Synthesis And Preliminary Activity Evaluation Of Histone Deacetylase-6 Inhibitors Against Alzheimer’s Disease

Alzheimer’s disease（AD）is a primary neurodegenerative disorder.The disease is histopathologically characterized by the presence of extracellular neuritic senile plaques（SP）and the intracellular neurofibrillary tangle（NFTs）.The senile plaques are formed by the accumulation of the amyloid β protein（Aβ,Aβ-peptide）while the neurofibrillary tangles are composed of hyperphosphorylated tau protein.AD becomes a major public health problem because of its long duration,increasing prevalence,and the high cost of care.At present,although anti-AD drugs can improve some pathological symptoms,it can not reverse the process of AD.Because the etiology of AD is rather complex and the causes for its pathogenesis are not very clear,the discovery of anti-AD drugs is much challenging.Oxidative stress plays an important role in the pathogenesis of AD.The imbalance of redox homeostasis in the body results in the production of several potentially toxic reactive oxygen species（ROS）that can cause neurotoxicity leading to neuronal death.The accumulation of oxidative stress also results in the Aβ accumulation and tau protein hyperphosphorylation,accelerating the progression of AD.AD is closely related to the presence of Aβ oligomers and their aggregates in the brain.Many causes can lead to the abnormality of Aβ metabolites,leading to over-production and lower degradation and clearance of Aβ.The excess Aβ aggregates to form the neurotoxic Aβ oligomers that are responsible for the development of inflammation,oxidative strese,and neuronal loss.In AD patients,some metal（Cu、Zn、Fe、Al）are acculated in the SP and NFT.Cu²⁺ forms a high-affinity complex with the Aβ peptide and promotes its aggregation and generates toxic ROS,further accelerating the oxidative stress.The levels of HDAC6 protein in the cerebral cortex and hippocampus of AD patients were significantly increased,indicating that HDAC6 may play an important role in the pathogenesis of AD.Hyperphosphorylation of tau decreases its affinity for microtubules,which leads to cytoskeleton disorganization,resulting in cell apoptosis.Aβ affects microtubules and consequently the related signaling pathways and the intracellular vesicular transport.These alterations lead to a deterioration of the traffic of mitochondria and neurotransmitters resulting in synapse degradation or synapse loss.HDAC6 inhibition could hyperacetylate a-tubulin and restore the mitochondrial transport.HDAC6 inhibitors can degradate tau and restore the Aβ-induced damages,and therefore,are hopeful drugs for AD therapy.Similar to other subtype HDAC inhibitors,the pharmacophore model of HDAC6 inhibitors can also be divided into three groups:a cap group（Cap）,a hydrophobic linking motif（Linker）,a zinc binding group（ZBG）.Usually,the cap group is an aromatic ring or a heterocycle.The linker motif is an alkyl chain or a substituted aromatic ring.The ZBGs are usually hydroxamic acid,thioalcohol.The surface binding domain of HDAC6 is larger than that of other HDAC isoforms,so a larger moiety can act as the Cap group.Moreover,the HDAC inhibitor for the treatment of AD must have the ability to pass through the blood-brain barrier.Therefore,we selected some pharmacophores,phenothiazine,10,11-Dihydro-5H-dibenz[b,f]azepine,memantine,often used in the drugs for the treatment of the central nervous system diseases,as the Cap group,connected different aromatic ring or aromatic heterocycles as the Liner,and used the hydroxamic acid as the ZBG,designed a series of novel HDAC6 inhibitors.A total of 20 new compounds were synthesized and evaluated for their inhibitory activities against HDAC6.The most potent HDAC6 inhibitors W1 and W3 were tested for their selectivity over other HDAC isoforms.The inhibition activities of some synthetic compounds against the SH-SY5Y and PC 12 cells,and the neuroprotective effects on H₂O₂-induced oxidative damage in PC 12 cells were determined by colorimetric MTT assay.To further investigate the effects of some selected compounds for their effects on Cu²⁺-induced Aβ aggregation and the disaggregation Cu²⁺-Aβcomplexes,Wl,W3,W8 and W15 were evaluated by a ThT-binding assay.The results showed that W3 is the most potent HDAC6 inhibitor,with an IC50 of 10.6 nM.W3 also has some selectivity over HDAC1,HDAC8 and HDAC 11（SI = 6.6,54,37）.All the compounds tested have no effects on the growth of SH-SY5Y and PC 12 cells,indicating their low cytotoxicity.W1 and W3 exhibited significant neuroprotective effects against H₂O₂-induced PC 12 cell damages at low micromolar concentrations,were more potent or comparable potency to that of the positive control Trolox.In the ThT-binding assay,W1,W3,W8 and W15 could inhibit Cu²⁺-induced Aβ aggregation effectively through chelating with Cu²⁺,and all the four compounds showed superior or comparable potency to the positive control clioquinol（CQ）.Among them,W1 and W3 inhibited Cu²⁺-induced Ap aggregation potently with an inhibition rate was 60%and 58%,respectively.In addition,W1 had comparable potency to disaggregate the Cu²⁺-induced Aβ aggregation as that of CQ with a disaggregation rate of 33%,while W3 showed superior potency with a disaggregation rate of 75%.Therefore,these novel HDAC6 inhibitors can inhibit Cu²⁺-induced Aβ aggregation,promote the disaggregation of Cu²⁺-induced Aβ aggregates,have anti-oxidative stress potency,target multifacet of AD,are worthy of further evaluation for anti-AD activity.