| Traditional Chinese medicine(TCM)has a long history and been widely used in prevention and treatment of epilepsy in China.The use of Chinese herbal compound treatment of the disease has accumulated rich experience in the theory and practice,especially the combination with Acorus tatarinowii.The innovative pharmaceutical research and development of lead compounds from traditional Chinese medicine has broad space and great opportunity in the future.Today,the treatment of intractable epilepsy(eg.Dravet)remains a thorny issue in the current field of medicine,and curing is a challenging and competitive job.In this paper,inspired by the traditional Chinese antiepileptic herb pair of Polygala tenuifolia-Acori Tatarinowii,33 novel substituted cinnamic ?-asaronol esters and analogues were designed by the strategy of Combination of Traditional Chinese Medicine Chemistry(CTCMC),synthesized and tested not only for anticonvulsant activity against several mice models but also LDH inhibitory activity.Thereinto,C14-C16 displayed excellent and broad spectra of anticonvulsant activities with modest preventing of neuropathic pain and lower neurotoxicity.Furthermore,C14-C16 exhibited good LDH1 and LDH5 inhibitory activity with noncompetitive inhibition type,which were at least 4 times higher than that of stiripentol.Thus,C14-C16 could be developed into promising candidates for AEDs,especially for treatment of some refractory epilepsies such as Dravet syndrome.Moreover,to further rationalize the biological results,molecular docking studies on candidate active compounds have also been performed.In this study,the experimental results fully affirmed that CTCMC drug design strategies could enhance rationality,veracity and efficiency,and time-saving,which could be applied successfully in novel drug design and development processes.The goal of CTCMC is to simplify the components of TCM fufang,to outstand combination of active components and optimize efficacy of integrate molecules.The CTCMC may provide some scientific basis for the development of clinical refractory epilepsy drugs,and thus,it is a new strategy for the modernization research and development of compound Chinese medicine.The dissertation was drafted as 4 chapters among which the author contributed greatly in following contents:1.In this paper,we have enriched and developed the CTCMC drug design strategies,and blazed new "Zhenghe(combination)-Jiehe(blending)-Ronghe(infusion)" trails of "He Ce Lue(Harmony strategy)" in novel rapid molecular designing and building.This strategy has a feature of rationality,efficiency easy-to-use,and time-saving,and be applied successfully in combination the Chinese herbal medicine active ingredients and/or metabolic effector molecule into a new molecular entity.It can be considered as secondary development of traditional Chinese medicine effective components and provide new practice reference for the modernization of Compound-TCM.2.Inspired by the traditional Chinese antiepileptic herb pair of Polygala tenuifolia-Acori Tatarinowii,33 novel substituted cinnamic a-asaronol esters and analogues were designed and synthesized according to the strategy of Combination of Traditional Chinese Medicine Chemistry(CTCMC).Additionally,29 intermediates were also synthesized in this study.Their structure is characterized by 1H NMR,13C NMR and HRMS(+ESI).3.The anticonvulsant activities of compouds C1-C33 were evaluated by the antiepileptic drug development program implemented by the USA National Institutes of Health.Initially,we selected mice-MES test and rotorod test as basic evaluation of anticonvulsant activity and neurotoxicity of all final products.Then,we selected mice-scPTZ and sc-3-MP seizure threshold tests to further evaluate the anticonvulsant activities of candidate active compounds and speculated their mechanism.Besides,the anti-neuropathic pain of candidate active compounds was also evaluated using formalin test.The ED50,TD50 and PI of the excellent potencies of the selected compounds in aforementioned anticonvulsant activity tests were calculated and termed using the mice-MES,-PTZ,-3-MP,-Tox,and-formalin test.We investigated the anticonvulsant activities of active compounds on behavior changes and epilepsy-related gene C-fos expression of PTZ-induced zebrafish.The results showed that compoud C10-C11,C14-C16 and C21 emerged as promising candidates with significant and broad spectrum of anticonvulsant activity,anti-neuropathic pain and high safety.4.Seizures may be controlled by inhibiting LDH of nervous system.The LDH inhibitory activities of compouds C1-C33 were measured by standard enzyme kinetic experiments on human LDH1 and LDH5 purified isoforms using Uv spectrophotometry.The results showed that rimethoxyl-substituted and tetramethoxyl-substituted a-asaronol cinnamate(C10-C11 and C13-C17)showed moderate to good inhibitory activity(8%?27%inhibition)both on LDH1 and LDH5 without marked selectivity.Especially,in the same molar concentration,the LDH inhibitory activity of C14-C16 was greater than STP(the first "orphan drug" approved in Europe,Canada and Japan for the treatment of Dravet syndrome).The active compounds C14-C16 on reaction product content from pyruvic acid to lactic acid catalyzed by LDH1 and LDH5 was determined using triple quadrupole LC/MS/MS system.On the basis of good LDH inhibitory activities of tri-and tetramethoxyl-substituted a-asaronol cinnamate,compounds C11,C14 and C15 were selected for a complete enzyme kinetic analysis to verify their type of inhibition versus pyruvate.In addition,their Ki values were also determined from Lineweaver-Burk plots.From secondary and Lineweaver-Burk plots,compounds C11,C14 and C15 were all found to be noncompetitive inhibitors of LDH1 and LDH5 in the conversion of pyruvate to lactate catalyzed by this two enzymes.Besides,the binding modes of C10-C11 and C14-C16 with the active site of LDH molecular docking was performed using Surflex-Dock,and the results were found to be consistent with the experimental data. |
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