Lead Compounds Design And Anti-tumor Activity Research Based On Indole Alkaloids From Gelsemium Elegans

As the second most fatal disease,cancer is a serious threat to the life and safety of humankind.In recent years,the incidence of lung cancer,such as non-small cell lung cancer increased year by year,and it has trended to be younger.Therefore,to research and develop leading anti-cancer drugs with novel structure and low side effects is urgent.As the wisdom crystallization of ancient Chinese Medicine,traditional Chinese medicine(TCM)has accumulated a lot of clinical experience in fighting cancer and other diseases.Therefore,simplifying and modifying the structure of the main active components in TCM is undoubtedly one of the effective ways to discover new lead compounds.Under this background,based on the traditional Chinese medicine Gelsemium elegans as the starting point,the simplification strategy and combination method of pharmaceutical chemical skeleton,this paper focuses on the discovery,design,preparation and screening of Gelsemium indole alkaloids,in order to screen new lead compounds which can act on the inhibition or treatment of non-small cell lung cancer.Objectives:the chemical study of modern TCM shows that the main active component in Gelsemium elegans is indole-fused cyclic or spirocyclic indole alkaloids with complex structure.In this paper,focusing on Gelsemium indole alkaloids,based on the design principle of pharmaceutical chemical lead compounds,a series of indole and spirocyclic compounds were prepared by the method of organic synthesis,and the library of Gelsemium indole alkaloids and spiro analogues was preliminarily constructed.In order to screen new lead compounds with novel structure and good clinical curative effect,the bioactivity of Gelsemium indole alkaloids against non-small cell lung cancer and other tumor cells in vitro was further evaluated.Methods:1.Using the cheap and easily available indoleimines,substituted benzaldehyde and malononitrile as substrates,Gelsemium indole alkaloids cyclic analogues fused a carboline were efficiently synthesized by three-component[3+2+1]cycloaddition reaction.The structures of the corresponding compounds were characterized by NMR and HRMS,and the structures of the representative compounds were confirmed by X-ray single crystal diffraction.2.Using the newly designed azoxyallyl cation precursor containing oxindole skeleton as the substrate,it reacts with various sulfonium salts under alkaline conditions through[3+1]cyclization reaction.Efficient introduction of quaternary?-lactam spiro-center scaffold at indole C3 position.In addition,using cinnamaldehyde as the substrate,using stereodivergent synthestic strategy,through the series cyclization process catalyzed by secondary amine and carbene,the five-membered cyclopentane skeleton was introduced into the C3 position of indole,and the spirocyclic analogues of Gelsemium indole alkaloids were prepared.The structures of the corresponding compounds were characterized by NMR and HRMS,and the structures of the representative compounds were confirmed by X-ray single crystal diffraction.3.Screening the in vitro anti-tumor activities of Gelsemium indole alkaloid tricyclic compound analogues containing various substituted groups were conducted by acid phosphatase assay,and the compounds with the highest activity was revealed.We further investigate the mechanism by Western Blotting protein expression and GST pull-down assay and other methods.Results:1.Through the three-component[3+2+1]cyclization of indoleimines,substituted benzaldehyde and malononitrile,we efficiently synthesized 25 Gelsemium indole alkaloids tricyclic compound derivatives with potential hydrogen bonding sites.Most of the yields reached 99%.The structure of the target product was confirmed by X-ray single crystal diffraction.2.Based on the[3+1]cyclization reaction mediated by sulfur ylide,we prepared a series of spiro[?-lactam]-oxindole derivatives with excellent diastereoselectivity.The highest yield was 91%,and the diastereoselectivity was more than 20:1.Their relative configurations were confirmed based on X-ray single crystal diffraction.At the same time,we adopted stereodivergent synthestic strategy and based on the series sequential reaction process under relay catalysis of the secondary amine and the NHC,the spirocyclopentaneoxindole products with different absolute configurations were prepared with excellent yield and stereoselectivity,and their absolute configurations were confirmed by X-ray single crystal diffraction.4.Through the tumor cytotoxicity screening of Gelsemium indole alkaloid tricyclic compound derivatives in vitro,2-3o were proved to be the most effective compound which could effectively inhibit the proliferation of non-small cell lung cancer cell line A549.The IC₅₀ values for Ral A and Ral B were 0.61?M and 0.96?M respectively,and induced tumor cell apoptosis in a dose-dependent manner.Mechanism investigation suggested that the most active compound 2-3o could inhibit the Ral A/B activation of A549,down-regulates Bcl-2,stimulates Cytochrome C and PARP cleavage,as well as induces cell apoptosis.Conclusions:1.Focusing on Gelsemium indole alkaloids,combined with pharmaceutical chemical skeleton simplification strategy and combination method,we succeed in preparing the Gelsemium indole alkaloid tricyclic and spirocyclic compounds by means of organic synthesis,and the Gelsemium indole alkaloid analogue library was preliminarily established.2.Gelsemium indole alkaloid and ring derivative 2-3o showed good anti-proliferation activity on non-small cell lung cancer cells through inhibiting Ral A-induced apoptosis,which could be used as a promising anti-tumor lead compound.