Design,synthesis And Biological Activity Study Of Lappaconitine And Panaxadiol Derivatives

In recent years,natural products have received widespread attention in the development and research of new drugs.Over the past 30 years,the drugs obtained from natural products or derivatives represent 62%of all small molecule drugs approved by the FDA.Natural products have become an integral part of drug discovery.Among them,alkaloids and terpenoids have become the molecules with the most vitality and development potential due to their structural diversity and extensive biological activity.In this paper,105 novel derivatives were designed and synthesized using diterpenoid alkaloid lapaconitine and tetracyclic triterpenoid 20(R)-panaxadiol as lead compounds.The structures of target compounds were confirmed by1H and 13C NMR,HR-MS spectra.Lapp aconitine(LA)is a natural compound with novel C18-diterpene alkaloid skeleton,which has a wide range of biological activity.However,The research on the pharmacological activity and structural modification of lappaconitine is very limited.So far,most of the pharmacological activity studies have focused on anti-tumor and analgesic effects,and some of its derivatives have been designed and synthesized.The anti-inflammatory effects of lappaconitine derivatives have not been reported.Hence,lappaconitine was selected as a lead compound.Based on Based on the principle of structural combination in new drug design,novel lappaconitine derivatives containing structural fragments of 1,2,3-triazole,cinnamic acid,indole,amino acid,aminophosphonate,benzyl and phenylacetamide were designed and synthesized.The anti-inflammatory activity,mechanism and pharmacokinetics of lappaconitine derivatives were systematically evaluated by inflammatory models in vitro and in vivo.The main contents and results of this chapter include:(1)Anti-inflammatory activity screening of target compounds in vitro.The cytotoxicity of all the target compounds to mouse RAW264.7 macrophages was evaluated by MTT method at the concentration of 30?M,and 17 non-cytotoxic compounds were screened for subsequent anti-inflammatory research.The target compounds against NO production were evaluated and compared by Griess analysis in order to screen the molecules with the best anti-inflammatory activity.The results showed that all the target compounds,except compound E2,exhibited excellent inhibitory ability against NO relative to that of lappaconitine.In particular,compound A4 showed the most potent inhibitory activity against NO,and with IC50 of 12.91 ?M.Based on the NO inhibitory activity of the target compound,the elementary structure-activity relationships(SARs)indicated that replacement of the benzene ring with an electron donating group could improve the anti-inflammatory efficacy.To further evaluate its the anti-inflammatory activity,compound A4 was evaluated for inhibitory ability against TNF-? and PGE2 production.As expected,compound A4 could effectively reduce the levels of inflammatory cytokines TNF-? and PGE2.(2)study on the anti-inflammatory mechanism of compound A4.Western blot was used to explore the effects of compound A4 on inflammatory pathway proteins including iNOS,COX-2,p-NF-?B p65,NF-?B p65,p-IKBa,I?Ba,p-ERK and ERK,so as to reveal the anti-inflammatory mechanism of compound A4.The results showed that compound A4 decreased iNOS and COX-2 expression,and down-regulated relative protein expression of p-NF-?B p65/NF-?B p65,p-I?Ba/I?Ba,and p-ERK/ER.Therefore,the anti-inflammatory mechanism of compound A4 may be related to the reduction of NO,and PGE2 production by inhibiting NF-?B and MAPK signal pathways.(3)the anti-inflammatory activity in vivo of compound A4.The model of LPS-induced acute lung injury was used to evaluate the anti-inflammation in vivo after intragastric administration of compound A4 at 5 mg/kg,10 mg/kg and 15 mg/kg.The results showed that compound A4 could effectively inhibit the LPS-induced increase in W/D ratio,total protein concentration,TNF-?,NO and MPO.Lung pathological analysis showed that A4 attenuates LPS-induced histopathological changes including inflammatory cell infiltration,interalveolar septal thickening,lung edema,alveolar structure damage,and alveolar hemorrhage.In addition,the above results were also confirmed by CD68 immunohistochemical analysis of lung tissue.Therefore,compound A4 exert an important therapeutic effect on acute lung injur induced by LPS in vivo.(4)preliminary pharmacokinetic evaluation of compound A4.SD rats were selected.Compound A4 was administered via tail vein injection at 2 mg/kg.The concentration of compound A4 in plasma was analyzed by LC-MS/MS.The Preliminary PK parameters and semi logarithmic plasma concentration-time profile of compound A4 were obtained by pharmacokinetic analysis20(R)-panaxadiol(PD)is a tetracyclic triterpenoid compound obtained by hydrolysis of dammarane-type ginsenoside isolated from Panax ginseng C.A.Mey.In recent years,20(R)-panaxadiol has attracted much attention due to its wide biological activity.Recent studies have shown that panaxadiol has excellent anti-AD activity in cells and animal models related to Alzheimer's disease(AD).Therefore,20(R)-panaxadiol was used as the lead compound to modify the 3-OH and A ring.20(R)-Panaxadiol derivative bearing structural fragments of carbamate,1,2,3-triazole,cinnamic acid,amino acid,secondary amine heterocycle,bisphenyl phospholipid,pyrazole ring,indole ring,isoxazole ring,seven-membered lactam ring,seven-membered alicyclic ring and hydrazine were designed and synthesized.The in vitro AD model was used to evaluate the anti-AD activity of the target compounds,and to preliminary explore the anti-AD mechanism of the target compounds.The research contents and results in this chapter include:(1)Screening of anti-AD activity of target compounds in vitro.The MTT method was used to evaluate and compare the protective effect of the target compound on A?25-35-induced PC 12 cell damage in order to screen the molecules with the best anti-AD activity.The results showed that among the screened compounds,the compound N1 bearing a benzyl substituted carbamate structural fragment showed the most significant neuroprotective activity.Notably,compound N1 showed excellent neuroprotective activity at a concentration of 7.5?M.With the increase of concentration,its activity was significantly enhanced in a concentration-dependent manner.Compound N1 show better neuroprotective effect relative to that of 20(R)-panaxadiol.In addition,compound N1 did not produce cytotoxicity to PC 12 cells at concentrations ranging from 7.5 to 120?M.(2)Preliminary study on the anti-AD mechanism of compound N1.The effects of compound N1 on the proteins including p-Tau(Thr 181),Total-tau,p-P38,P38,p-ERK,ERK,and IL-1? were explored by western blot to reveal the anti-AD mechanism of compound N1.The results showed that compound N1 reduced inflammatory cytokines IL-1? and down-regulated the relative protein expressions of p-Tau(Thr 181)/Total-tau,p-P38/P38,and p-ERK/ERK.Therefore,the anti-AD mechanism of compound N1 may be to inhibit the activation of MAPK pathway,reduce inflammatory cytokines and abnormal phosphorylation of Tau protein,and thus produce neuroprotective effect.In summary,two potential active molecules,compound A4 with anti-inflammatory activity and compound N1 with anti-AD activity,were selected from the design and synthesis of lappaconitine and 20(R)-panaxadiol derivatives.This will provide a favorable reference for natural products in the development of new drugs,and also provide a certain experimental basis for the research and develop of anti-inflammatory or anti-AD drugs.