Design,Synthesis And Anticancer Evaluation Of Ring-contracted Artemisinin Dimers And PI3K? Mutant Inhibitors

Cancer is one of the leading causes of deaths worldwide,thus the development of new drugs for treating cancer is one of the most active research areas.Due to the diversity and complexity of cancers,most existing anticancer drugs display strong side-effects and/or toxicity,drugs directly targeting cancers or with high selectivity over normal cells are urgently needed.Based on these considerations,this thesis focused on the discovery of new anticancer agents.Firstly,we modified and developed a series of novel compounds with improved anticancer activities from artemisinin,a natural product with very low toxicity.Secondly,we identified two new lead compounds by high-throughput screening,which directly inhibited a specific mutant PI3K?.Systematic chemical modifications were employed to develop structurally novel inhibitors.Since these inhibitors specifically target the mutant protein,they possessed potent anticancer activities and a high selectivity.This thesis consists of the following parts.?.Ring-contracted artemisinin dimersArtemisinin is a novel trioxane-containing sesquiterpene with diverse biological activities.Currently,artemisinin and its derivatives are the first-line anti-malaria drugs,which exhibit low toxicity and high efficacy.Artemisinin and its derivatives have recently been studied for treating anticancer.However,their low anticancer activity and poor metabolic stability limited their clinical applications.In our studies,we designed and synthesized a series of novel ring-contracted artemisinin dimers,and then screened them against five cancer cell lines as well as one human normal cell line.The structure-activity relationships for this series of compounds were established,and the compound with the strongest biological activity was chosen and explored its biological roles in cancer cell lines and metabolic stability.Compared to artemisinin,most of the newly prepared artemisinin dimmers exhibited improved antiproliferative activities.Especially,compound A-8b showed the most pronounced anti-cancer activity for PC12 cancer cells with an IC₅₀ value of 1.56?M.Thus,PC12 cancer cells were used to further investigate the mechanism of anti-proliferation for this series of compounds.Compound A-8b arrested cell cycle at G1phase and induced cell apoptosis via up-regulation of Bad,Bax,caspase-3 and caspase-9protein expressions while inhibiting the expression of Bcl-xL.In addition,compound A-8b exhibited an extended half-life of metabolic stability and decreased intrinsic clearance in human liver microsomes,suggesting a better metabolic stability for compound A-8b.Taken together,the present study is the first to synthesize the ring-contracted artemisinin dimers and showed that these dimers had potent anti-tumor activities against several cancer cell lines and improved metabolic stability.?.PI3K?mutant inhibitorsThis part our studies concerns the rational design of novel and selective inhibitors targeting abnormal over-expressed PIK3CA mutant in cancer cell.These small molecules inhibitors specific target the PI3K protein in cancer cells,thus displayed high selectivity and less cytotoxicity as compared to common chemotherapeutic agents.The PI3K pathway is involved in many cellular functions including cell growth,metabolism and transformation.The frequent activation of this pathway in cancer development has made it a promising target for cancer treatment.In recent years,a number of PI3K inhibitors have been studied in clinical trials,which included dual inhibitors of PI3K and mTORC,isoform-specific inhibitors of PI3K isoforms,pan-PI3K inhibitors,and allosteric and catalytic inhibitors of AKT and so on.However,most of the candidates showed significant side-effects and/or toxicity.Inspired by these observations,we herein aimed to develop specific inhibitors which directly target PI3K?,with the aim of discovering potent anticancer activity and low toxicity.Through a high throughput screening?HTS?campaign by using a homogenous time-resolved fluorescence assay technique against PI3K??H1047R?mutation,two unique scaffolds were identified and selected for further structural modifications.The first series of compounds containing an acetophenone skeleton?Hit-01?were synthesized and showed good inhibition on mutation kinase and moderate inhibition on PIK3CA mutated cancer cell line HCT116.Among these,compound B-7h showed the most potent anti-cancer activity for HCT116 cancer cells,with an IC₅₀ value of 10.91?M,over 10-fold more potent than Hit-01,and potently inhibited PI3K??H1047R?mutant with an EC₅₀ of 0.555?M,over 200-fold more potent than Hit-01,respectively.Thus,HCT116 cell line was used to investigate the mechanism of anti-proliferation for this series of compounds.Compound B-7h arrested cell cycle at G2/M phase without induction of cell apoptosis.Further exploration showed that the compound induced autophagy effect of cancer cells.Western blot analysis was performed to study the expression levels of autophagy and several typical PI3K related proteins.Our results showed that B-7h activated cell autophagy and blocked PI3K pathway.The second series of compounds containing a quinoxaline scaffold?Hit-02?showed excellent inhibitory activity on mutatant kinase and related cancer cell lines.Among all synthesized compounds,compound C-7b showed superior inhibitory activity for PI3K?mutation kinase(EC₅₀=0.137?M),over 500-fold more potent than Hit-02.Moreover,it also exhibited good antiproliferative activity on HCT116 cell line.HCT116 cell line was selected to reveal the mechanism of anti-proliferation for C-7b.As a result,C-7b arrested cell cycle at G1 phase and induced cell apoptosis via up-regulation of capase-8,caspase-3and caspase-9 protein expressions.It also showed significant inhibitory effect on the expression of p-AKT.These result suggested that C-7b could be a potential PI3K?inhibitor.At last,we investigated the selective profile of these two representative compounds?B-7h and C-7b?for wild PI3K?,mutant PI3K?and other subtypes of PI3K kinase?PI3K?,?,??and mTORC1.Both compounds inhibited wild and mutant PI3K?,but showed little effect on other isoforms,indicating the two compounds are selective for PI3K?.Collectively,this thesis was most focus on the design of new compounds with good anticancer activities,while decreasing serious of side-effect and/or off-target effect.We obtained the lead compounds from a natural product or a high-throughput screening campaign.Then,chemical modification and structure-activity relationship analysis enabled us to discover three compounds?A-8b,B-7h and C-7b?with potent anticancer activities.Furthermore,the detailed biological mechanism of these anticancer compounds,including cell cycle block,cell apoptosis,cell autophagy and the expression of relative apoptosis factor were investigated.We hope we can supply and show some valuable and useful preliminary results,which can shed some light for future work or pre-clinical trial of this related work.