Design,Synthesis And Biological Evaluation Of Anti-alzheimer’s Agents Based On The Metabolism-activated Multitargeting Drug Design Strategy(Ⅰ)

Alzheimer‘s disease（AD）is an irreversible chronic neurodegenerative disease,characterized by cognitive dysfunction,personality change and behavioral abnormality.The occurrence of AD is hard to detect and many factors play key roles in its pathogenesis,including oxidative stress,abnormal deposition ofβ-amyloid protein,hyperphosphorylation and aggregation of tau protein,decrease of acetylcholine level,neuroinflammation and imbalance of metal ion homeostasis.Currently,most of the anti-AD drugs used in clinical are designed for single target,these drugs include acetylcholinesterase inhibitors（Donepezil,Galanthamine and Rivastigmine）,N-methyl-D-aspartate receptor antagonist（Memantine）and gut microbiota regulator（Sodium oligomannate）.Unfortunately,the drugs described above can temporarily alleviate the symptoms of AD but failed to reverse the process because of the complex pathological nature of AD.Therefore,anti-AD drugs which can interact with multiple targets in AD pathogenic net may have ability to modify the progression of AD while relieving symptoms.The multi-target drug design strategy commonly used by researchers currently often combines different pharmacophores by linking,fusing and merging.However,the obtained compounds often possess increased molecular size and unfavorable physicochemical properties,which lead adverse effects and/or an inappropriate ADME（absorption,distribution,metabolism,and excretion）profile.In recent years,researchers described a novel multi-target drug design strategy:Metabolism-activated multitargeting（MAMUT）.Through careful design by the drug design strategy,the parent MAMUT compounds possess different pharmacological activities with their metabolites,and its multi-function can be achieved through the combination of synergistic effects of the parent drug and its active metabolite（s）.Because the parent drug and metabolites share the same molecular scafford,MAMUT drugs often have smaller molecular size and appropriate ADME properties.Based on“MAMUT”drug design strategy,we designed and synthesized three series compounds,including 3-（aminoalkyl/piperidinealkyl thio）phthalide compounds（I）,3-（aminoalkyl/piperidinealkyl disulfaneyl alkyl）phthalide compounds（II）and3-（aminoalkyl/piperidinealkyl thio）-2-thiophthalide compounds（III）by using phthalide alkyl tertiary amine derivatives and piperidine alkyl phthalide derivatives as lead compounds and using aminoalkyl mercaptan,aminoalkyl disulfide,aminoalkyl sulfonic acid and gas transmitter hydrogen sulfide（H₂S）as pharmacophores.The structures of target compounds were shown as follow:For the synthesis of target compounds I,the intermediates of aminopropyl Bünte salt,aminobutyl mercaptan,aminohexyl mercaptan with different substituents and4-benzylpiperidine alkyl mercaptan with different carbon chain lengths were prepared firstly,then the condensation of these intermediates with 3-hydroxyphthalide（4）or3-hydroxy-5,6-dimethoxy phthalide（5）under acidic conditions afforded3-（aminoalkyl/piperidinealkyl thio）phthalide compounds（I）.The synthesis of target compounds II requires the preparation of amine ethyl mercaptan and amine butyl mercaptan intermediates containing a variety of tertiary amine substitutions and4-benzylpiperidine alkyl mercaptan with different carbon chain lengths.In addition,it also needs to prepare the intermediates of benzenesulfonates at the 3-position of phthalide with different carbon chain lengths,and then the condensation of the intermediate of benzenesulfonates with the intermediates of mercaptan at room temperature afforded 3-（aminoalkyl/piperidinealkyl disulfaneyl alkyl）phthalide compounds.The synthetic method of compound III is similar with the synthetic method of compound I.Firstly,the intermediates of aminopropyl Bünte salt,aminohexyl mercaptan and 4-benzylpiperidine propyl mercaptan were prepared,and then 3-hydroxy-5,6-dimethoxy-2-thiophthalide was prepared.The condensation of these two kinds of intermediates afforded 3-（aminoalkyl/piperidinealkyl thio）-2-thiophthalide compounds（III）.After the preparation of the target compounds,the metabolites of selected target compounds were also synthesized,including aminoalkyl mercaptan,aminoalkyl disulfide,aminoalkyl sulfonic acid,3-（mercaptoalkyl）phthalide and 3-（sulfonic acid alkyl）phthalide.Totally,76 target compounds and 32 metabolites were synthesized,all the target compounds and most of metabolites were not reported by literature previously,and all the target compounds and metabolites were characterized by ¹H NMR,most of them were further characterized by ¹³C NMR and ESI-MS.To figure out the MAMUT anti-AD activity of these target compounds,we screened anti-AD activities of these target compounds and metabolites in vitro,and conducted further pharmacokinetics study and pharmacodynamics study of representative compounds in vivo.The results indicated that 3-（aminoalkyl/piperidine alkyl thio）phthalide compounds（I）generally showed good MAMUT anti-AD activities.Compounds I-4-2c and I-2-2g raised particular interest because of their outstanding anti-AD activities.I-4-2c showed extremely excellent and selective ACh E inhibition(ee ACh E:IC₅₀=2.60×10^-4μM;hu ACh E:IC₅₀=3.84×10^-4μM),the representative compound I-2-2g also displayed good ACh E inhibitiory activity(ee ACh E:IC₅₀=0.27μM;hu ACh E:IC₅₀=0.24μM),their aminoalkyl mercaptan metabolites:4-（ethyl（2-methoxybenzyl）amino）butane-1-thiol（D-1-2g）,3-（1-benzyl piperidin-4-yl）propane-1-thiol（D-1-4c）,and aminoalkyl disulfide metabolites:4,4’-dis ulfanediylbis（N-ethyl-N-（2-methoxybenzyl）butan-1-amine）（D-3-2g）,1,2-bis（3-（1-benzylpiperidin-4-yl）propyl）disulfane（D-3-4c）also showed decent and selective ACh E inhibitory activity.In addition,the aminoalkyl mercaptan metabolites（D-1-2g and D-1-4c）and aminoalkyl disulfide metabolites（D-3-2g and D-3-4c）also possess good self-induced Aβaggregation inhibitory activity and antioxidant activity,which were far more excellent than corresponding parent drugs I-2-2g and I-4-2c.Further study showed that aminoalkyl mercaptan metabolites（D-1-2g and D-1-4c）and their parent drugs（I-2-2g and I-4-2c）had good metal chelating abilities,and they can also inhibit the oxidative stress induced by Cu²⁺.Parent drugs（I-2-2g and I-4-2c）and aminoalkyl mercaptan metabolites（D-1-2g and D-1-4c）also showed appropriate brain blood barrier（BBB）permeability in vitro.In addition,I-2-2g and I-4-2c also exhibited good anti-neuroinflammatory activities in vitro,they can inhibit the activation of BV-2 glial cells induced by lipopolysaccharide（LPS）,and suppress the expression of NO and TNF-α.In vitro enzyme cleavage studies showed that I-2-2g and I-4-2c were stable in phosphate buffer saline（PBS,p H=7.4）,but can be hydrolyzed to aminoalkyl mercaptan metabolites and 3-hydroxyphthalide（4）or3-hydroxy-5,6-dimethoxy phthalide（5）by porcine liver esterase.The pharmacokinetic study in mice further confirmed that I-4-2c can cross the BBB and can be hydrolyzed to 3-（1-benzylpiperidin-4-yl）propane-1-thiol（D-1-4c）and 5,the D-1-4c would be oxidized to 3-（1-benzylpiperidin-4-yl）propane-1-sulfonic acid（D-2-4）subsequently.The pharmacodynamics study in vivo revealed that I-2-2g and I-4-2c can attenuate the central cholinergic disturbance induced by scopolamine,reduce the oxidative stress induced by Na NO₂,and significantly improve the cognitive ability in mice.To further improve the MAMUT activities of 3-（aminoalkyl/piperidinealkyl thio）phthalide compounds（I）,we introduced disulfide bond to the side chain in the3-position of tertiary amine alkyl phthalide derivatives and piperidine alkyl phthalide derivatives,designed and synthesized a series of 3-（aminoalkyl/piperidinealkyl disulfaneyl alkyl）phthalide compounds（II）.The in vitro biologic screening results indicated that these target compounds generally possessed good MAMUT anti-AD activities.The representative compound II-2-1c raised particular interest because of its outstanding anti-AD activities.II-2-1c displayed good and selective ACh E inhibitory activity(ee ACh E:IC₅₀=0.025μM;hu ACh E:IC₅₀=0.022μM),decent inhibitory activity against Aβaggregation（33.9%inhibition at 25μM）and disaggregation ability on Aβfibrils（23.5%disaggregation at 25μM）.Moreover,its metabolite（D-4-1a）showed comparatively potent antioxidant activity（ORAC=0.72Trolox equivalents）,which is better than the parent drug II-2-1c（ORAC=0.34Trolox equivalents）.Further study revealed that both II-2-1c and 3-（mercapto alkyl）phthalide（D-4-1a,D-4-1b）have good biometal chelating abilities and can form the complex with Cu²⁺at a mole fraction of 2:1,II-2-1c and D-4-1a can also inhibit the oxidative stress induced by Cu²⁺.Further study showed that II-2-1c possessed appropriate brain blood barrier（BBB）permeability in vitro.In vitro cleavage studies showed that II-2-1c was stable in PBS（p H=7.4）,and can be decomposed into3-（1-benzylpiperidin-4-yl）propane-1-thiol（D-1-4c）and 3-（mercaptomethyl）-5,6-di methoxyphthalide（D-4-1a）in the presence of glutathione,and this decomposition mechanism of II-2-1c was further confirmed by the animal study in mice.Unfortunately,we detected II-2-1c in plasma but did not detect it in the brain after oral administration in mice,so we did not perform the pharmacodynamics study in vivo.Based on the study of 3-（aminoalkyl/piperidinealkyl thio）phthalide compounds（I）,we designed and synthesized 3-（aminoalkyl/piperidinealkyl thio）-2-thiophthalide compounds（III）by replacing the phthalide moiety of target compounds I by2-thiophalide.The in vitro biologic screening results indicated that these target compounds generally showed good MAMUT anti-AD activities.Among them,compound III-3c was selected because of its outstanding anti-AD activities.III-3c displayed good and selective ACh E inhibitory activity(ee ACh E:IC₅₀=0.084μM;hu ACh E:IC₅₀=0.072μM),decent inhibitory activity against self-induced Aβaggregation（29.4%inhibition at 25μM）and disaggregation ability on Aβfibrils（14.9%disaggregation at 25μM）.Compound III-3c also showed antioxidant activity（ORAC=0.32 Trolox equivalent）and appropriate BBB permeability in vitro.In vitro enzyme cleavage studies showed that III-3c was stable in PBS（p H=7.4）,and cannot be decomposed by porcine liver esterase,acetylcholinesterase,butyrylcholinesterase and cysteine,but by primary amines.The pharmacokinetic study in mice further revealed that III-3c can cross the BBB and be hydrolyzed to3-（1-benzylpiperidin-4-yl）propane-1-thiol（D-1-4c）and 3-hydroxy-5,6-dimethoxy phthalide（5）,the D-1-4c would be oxidized to 3-（1-benzylpiperidin-4-yl）propane-1-sulfonic acid（D-2-4）subsequently.The pharmacodynamics study in vivo revealed that III-3c can attenuate the central cholinergic disturbance induced by scopolamine,reduce the oxidative stress induced by Na NO₂,and significantly improve the cognitive ability in mice.Overall,various of pharmacological studies in vitro and in vivo showed that I-2-2g、I-4-2c、II-2-1c and III-3c were the most promising candidates in this thesis,and the follow-up studies referring to the further pharmacodynamics study,mechanism study and structure optimization are still in progress.Therefore,the work presented in this thesis laid a foundation for the development of novel anti-AD drugs.