Design,synthesis,and Neuroprotective Activity Of Novel HDAC6 Inhibitors

With the development of aging society,Alzheimer’s disease（AD）,has become one of the major diseases especially for the elderly.However,the pathogenesis of AD is not yet clear,and marketed anti-AD drugs can only alleviate symptoms.Therefore,it is urgent to develop safer and more effective anti-AD drugs.Research shows that Histone deacetylase 6（HDAC6）is over expressed in the cerebral cortex and hippocampus of AD patients,which is associated with a series of AD related processes,such as tau protein hyperphosphorylation,Aβaggregation,neuroinflammatory response,and oxidative stress.A lot of study showed that HDAC6 inhibitor（HDAC6i）can significantly inhibit these pathological and physiological processes.Hence,HDAC6 has become one of the potential targets in the development of anti-AD drugs.Quinazoline structure exists in many natural product compounds and displays various pharmacological activity.Based on the classic design idea of selective HDAC6i,we used 4-phenylquinazoline as the surface binding domain（Cap）,methylene phenyl as the linker group,and hydroxamic acid as the zinc binding group（ZBG）and synthesized a series of quinazoline-based derivatives.Molecular simulation results showed that lead compound 4a can effectively bind to the catalytic site of HDAC6.Subsequently,the cap and linker part of 4a were further modified,resulting in a total of 13 compounds（series I）.Then,scaffold hopping strategy was adopted to break the quinoline ring and obtain 7 compounds as series II and 1 compound as series III.In vitro,all compounds showed obvious inhibitory activity against HDAC6 except for series III compounds.Among all,4a and 15b are the most potent compounds in series I and II,with IC₅₀ of 2.36 n M and 2.12 n M,respectively.The preliminary structure-activity analysis shows that linker group can significantly affect the selectivity of the compound towards HDAC6.To investigate the neuroprotective effect of all target compounds,we established a mouse microglioma（BV-2）induced by lipopolysaccharide（LPS）as an in vitro neuroinflammation model and evaluated anti-neuroinflammatory activity of the designed HDAC6i.Firstly,all compounds did not injure the cell viability of BV-2 within a certain concentration range.Secondly,the release of inflammatory biomarkers nitric oxide（NO）and the expression of pro-inflammatory marker proteins inducible nitric oxide synthase（i NOS）and cyclooxygenase-2（COX-2）were used as indicators to evaluate the anti-neuroinflammatory activity of all compounds.The results showed that compounds with low selectivity and strong HDAC6inhibition could effectively reduce NO release.The 4a and 15b with the strongest inhibitory activity against HDAC6 can significantly reduce the expression level of i NOS.Finally,in order to explore the molecular mechanism of anti-neuroinflammatory activity,HSP90acetylation level was measured in neuroinflammation models treated with 4a and 15b.The result showed that the HSP90 acetylation level had a significant increase.Based on literature research,it is preliminarily speculated that the acetylated HSP90 regulates the expression of i NOS,leading to the anti-neuroinflammatory effect of HDAC6 inhibitors.In summary,this paper provided a theoretical basis for the design of new anti-AD drugs based on HDAC6i,and lay a foundation for the next exploration of the neuroprotective mechanism of HDAC6i in anti-neuroinflammation.