Discovery And Optimization Of Multi-targeting Antitumor And Antifungal Lead Compounds

Recently,the mortality and morbidity of cancer and invasive fungal infections（IFIs）has been significantly increased,which represents a great threat to human health.Single target drugs might not achieve the desired therapeutic effects for the treatment of complex diseases such as cancer.The underlying reason is that the dysregulation of multiple signaling pathways is a hallmark of cancer development and progression,and the existence of compensating signaling pathways has reduced the efficacy of single target drugs.Specific drug combinations have expanded the treatment to achieve more durable disease control.However,the employment of drug cocktails often complicates the doses/schedule and negatively impacts patient compliance.It can also introduce the unpredictable pharmacokinetic（PK）profile and drug-drug interactions.In contrast,a single multi-targeting molecule has the advantages of higher synergistic effect,more predictable PK profile,reduced compliance difficulties and so on.Despite development of more effective new antifungal agents,fluconazole（FLC）is still widely used in clinic because of its efficacy and low toxicity.However,with the broad use of FLC,FLC-resistant Candida albicans isolates emerged more frequently,leading to a huge human mortality and morbidity for immmunocompromised populations.Various approaches have been proposed to increase the susceptibility of C.aibicans to FLC in order to cope with treatment failures,among which is the combination of FLC with different classes of non-antifungal agents such as calcineurin inhibitors,heat shock protein 90 inhibitors,calcium homeostasis regulators and traditional Chinese medicines.With an aim to discover novel multi-targeting antitumor and antifungal drugs,this thesis mainly includes four parts:（1）Discovery of Janus kinase 2（JAK2）and histone deacetylase（HDAC）dual inhibitors as a novel strategy for combinational treatment of leukemia and invasive fungal infections;（2）Discovery of novel evodiamine derivatives as dual inhibitors against topoisomerase（Top）and HDAC;（3）Rational design,synthesis and biological evaluation of nicotinamide phosphribosyltransferase（NAMPT）/DNA dual-targeting inhibitors.Besides,the discovery and optimization of new spleen tyrosine kinase（Syk）inhibitors through structure-based virtual screening was performed,laying the foundation for further multi-targeting drug design.1.Discovery of JAK2 and HDAC dual inhibitors as a novel strategy for combinational treatment of leukemia and invasive fungal infectionsClinically,immunocompromised leukemia patients often suffer from limited efficacy of chemotherapy and bone marrow transplantation therapy,and are highly risks to be infected by invasive fungal pathogens.Herein a novel therapeutic strategy was developed that a small molecule can simultaneously treat acute myeloid leukemia（AML）and IFIs.Novel JAK2/HDAC dual inhibitors were identified to possess potent antiproliferative activity toward hematological cell lines and excellent synergistic effects with FLC to treat resistant C.albicans infections.In particular,compound B18a,a highly active and selective JAK2/HDAC6 dual inhibitor(IC₅₀=8.4 nM and 71 nM,respectively),showed excellent in vivo antitumor efficacy in several AML models,and synergized with FLC to extend the survival of mice infected with resistant C.albicans.This study highlights the therapeutic potential of JAK2/HDAC dual inhibitors in treating AML and IFIs and provides a promising lead compound for multi-targeting drug discovery.2.Discovery of novel evodiamine derivatives as Top/HDAC dual inhibiotrs with in vivo antitumor activityRecently,multi-targeting drug design on the basis of epigenetic target HDAC has become a hot research area.HDAC-based multi-targeting antitumor agents have several advantages such as improved efficacy,reduced toxicity and overcoming drug resistance.Based on the chemical structures of evodiamine and HDAC inhibitors,a series of novel Top/HDAC dual-targeting inhibitors were designed,synthesized and evaluated.Most of them showed good inhibitory activities against Top and HDAC,and exhibited good to excellent antitumor activities against various human cancer cell lines.Moreover,compound C23a showed potent antitumor activity against HCT-116 cell line(IC₅₀=0.94μM),and can effectively induce the apoptosis and the cell cycle arrestment in HCT-116cells.Particularly,compound C23a was orally active and showed good antitumor efficacy in HCT-116 xenograft model（TGI=75.2%）,which was more potent than SAHA（TGI=36.0%）,evo（TGI=45.6%）and their combinational therapy（TGI=54.5%）.Taken together,this study provided an efficient approach for discovering novel multi-targeting antitumor agents based on Chinese traditiaonal medicine.3.Rational design,synthesis and biological evaluation of NAMPT/DNA dual-targeting inhibitorsNAMPT is the rate-limiting enzyme that catalyzes the first step in the mammalian nicotinamide adenine dinucleotide（NAD）salvage pathway.Aberrant NAD metabolism was associated with oncogenic sigal transduction,suggesting the critical roles of NAMPT in tumorgenesis and metastasis.Inhibition of NAMPT activity by small molecule inhibitors has demonstrated antitumor activities in multiple in vitro and in vivo models.However,no objective responses of NAMPT inhibitors（such as FK866）were seen in clinical trials of patients with advanced solid tumors.Drug combinations were proven to be the promising strategy to enhance the effectiveness of NAMPT inhibitors.The simultaneous increase NAD consumption caused by the activation of PARP induced by DNA damaging agents and the inhibition of NAD regeneration by NAMPT inhibitors results in catastrophic NAD depletion and accumulation of unrepaired DNA,leading to tumor cell death.Inspired by the results,a series of novel NAMPT/DNA dual-targeting inhibitors were designed and synthesized using the NAMPT inhibitor FK866 and DNA damaging agent chlorambucil as initial templates,aiming to enhance the efficacy of FK866.Most of them showed good to excellent antitumor activities against various human cancer cell lines,especially compounds D9a and D9b.Moreover,in the CT-26 xenograft model,compounds D9a and D9b showed more potent in vivo antitumor efficacy in BALB/c mice than in BALB/c-nude mice,suggesting that they might activate the immune system.In summary,this study provided an efficient approach for discovering novel antitumor agents,and further investigation of the lead compounds D9a and D9b is in progress.4.Discovery and optimization of new Syk inhibitorsSyk is an attractive target for the discovery of new treatments for inflammatory and autoimmune disorders.Structure-based virtual screening was performed for identifying novel scaffolds of Syk inhibitors.A total of 16 hits were discovered in the enzyme assay and 8 compounds had an IC₅₀ value lower than 10μM.In particular,the indazole scoffold compound E8(IC₅₀=3.2μM)was active in the cellular Syk assay and could inhibit lymphocytes proliferation in a dose-dependent manner.Next,the structure-activity relationship of the compound E8 was further investigated,and a series of novel Syk inhibitors were designed and synthesized.Several new inhibitors demonstrated potent activity against Syk.In particular,compound E33c showed better Syk inhibitory activity(IC₅₀=1.2μM)than compound E8,represented a good lead compound for Syk-based drug discovery,and laid the first stone for further multi-targeting drug design.