Design And Anti-tumor Evaluation Of Glaucocalyxin A Derivatives And Artemisininanalogues, And Synthesis-free PET Imaging For Tumor And Brown Adipose Tissue

This thesis includes two sections.Part I:Design,synthesis and biological evaluation of novel Glaucocalyxin A derivatives and Artemisinin analogues as potential anti cancer agents.Cancer is a serious disease that threatens human health.Currently,chemotherapy is one of the main strategies for the treatment of cancer,however the effectiveness of the chemotherapeutic drugs is limited by their non-specificity toward tumor cells.Discovery of anticancer active ingredients with high efficacy and low toxicity from the natural animal,plants and minerals has been the efforts of many drug companies and research scientists.Because of their excellent therapeutic potential and unique mechanism of action,natural products play a significant role in anti-cancer drug discovery and development.Rabdosia japonica is Labiatae Isodon plants distributed in Northeast China,North China and North Korea,Japan and the former Soviet Union in the Far East,especially rich in resources in Jilin province.R.japonica was used for the treatmentof gastritis,hepatitis,mastitis,cold fever,joint pain and other diseases.As an important member of ent-kaurane diterpenoids,Glaucocalyxin A?GLA?has been reported to exhibit a wide range of biological activities,including antitumor effects,antibacterial activity and cardiovascularsystem protecting activity.As an anti-malarial drug,Artemisinin was separated from Artemisia annua Land characterizedin 1972.It is a sesquiterpene with an unique endoperoxide pharmocophore,and also exhibits potent anti-cancer,immunoregulation and anti-bacterial activity.Herein,we demonstrate the design and synthesisof derivatives of Glaucocalyxin A?GLA?and analogues of artemisinin,with improved inhibitory activity against both drug-sensitive and drug-resistant cancers and enhanced selectivity.Chapter I:Design,synthesis and biological evaluation of novel Glaucocalyxin A derivatives as potential anti cancer agentsGlaucocalyxin A?GLA?has been reported to exhibit a wide range of biological activities,however clinical application of GLA was limited by its high metabolic rate and poor bioavailability.So far,no research effort was reported to address these issues.In this report,a series of Mannich base type derivatives of Glaucocalyxin A?GLA?were designed and prepared.The cytotoxicity of these compounds was evaluated against six tumor cell lines?SMMC-7721,B16,SGC-7901,A549,KB,HL-60?.Most compounds exhibited potent antiproliferative effects with low micromolar IC₅₀ values.Compound 1 with4-methylbenzylamino moiety and compound 16 with cyclohexylamino moiety displayed the highest inhibition efficacy.Significantly,the cytotoxicity of compound 1 was much lower than GLA against the normal human liver cell?HL-7702?.The in vitro stability assay revealed that transformation of GLA to Mannich base type derivatives improved the compound stability in rat plasma.Finally,decomposition product analysis supported that compound 1 could act as a prodrug and release GLA in the intracellular environment.Chapter II:Design,synthesis and biological evaluation of novel Artemisinin analoguesas potential anti cancer agentsThe anti-malarial drug Artemisinin?Qing hao su?was proven to exhibit potent anti-cancer activity both in vitro and in vivo.Although the precise antitumor mechanism is still unclear,current consensus believes that the ferrous iron mediated cleavage of the endoperoxide group to release reactive oxygen species?ROS?and/or carbon centered radicals,which play an important role in inducing DNA damage,mitochondrial depolarization and apoptosis.The first generation of EGFR inhibitors are structurally characterized with a 4-anilinoquinazoline core scaffold.Although both drugs were effective for NSCLC patients harboring somatic mutations L858R and delE746_A750,acquired drug resistance was detected in approximately 50%of the NSCLC patients after approximately one year of treatment due to the secondary mutation T790M.To overcome resistance caused by T790M,in the present study,endoperoxide and 4-anilinoqnazoline were conjugated to obtain a series of compounds.These conjugates exhibited high antiproliferative potency against a number of cancer cell lines,including the epidermal growth factor receptor?EGFR?L858R/T790M mutant cell.Further experiments revealed the conjugate's reactive oxygen species?ROS?generating ability,apoptosis inducing activity and involvement in EGFR downstream signaling pathways.Part II:Synthesis-free PET imaging for tumor and brown adipose tissue.PET imaging is a widely applicable,but very expensive technology.Strategies that can significantly reduce the high cost of PET imaging are highly desirable both for research and commercialization.On-site synthesis is one important contributor for the high cost.In this report,we designed and prepared two types of ⁶⁴Cu labeled PET tracer,which could be usedfor synthesis-free PET imagingfor tumor and brown adipose tissue.Compared with traditional PET imaging,synthesis free method could remarkably reduce the cost of PET tracer development.Chapter I:Pseduo-synthesis-free PET imaging of?v?3 integrinIn this report,we demonstrated that pseduo-synthesis-free PET imaging of?_v?3integrin is feasible.To achieve this goal,we utilized a three-in-one function of ⁶⁴Cu to design PET tracers,in which ⁶⁴Cu is not only responsible for PET signal,but also crucial for proper functioning of the ligand.We designed GHRGDHG-⁶⁴Cu for imaging of?_v?3integrin in U87 tumors.In the designed probe,⁶⁴Cu bears three functional roles that include generation of PET signal,coordination with two GHs moieties,and cyclizing the linear peptide into an active cyclo-RGD form.Our in vitro and in vivo data indicated that GHRGDHG-⁶⁴Cu had excellent specificity for?_v?3 integrin,fast clearance from the body,and high uptake in tumors.Moreover,duo tothe facts that the linear peptide is a weak binding form,but the cyclo-RGD is the strong binding form for?_v?3 integrin,purification of the ⁶⁴Cu complex was not necessary,therefore we considered this method as a pseudo-synthesis-free PET imaging.We believe that our design strategy and pseudo-synthesis-free imaging method can be extended to other imaging targets as well.Chapter II:Combination of Disulfiram and ⁶⁴CuCl₂ as a synthesis-free method for PET imaging of brown adipose tissue and TSPO.In this report,we demonstrated the feasibility of a synthesis-free method for PET imaging of brown adipose tissue?BAT?and translocator protein 18 kDa?TSPO?via a combination of Disulfiram,an FDA approved drug for alcoholism,and ⁶⁴CuCl₂(termed as⁶⁴Cu-Disulfiram).In this method,a step-wise injection protocol of ⁶⁴CuCl₂ and Disulfiram was used to accomplish the purpose of synthesis-free.In this protocol,Disulfiram,an inactive ⁶⁴Cu ligand,was first injected to allow it to metabolize into diethyldithiocarbamate?DDC?,a strong ⁶⁴Cu ligand,which can chelate ⁶⁴CuCl₂ from the following injection.The“synthesis-free”imaging in this case means that the active tracer is being formed in situ through the chelation of ⁶⁴CuCl₂ and DDC in vivo.Our PET images produced distinct contour of the triangular shape of BAT,and excellent BAT selectivity over WAT in mice.Our blocking studies,Western blot,and immunohistology suggested that the observed BAT contrast was due to ⁶⁴Cu-Dis binding to TSPO,which was further confirmed as a specific biomarker for BAT imaging using[¹⁸F]-F-DPA,a TSPO-specific PET tracer.Our studies,for the first time,demonstrated that TSPO could serve as a potential imaging biomarker for BAT.Furthermore,since imaging contrast obtained with both ⁶⁴Cu-Dis and[¹⁸F]-F-DPA was not dependent on BAT activation,these agents could be used for reliable imaging of BAT mass.Additional value of our synthesis-free approach could be applied to imaging TSPO in other tissues as it is an established biomarker of neuro-inflammation in activated microglia and plays a role in immune response,steroid synthesis,and apoptosis.Although here we applied ⁶⁴Cu-Dis for a synthesis-free PET imaging of BAT,we believe that our strategy could be extended to other targets while significantly reducing the cost of PET imaging.