Design,Synthesis And Bioactivity Study Of Novel Anti-stroke N-Hydroxypyridone Derivatives And Acyl-hydrazine Based PfHDAC Inhibitors,and Curcumin-based Ratiometric Lysosomal Polarity Fluorescent Probes

This doctoral dissertation consists of 3 parts.Part 1.Design,synthesis and bioactivity study of novel N-hydroxypyridone derivatives as potential anti-ischemic stroke agents.Ischemia-reperfusion(I/R)injury aggravate brain damage of stroke patients,the administration of neuroprotective agents can attenuate I/R injury and promote prognosis.In our previous studies,in vivo&in vitro results had confirmed the effectiveness of ciclopirox(CPX)for ischemic brain injury.Whereas CPX possesses mediocre activity and poor metabolic stability,in this study,its structure is optimized to afford better neuroprotective activity and improve pharmacokinetic properties.N-hydroxyl pyridone derivatives A1-A31 were designed and synthesized by replacing the hydrogens or substituents at position 1,3,4,5 and 6 of CPX with different substituents.The neuroprotective effect of these compounds was evaluated by Oxygen-Glucose Deprivation(OGD)experiment on SH-SY5Y cell line.7 of these derivatives show good neuroprotective activity on 1 μM,we subsequently carried out another multi-doses OGD experiment,blood-brain barrier(BBB)permeability assay and oxygen radical scavenging assay to further evaluate these 7 compounds.Among them,A10,6-endo-norbornane derivative,demonstrates the best neuroprotective activity in OGD model,and it also has good BBB permeability ability and better antioxidants capability compared to CPX.Thus,compound 11 was selected for further studies.The complex of compound A10 and olamine—A10·Ola processed good water-solubility(10.90 mg/mL),which is better than that of CPX olamine.A10·Ola could attenuate oxidative damage in SH-SY5Y cells and glutamate-induced toxicity in HT22 cells.A10·Ola also reduce the cell apoptosis and protect the cell morphology against OGD injury.In MCAO(middle cerebral artery occlusion)in vivo experiment,0.3 mg/kg dose of compound A10·Ola afford the same reduction on infarct volume compare to CPX olamine,also,effects of 11·Ola at the dose of 1mg/kg on mNSS(modified neurological severity scores)score was comparable to that of CPX olamine at 3 mg/kg.Considering that the cytotoxicity of compound A10·Ola and CPX olamine is at the same level(IC50 on MRC-5 cell line～23 μM),the treatment with lower doses of A10·Ola possesses superior safety window compared to CPX olamine.Furthermore,A10·Ola showed superior metabolic stability compared to CPX olamine.Probe AP1,AP2 and AP3 were designed and synthesized to study the mechanism of A10 and CPX.AP1 and AP2,which were obtained by attaching biotin-linker moiety to position 1 and 3 of CPX,showed no activity.Thus,AP3 which bears diazirine and alkyne were subsequently synthesized,it can specifically label protein through click reaction and photo-crosslinking reaction.However,AP3 pull-down experiment failed.Therefore,we proceeded the mechanism study through other strategies.We carried out bSDTNBI in silico prediction and iron chelating experiments,the results of these experiments suggested that the neuroprotective activity of A10 and CPX might correlate with iron chelation and stabilization of HIF1-α.Besides,the radical scavenging ability of A10 and CPX may contribute to their neuroprotective activity.In brief,part 1 of this dissertation presents the design and synthesis A1-A31 and the study of their in vivo and in vitro neuroprotective activity and drug-like properties.Among these derivatives,A10 exhibits significant neuroprotection against oxygen glucose deprivation(OGD)and oxidative stress induced injuries in neuronal cells.It also possesses good BBB permeability and superior antioxidant capability.In addition,its complex with olamine-A10·Ola possesses good water solubility,negligible hERG inhibition and superior metabolic stability.A10·Ola significantly reduces brain infarction and alleviate neurological deficits in MCAO rats.Hence,A10·Ola is identified in our research as a prospective prototype in the innovation of stroke treatment.Part 2.Design,synthesis and bioactivity study of acyl-hydrazine based PJHDAC inhibitors.PfHDAC(P.falciparum histone deacetylase)inhibitor,of which mechanism of action is different with that of artemisinin,is expected to be used in the treatment of artemisinin-resistant malaria.In our previous study,based on the structure of quisinostat(an HDAC inhibitor for the treatment of cancer),we developed a series of pfHDAC inhibitor through replacing the 4-amomethylpiperidine linker and 1-methylindole moiety,keeping the zinc ion binding group isohydroxamic acid unchanged.Among them,the selected compounds show lower toxicity,but still possess strong inhibitory activity against human HDAC,poor oral bioavailability and metabolic stability.Zinc is an essential cofactor for HDAC.In view of that the structure of catalytic centers of PfHDAC and human HDAC are different,and the hydroxyl in isohydroxamic acid is the key metabolic site,the structural modification of zinc binding group is the key to improve the selectivity and optimize the pharmacokinetic properties.Therefore,we designed and synthesized a series of novel PfHDAC inhibitor B1-B64 with novel zinc binding groups,in order to reduce the inhibition of human HDAC and improve the pharmacokinetic properties while maintaining the antimalarial activity.First,we replaced the isohydroxamic acid of quisinostat with different zinc binding groups to afford B1-B18.Among them,B1 and B2,which contain N1-acyl-4-fluoro-o-phenylenediamine and N1-acyl-o-phenylenediamine as zinc binding groups respectively,showed poor antimalarial activity.B3-B9,the other derivatives contains o-phenylenediamine,are inactivity.These findings indicated that these structures are not suitable for zinc ion binding groups as PfHDAC inhibitors.Thus,we subsequently designed and synthesized B10-B18 with acyl-hydrazine as zinc binding group.Among them,B11(contains N2-propyl-N1-acylhydrazine moiety)possessed better antimalarial activity,lower cytotoxicity,compared with quisinostat.Also,the inhibitory activity of B11 against various subtypes of human HDAC was significantly lower than that of quisinostat.Hence,the structure of N2-propyl-N1-acylhydrazine was fixed as the zinc ion binding group for the new generation of PfHDAC inhibitor with superior selectivity.The 4-amomethylpiperidine linker and 1-methylindole moiety of B11 were further replaced with other structures to obtain the derivative B20-364.Derivative B19-B23 were obtained through replacing 4-amomethylpiperidine with different aza-spiro ring,aliphatic chain and piperazine,however,the activity and selectivity of them were inferior to that of B11.We thereupon replaced 1-methylindole moiety with other substituents to afford B24-B64,while the linker of them is still 4-amomethylpiperidine.Derivatives B24-B36 bear substituted indoles or other aromatic rings which are similar to indoles.Among them,B32,B35 and B36 showed superior anti-malarial activity,Pf3D7 IC50 of B36 is around 25 nM.Derivatives B37-B51 bear aromatic dicyclic rings which are not similar to indoles,the activity of them is inferior to that of B11.Derivatives B52-B64 bear different aromatic monocyclic ring,aliphatic ring or other substituents,the activity of these derivatives is greatly reduced compared with that of B11.B36 showed the best antimalarial activity among all the acyl-hydrazine derivatives,also showed lower cytotoxicity and better selectivity,the SI293T/Pf3D7 of B36 is 63,which is far better than that of quisinostat and B11.Moreover,the inhibitory activity of B36 against human HDAC decreased by about 70 times compared with quisinostat,its hydrochloride-B36·HCl possesses higher oral bioavailability than that of isohydroxamic acid derivatives.In this study,PfHDAC inhibitors B1-B64 with novel zinc binding groups were designed and synthesized,and their antimalarial activity,cytotoxicity,selectivity and inhibitory activity against human HDAC were investigated.Among them,the selected compound B36 has superior antimalarial activity and lower cytotoxicity,significantly decreased inhibitory activity on human HDAC,significantly improved selectivity,and higher oral bioavailability.B36 has the potential to be developed as oral pfHDAC-targeted anti-malarial agent with better safety.Part 3.Study of curcumin-based ratiometric lysosomal polarity fluorescent probes.Various physiological and pathological processes are accompanied by lysosomal polarity changes,lysosomal polarity fluorescent probes can be a powerful tool for these research.Currently,a limited number of fluorescent probes can monitor lysosome polarity with a ratiometric response,the emission wavelength of them is not long enough.Thus,we presented a novel dual sites design strategy for curcumin-based polarity fluorescent probes.Curcumin possesses low polarity specificity and water insolubility.Therefore,probe C1-C5 was obtained through attaching galactose moiety and different amines to curcumin in order to increase the water solubility,regulate the probe working mode and improve polarity sensitivity.C1 and C2 exhibited preferable fluorescence responses to polarity,indicating that diethylamino moiety is essential to monitoring polarity.Moreover,we found the introduction of the galactose group effectively improved the water solubility and regulated the detection mode to polarity by comparing the optical properties between C1 and C2.C1 and C2 exhibited a good linear relationship between fluorescence ratio(C1:I602/I554;C2:I606/I545)and solvent parameter △f.Both of them specifically response to polarity change without significant interferences from pH,viscosity or some other common interfering species(metal ions,anion species and biothiols).Besides,C1 and C2 displayed good photostability.The analysis of 1D and 2D NMR spectra the ratio between 2 tautomer(β-diketone and keto-enol)of C1.In cell imaging experiments,C1 possesses the ability to localize in lysosomes.Since the ratio between the green channel fluorescence intensity and the red channel fluorescence intensity varies with the polarity in lysosomes of Cl stained cells,it possesses the potential ability to discriminate between cancer cells and normal cells and the ability to monitor the variations in lysosome polarity caused by DMSO and sucrose.C1 can be employed for the detection of lysosome polarity to study polarity-related biological processes.