1 Arctium Lignan Analog Synthesis And Detection Of Antitumor Activity 2 Preparation Of Dihydrofluorescein Probe And Its Use In Oxidative Stress Detection

Part one Studies on Synthesis and Cytotoxic Activity of the Analogues of Lignans from the Seeds of Arctium lappa L.BackgroundAs a wildly-used Chinese herbal medicine,the seeds of Arctium lappa L.contain large proportion of lignans which are the main active ingredients of Arctium lappa L.The structure and pharmacological activity of Arctium lappa L.components are diverse and have important research significance.However,the extraction,separation and purification of lignan compounds have complicated operation steps and low separation efficiency,which makes the preparation of large amounts of pure products and the in-depth study of pharmacological effects in vivo become difficult.ObjectivesThe purpose of this article is to study the synthesis of lignans and their analogs with great biological activity in the seeds of Arctiumlappa L.,to explore new synthetic methods,or to optimize the synthetic routes of lignin compounds in the seeds of Arctiumlappa L.that reported in the literature as follows:simplify the synthesis steps,improve the synthesis efficiency,reduce the difficulty of synthesis,use cheaper,easily available and safe synthesis reagents.It provides stable and feasible synthesis technology for the research and development of new lignan small molecule drugs such as anti-oxidation,anti-aging and anti-cancer,and lays the leading material basis and ideas for the development of new drugs.At the same time,the anti-tumor activity of synthetic lignan compounds was discussed.MethodsThis thesis uses organic chemical synthesis methods,combined with previous studies on the biological activity of each lignan component in the seeds of Arctiumlappa L.and its monomer structure.Through multi-step chemical reactions,extraction,normal-phase silica gel columns,and reverse-phase silica gel columns.A series of lignans contained in the seeds of Arctiumlappa L.were synthesized by using separation methods such as high-performance liquid phase（HPLC）chromatography and other separation methods,and using spectral analysis techniques（1H-NMR,13C-NMR,etc.）and MS analysis methods.Monomer,and structural identification of the synthesized monomer compounds.1.The synthesis of Lappaol A is synthesized by the following three steps:First,the demethylation reaction of boron tribromide is used to reduce the arctigenin to form the important intermediate matairesinol.Then the reduction ability of lithium aluminum hydride is used to reduce the Ethyl 4’-hydroxy-3’-methoxycinnamate to coniferyl alcohol.Finally,the synthesized mogrosol resin and coniferol produced Lappaol A under the catalysis of ferric chloride.2.When attempting to prepare matairesinol by using a total synthetic method,a vanillin was used as a material,and a condensing method was used to construct a matairesinol skeleton by acid or alkali-catalyzed esterification reaction and ether formation reaction.The five-membered lactone ring of matairesinol was constructed by reduction reaction and esterification reaction.However,in the last step,palladium on carbon hydrogenation was used to remove the protected benzyl group and reduce the two double bonds.After analysis and purification by TLC and suction filtration,passing through a forward silica gel column,HPLC,etc.,two compound monomers were obtained.The structure was analyzed by using spectral analysis techniques（1H-NMR,13C-NMR,etc.）and MS analysis methods to clarify the reduction order.3.Inspired by the synthetic route of matairesinol,it was found that the product produced by the aggregation method is similar to the lignan component.Secoisolariciresinol,which contained in the seeds of Arctiumlappa L.,so palladium carbon hydrogenation or borohydride sodium was used as a reducing agent.The secoisolariciresinol were obtained.4.SRB method was used to determine the effects of compounds on the proliferation of various tumor cells.ResultsThis article reports the synthesis of a series of lignans isolated from the seeds of Arctiumlappa L..In the process of experimental exploration,we not only synthesized the known compound lappaol A for the first time,but also obtained a new route and synthetic method.Known or completely new compounds,and studied the biological activity of each synthetic product through cell experiments.1.For the first time,an effective anti-allergic component of lignin in the seeds of Arctiumlappa L,Lapasol A was synthesized.2.For the first time,arctigenin was synthesized from arctigenin as raw material.3.When trying to synthesize matairesinol with vanillin as raw material,two new derivatives of matairesinol were obtained.4.Synthesize a lignin component in the seeds of Arctiumlappa L.secoisolariciresinol by a new,easy-to-operate and easy-to-obtain route—providing a more efficient and simple operation for the synthesis of secoisolariciresinol Synthetic method.5.In vitro anti-tumor activity test,the compound Lappaol A,intermediate 2-（4-hydroxy-3-methoxybenzyl）-4-（4-hydroxy-3-methoxyphenyl）-3-methylbutanoic acid which were synthesized by the new method and the natural extraction compound Lappaol F as comparison,the IC50 of these compounds on HeLa cell line is:48 p.M,more than 100μM and 28 μM.ConclusionThis article reports a synthetic method of lappaol A for the first time,which provides a synthetic basis for the development of lappaol A as an anti-allergic drug,and provides new ideas for drug development.The chemical synthesis method can greatly reduce the difficulty of preparing lappaol A,and provide a synthetic reference for the large-scale preparation of lappaol A.By using arctigenin as raw material,matairesinol was synthesized,so that two lignans present in seeds of Arctiumlappa L.were transformed.And through the total synthesis method,two brand-new matairesinol analogues were synthesized.The secoisolariciresinol was synthesized by a more convenient method,and the secoisolariciresinol analogues was synthesized by different reduction methods.It solves the problems that predecessors had long routes or complicated operations,the use of dangerous reagents,or the difficulty of reaching the reaction temperature in the synthesis of secoisolariciresinol.In terms of antitumor activity in vitro,the newly synthesized compound Lappaol A and natural Lappaol F both showed a certain inhibitory effect on Hela cells.Part Two Preparation of dihydrofluorescein probes and its application to oxidative stressBackgroundDesign and synthesis of advanced nano-sized or small molecule-based fluorephores for bioanalytical applications have been attracting increasing attentions for both industrial and research interests.Up to date,a large number of fluorescein based probes have been developed for bio-analyzing such as pH indicating,metal ions detection,cellular redox sensing,enzymatic activities monitoring,and even theranostics.Traditional approaches to directly synthesize DCFH?most of these methods suffer from one or more drawbacks,such as the requirements of expensive and strong reducing agent,harsh reaction conditions and sometimes extra steps to deal with the used metal-containing reagents.Therefore,the challenge for this reduction process to obtain desired dihydrofluoresceins lies on the development of mild and controllable strategies to reduce fluoresceins and their derivatives.ObjectivesIn combination with the previous deficiencies in the reduction of dihydrofluorescein acid or dihydrofluoresceinol,the aim is to develop a mild and controllable reduction method to reduce fluorescein to dihydrofluorescein acid or dihydrofluoresceinol.In addition,the method was used to reduce a variety of xanthene fluorescein,and the applicable range of the new reduction method was studied.Dihydrofluorescin and dihydrofluorescein were tested to evaluate their different reactivity to ROS.Based on this reduction method,the core structure of xanthene-type fluorophore is modified by using the required fiinctional groups to develop a fluorescent probe with dual factions.MethodsIn this thesis,organic chemical synthesis is used to systematically study the use of fluorescein as a model substrate and NaBH4/ h as the reducing conditions.By adjusting the reaction conditions: for example,the amount of NaBH4/l2 reducing agent used,reaction time,and temperature The Agilent HPLC 1260 was used to analyze the peak area integration of all tested reaction products to determine the yield of all tested reactions.An efficient,gentle and highly controllable reduction method was established.On the basis of this method,xanthene-type fluorophore dyes including fluorescein,dichlorofluorescein,and rhodamine B were reduced,and a series of tests were performed.In the test,we selected a standard reaction system for the generation of reactive free radical species: the HRP / H2O2 system,which was used to evaluate the reactivity of the ROS probe.And under the optimal reducing conditions,two kinds of targeting probes were synthesized,and the ability of the targeting probes to perform subcellular imaging of oxidative stress in living cells was verified by cell imaging experiments.ResultsA mild NaBH₄/I₂ reduction system is designed to reduce fluorescein to dihydrofluoresceinol and dihydrofluorescein acid easily.Using the HRP / H2O2 system as a model system for generating reactive free radical species,dihydrofluoresceinol is more reactive to oxidative stress sensing than the dihydrofluorescein form.Finally,two dual-function probes were synthesized using our optimized reduction method,and these probes show the great potential in the fluorescence imaging of subcellular oxidative stress.