoxicity by activating the PGC1?-Nrf2-ARE pathway and upregulating the HO-1 expression.2)CT-006 prevents neurotoxin-induced induced neurotoxicity because of the multi-functional mechanisms:(1)both directly scavenging free radicals and inhibiting the production of ROS and RNS induced by the neurotoxin;(2)inhibiting glutamate-induced Ca2+ influx;(3)preventing MPP+ induced neurotoxicity by activating the PI3K-Akt and inhibiting GSK3? pathways;(4)directly activating the MEF2D-PGC1? and Nrf2-ARE pathway in DA neurons derived from i PS cells.3)CT-006 reduces memory deficits in APP/PS1 transgenic mice and ameliorates MPTP-induced behavioral impairments in mice.CT-006 protects against MPTP-induced loss of substantia nigra pars compacta(SNpc)DA neurons and reduction of dopamine(DA)and its main metabolites,3,4-Dihydroxyphenylacetic acid(DOPAC)and homovanillic acid(HVA),the preliminary mechanism of the CT-006's neuroprotection is inhibiting CDK5 and activating the MEF2D-PGC1? pathway.Conclusion Activating the MEF2D-PGC1? and Nrf2-ARE pathway would be the new therapeutic targets for the treatment of neurodegenerative diseases.CT-011 and CT-006,with potent neuroprotection of multi-functional activities,would open new avenues for the treatment of neurological diseases including AD and PD.