The Design,synthesis And Biological Evaluation Of 1,4-dihydroquinoline Prodrugs Of Scutellarin And The Design,synthesis And Biological Evaluation Of Twin Drugs Of Tetramethylpyrazine Active Metabolite And Scutellarein

This thesis includes two sections.The first section: The design,synthesis and biological evaluation of 1,4-dihydro-quinoline prodrugs of scutellarin.Reactive oxygen species are closely associated with oxidative metabolism,they are highly active because of the presence of unpaired electrons.In some specific physiological processes,reactive oxygen species show a beneficial and necessary role.However,when a large number of species are enriched in a specific region,oxidative stress will be caused,which can directly cause necrosis of cells,and can also induce cell necrosis,aging and apoptosis by activating the redox signal pathway.Ischemic brain disease can produce a large number of lactic acid,which induce peroxidation process,accelerate the production of activity of reactive oxygen species in short time,and consequently,oxidative stress will be caused.The use of free radical scavengers can effectively prolong thetreatment time for ischemic,so research and development of free radical scavenger have great significance.Erigeron is mainly distributed in the southwest of China,such as Yunnan and Guizhou.Breviseapine is the main flavonoid of Erigeron breviscapus.Over 95% of breviseapine is Scutellarin.Scutellarin exhibited its antioxiditive effect by scavenging free radicals,hydrogen peroxide and superoxide anion.It is widely used to treat the deficiency of cerebral blood supply,cerebral hemorrhage caused sequelae,hyperviscemia,cerebral thrombosis,coronary heart disease and angina pectoris.The mechanism for the treatment of cerebrovascular disease is believed to remove the excess reactive oxygen species caused by inflammation.However,the poor solubility,short half-life in the body,and low bioavailability limited the further use of scutellarin.Redox-based chemical delivery system can enhance the ability of drugs to penetrate the blood brain barrier and enrich the drug concentration in the brain,based on this theory,fifteen new 1,4-dihydroquinoline prodrugs of scutellarin were designed and synthesized.The target compounds were studied on their physicochemical properties,in vitro stability of PBS buffer,in vitro scavenging activity of DPPH,in vitro blood-brain barrier permeability and protective effect on cerebral ischemia/reperfusion in the middle cerebral artery occlusion(MCAO)model.Our experimental results indicated all fifteen compounds displayed improved water solubility in varying degrees,their stability in PBS(p H7.4)buffer system increased,and the half-life increased from 12.1 hours to 72 hours.Except compounds 5,6,7,8,10,the other compounds showed good free radical scavenging activity at tested concentrations.The compounds(13,15,16,17,18,19),with good physicochemical properties and free radical scavenging activity were selected to study on the permeability across blood brain barrier.The results show that the transfer amount of test compounds increased with elevated drug concentration and transport time,and the transfer amount was significantly increased than that of scutellarin at the same time and at various concentrations.It is suggested that the 1,4-dihydroquinoline group can enhance the membrane permeability of scutellarin.The efflux rate of tested compound is less than 1,it is presumed that the tested compounds may not be affected by the efflux protein.Finally,compound 17 with the optimal physicochemical properties,strong scavenging activity and BBB permeability,was selected to study the protective effects on cerebral ischemia/reperfusion injury in rats.The results showed that compound 17 could significantly improve the neurobehavioral characteristics of rats after ischemia/reperfusion injury and reduce the volume ratio of cerebral infarction.To clarify the protective mechanism,the SOD activity,the content of MDA and the activity of GSH-Px in the serum of rats with cerebral ischemia/reperfusion injury were further studied.The results showed that theactivity of SODand the activity of GSH-Px in the serum of the rats treated with scutellarin and compound 17 were significantly increased,and the content of MDA decreased.The result from Hematoxylin Eosin staining showed that the nerve cell structure of the rats in the treatment group was clear,only a small amount of cells were necrotic,deep stained and the cell lesion was lighter.Immunohistochemical result showed that the expression of Beclin-1 protein and Caspase-3 protein in the treatment groups was significantly lower than that in the model groups.Compared with scutellarin group,the Beclin-1 and Caspase-3 protein levels in rats treated by compound 17 were significantly decreased.It is presumed that the mechanism of the effect of scutellarin and its derivatives may be related to the anti free radical damage and the regulation the expression of autophagy and apoptosis proteins.The second section: The design,synthesis and biological evaluation of twin drugs of tetramethylpyrazine active metabolite and scutellareinStroke can be divided into ischemic stroke and hemorrhagic stroke,of which ischemic stroke is more common.Brain tissues rely on blood circulation to provide oxygen and other nutrients needed for normal body.Metabolic products are also excreted by blood circulation.When cerebrovascular thrombosis occurs,normal circulation is blocked,resulting in the occurrence of ischemic stroke.Therefore,thrombolytic therapy is the primary task for the treatment of ischemic stroke.Ischemic stroke leads to a cascade of cell biochemical reactions that eventually lead to a large number of reactive oxygen species.Normal body tissues have a certain defense mechanism for the species,but ischemic stroke breaks the system and accelerates the release of reactive oxygen species,leading to irreversible apoptosis of nerve cells.Therefore,simple thrombolysis is not the best treatment for ischemic stroke,while reducing the damage caused by reactive oxygen species is considered as an important strategy for the treatment.Tertramethylpyrazine is an alkaloid extracted from the dry rhizome of Ligusticum Chuanxiong.It has the effect of preventing cerebral ischemia,improving microcirculation,antithrombotic,protecting coronary artery,protecting myocardial cells and preventing the ischemia.Tertramethylpyrazine can easily penetrate blood brain barrier and be enriched in the brain,especially in brain stem tissue.In clinical application,tertramethylpyrazine has the disadvantages such as fast metabolism,short half-life and low bioavailability.One of the main reasons for this is that the methyl could be easily oxidized,resulting in the production of polar and water-soluble metabolites,which are rapidly expelled from the body,that limits its application.(3,5,6-trimethylpyrazin-2-yl)methanol,3,5,6-trimethylpyrazine-2-carboxylic acid are the active metabolite of tetramethylpyrazine,and their toxicity is lower.Scutellarin is mainly absorbed by the glycosides in the small intestine.Compared with scutellarin,scutellarein can be absorbed more easily and has higher metabolic stability in vivo,its relative bioavailability is 301.8%(relative to scutellarin).Studies using rat cerebral ischemia infarction area indicated the effect of scutellarein was nearly 1.5 times higher than that of scutellarin.But problems such as poor solubility in water,poor stability,short half-life and poor bioavailability,still existed.Based on combination principle,twelve new twin drugs of scutellarein and tetramethylpyrazine active metabolites were designed and synthesized,the physicochemical properties,in vitro stability,protective effects on antioxidant injury,anticoagulation and protective effect on cerebral ischemia/reperfusion in the middle cerebral artery occlusion(MCAO)model were studied.The result indicated that,except compound 22,the rest of the target products displayed improved water solubility at different degree.Especially,when the linker is L-amino acid,the solubility of the target compounds increased significantly.The stability in PBS(p H7.4)buffer increased and the half-life changed from 14.3 hours to 72 hours.In vitro plasma incubation results showed that,after incubation of 30 min,all tested compounds could release scutellarein and(3,5,6-trimethylpyrazin-2-yl)methanol,3,5,6-trimethylpyrazine-2-carboxylic acid,this results also prove the feasibility of the twin design.Subsequently,target compounds were screened for their antioxidant activity.All groups with different concentrations exhibited increased cell viability to different degree,the activity of SOD in damaged cells increased,and the intracellular LDH release rate and MDA content decreased.The results of blood coagulation time examination showed that the tested compounds(30,44,46,47)could extend TT,APTT and PT at different degrees,and significantly decreased the content of FIB in plasma.Based on the above results,compound 44 is selected to study the protective effect using ischemia/reperfusion injury rats model.The results showed that compound 44 could significantly improve the neurobehavioral characteristics of injured rats and reduce the volume ratio of cerebral infarction.To further clarify the protective mechanism,the SOD activity,MDA content and GSH-Px activity in the serum of ischemia/reperfusion injury ratswere further studied.The results showed that the activity of SOD and the activity of GSH-Px in the serum of scutellarein and compound 44 rats increased significantly,and the content of MDA decreased.The results of Hematoxylin-eosin staining staining showed that compared with MCAO group,the neurons in the treatment group had clear structure,and only a small amount of cell necrosis,deep staining,and mild cytopathic effect.To study the effect of target compounds on proteins related to autophagy and apoptosis,immunohistochemical technique was used to test the expression of beclin-1 and caspase-3 in rat hippocampal CA1,theresults showed that the expression of beclin-1 and caspase-3 in treatment groups was significantly reduced,compared to the scutellarein group.The expression of beclin-1 and caspase-3 in treatment groups(0.70mmol·kg-1 and 1.40 mmol·kg-1)decreased significantly.