Design And Activity Mechanism Of Evodiamine-based Lead Compounds As Anti-colorectal Cancer Agents

Background:The increasing incidence and mortality of cancer have become a significant public health problem globally.Therefore,developing novel anti-tumor drugs with high efficiency,low toxicity,and safety has attracted much more attention from researchers.It is an effective means to prevent and cure many diseases with a long history of clinical practice.The treatment of diseases with TCM has the advantages and characteristics of multi-target and multi-pathway integrated regulation.It thus shows unique benefits in treating multi-factor diseases,such as cancer.However,there are many kinds of chemical components in TCM with complex structures and some shortcomings,such as the high difficulty of extraction and separation,low content of active ingredients,poor medicinal properties,etc.These hinder the development and clinical application of innovative anti-tumor drugs for TCM.Based on the structural simplification of the core skeleton of TCM effective constituent,the construction of a library of active substances of TCM with skeleton,functional groups,and chiral centers diversities is a crucial method for anti-tumor drug discovery.Evodiamine is the main active ingredient of Euodiae Fructus,which exhibits good anti-tumor activity on the proliferation of many kinds of cancer cells.However,the low content,the complex extraction and separation procedures,the poor water solubility and bioavailability make Evodiamine limited in the application and drug development.Therefore,with the strategy of structural simplification,modifying the structure of Evodia alkaloids,designing and constructing evodiamine analogs is an effective way to promote the discovery of anti-tumor lead compounds of Euodiae Fructus.Objective:Based on the above background,this paper takes Evodiamine as the research object,through the strategy of structure simplification,with the aid of computer-aided drug design and asymmetric organocatalytic synthesis,to construct a library of evodiamine derivatives with the diversity of functional groups and chiral centers,and to study its antitumor activity and structure-activity relationship from the aspects of pharmacology,molecular biology,and proteomics of TCM.It provides the material basis for screening antineoplastic drugs in TCM.Furthermore,it is expected to clarify the molecular mechanism of evodiamine in treating malignant tumors and provide ideas for discovering innovative lead compounds in TCM.Methods:Part One:Based on the reasonable drug design and the strategy of simplifying the structure of active ingredient of TCM,a class of potential selective Akt1inhibitors based on simplified Evodiamine derivatives 3 was designed.Under mild conditions,the preparation of the simplified derivatives 3 was achieved with indole fragment 1 and nitroolefin MBH acetate 2 as substrates.The inhibitory activity and selectivity of the prepared evodiamine analogs 3 on Akt1 protein kinase were tested by kinase activity test in vitro,and the tumor activity and molecular mechanism of the selected compounds were studied by means of cytotoxicity test,flow cytometry,immunofluorescence,transmission electron microscope,and molecular docking.Part Two:Based on the pharmacophore hybrid strategy,a kind of chiral spirocycle evodiamine analogs 8 was designed,and the conditions and methods for the construction of evodiamine analogs 8 were studied by using indole fragment 1 and indirubin derivative 6 as substrates.The antitumor activity and mechanism of the compounds were screened and studied by cell proliferation experiment,western blot,immunofluorescence,transmission electron microscope and flow cytometry.Results:Part One:Under mild conditions,using indole fragment 1 and nitroolefin MBH acetate 2 as substrates and tertiary amine as the catalyst,27 simplified Evodiamine analogs 3 with different functional groups were quickly and efficiently prepared by[3+3]cyclization reaction,and their structures were identified by ¹H/¹³C NMR,high-resolution mass spectrometry and X-ray single-crystal diffraction.Through the kinase activity test in vitro,it was confirmed that derivatives 3 is an effective selective Akt1 inhibitor,among which 3r has the best anti-tumor activity,and the IC₅₀value for HCT116 cells is 2.04μM.preliminary studies show that inducing autophagy and apoptosis may be the mechanism of 3r’s anti-tumor cell proliferation.Part Two:Using indole fragment 1 and indirubin derivative 6 as substrates and chiral tertiary amine derived from cinchona base as the catalyst,the[3+3]cyclization reaction platform was successfully established,and the efficient construction of spirocycle evodiamine analogue 8 with multi-functional groups and multi-chiral centers was realized.24 new compounds were obtained,and their structures were identified by¹H/¹³C NMR,high-resolution mass spectrometry and X-ray single-crystal diffraction.Through the screening of anti-tumor activity,it was found that evodiamine analogues had the best anti-proliferation activity of colon cancer cells at 8h,and the IC₅₀ value for HCT116 cells was 0.857μM.preliminary mechanism studies showed that 8h could induce apoptosis and protective autophagy of tumor cells at the same time,and 8h combined with autophagy inhibitors could effectively exert an anti-tumor effect.Conclusion:Based on the strategy of structure simplification,using hydrogenated carboline,the core pharmacophore of Evodiamine,as a template,a drug material library of skeleton-functional group-chiral center diversity was successfully constructed,including 27 functional substituted acyclic evodiamine derivatives and 24 chiral spirocyclic evodiamine analogues.Through preliminary activity screening,Evodiamine-derivatived lead comound 3r and 8h with good anti-tumor activity were obtained,and their IC₅₀ values against HCT116 cells were 2.04μM and 0.857μM,respectively.Mechanism studies have shown that 3r can be used as an effective Akt1inhibitor to inhibit tumor proliferation by inducing apoptosis and autophagy,and apoptosis and protective autophagy can be induced in tumor cells at 8h.Combined with autophagy inhibitors provides a new strategy to improve the anti-tumor effect of evodiamine analogues for 8h.In this paper,the successful construction of a pharmacodynamic substance derivative library provides a material basis for the screening of evodiamine anti-tumor leaders.The establishment of the pharmacodynamic evaluation system of"pharmacological substance-target pathway-action mechanism"promoted the discovery of evodiamine anti-tumor lead compounds.