Designing An Anticancer Copper(?) Pro-drug Based On The Nature Of Cancer Cell And Human Serum Albumin(HSA) Carrier ?A Subdomain

Metallodrugs offer biological and chemical diversity that is distinct from that of organic drugs.Since the serependitous discovery of the cisplatin antiproliferative activity,many efforts have focused on the design of potent metal-based drugs for oncology therapies.A large number of metal compounds have been evaluated both in vitro and in vivo and some have reached clinical trials;however,the major challenge remains of how to improve tumor target ability,enhance the efficiency of metal drugs in killing cancer cells,and decrease their side effects in vivo.Excitingly,drug delivery system and the prodrug strategy have been promising for overcoming the aforementioned problems.Human serum albumin(HSA)-based drug delivery systems are promising because HSA is a nontoxic,nonantigenic,biocompatible,and biodegradable endogenous protein that lacks immunogenicity.Moreover,Cu-containing drugs may serve as promising next-generation anticancer agents because Cu is an essential element for human physiological functions because of its bioactivity and oxidative nature.To synergistically enhance the selectivity and efficiency of anticancer copper drugs,we proposed and built the models to develop anticancer copper pro-drugs based on the nature of HSA ?A subdomain and cancer cells.On the basis of the previous research,the following major studies were carried out in the thesis:(1)synthesizing a series of aroylhydrazone Schiff(ONO)base-derived Cu(?)compounds containing the one/two potential leaving groups;(2)providing solid evidence on the feasibility for developing Cu pro-drugs based on the nature of HSA carrier ?A subdomain and cancer cells;(3)determining whether the HSA carrier increased the targeting ability and therapeutic efficacy of Cu pro-drugs relative to that of Cu(II)compounds alone in vivo;(4)determining whether the HSA complexes was associated with lesser side effects than Cu(II)compounds alone in vivo,and(5)investigating the anticancer properties and possible anticancer mechanisms of the HSA Cu pro-drugs complexes.The whole dissertation includes five chapters:Chapters 1:The nature of cancer cell,current status of copper-based anticancer drugs,X-ray crystallography and the structures and properties of HSA were briefly introduced firstly.The development of anticancer prodrugs based on these special residues of HSA was reviewed.Chapters 2:Three copper(?)compounds of a 2-hydroxy-1-naphthaldehyde benzoyl hydrazone Schiff-base ligand in the presence pyridine,imidazole,or indazole ligands were synthesized(C1-C3).The structures of three HSA complexes revealed that the Cu compounds bind to the hydrophobic cavity in HSA ?A subdomain.Among them,the pyridine and imidazole ligands of Cl and C2 are replaced by Lys199,and His242 directly coordinates with Cu(II).The indazole and Br ligands of C3 are replaced by Lys199 and His242,respectively.Compared with Cu(?)compounds alone,the HSA complexes enhance cytotoxicity in MCF-7 cells approximately 3-5 fold,but do not raise cytotoxicity levels in normal cells(HL-7702)in vitro through selectively accumulating in cancer cells to some extent Moreover,our results revealed that the HSA pro-drug complex kill MCF-7 cancer cells possible through anti-cancer multi-mechanisms,inducing DNA damage,which results in activation of the p53 pathway,cell cycle arrest at the G2/M phase,and mitochondria-mediated apoptosis by regulating the expression of Bcl-2 family proteins.Chapters 3:We built a breast cancer(MCF-7)mouse model on developing anti-cancer copper(Cu)pro-drug based on the nature of IIA subdomain of HSA carrier and cancer cells.Thus,we first synthesized a new Cu(II)compound derived from tridentate(E)-N'-(5-bromo-2-hydroxybenzylidene)benzohydrazide Schiff base ligand(HL)containing 2 potential leaving grops[indazole(Ind)and NO3-],namely,[Cu(L)(Ind)NO3](C4).Structural analysis of the HSA complex revealed that[Cu(L)(Ind)NO3]could bind to the hydrophobic cavity of the HSA ?A subdomain.Lys199 and His242 coordinate with Cu(?)by replacing the indazole and NO3 ligands of Cu(L)(Ind)(NO3).The release behavior of the Cu compound from the HSA complex could be regulated at different pH levels.[Cu(L)(Ind)NO3]can enhance cytotoxicity by 2 times together with HSA specifically in cancer cells but no effect on normal cells in vitro.Inportantly,our in vivo results showed that the HSA complex showed increased the selectivity and capacity to inhibit tumor growth and was less toxic than[C u(L)(Ind)NO3]alone.Chapters 4:The types and numbers of coordinated ligands in metal agent can be modified to enhance its anticancer activity.To further improve delivery efficiency,anticancer activities and selectivity of copper agents in vivo,we proposed to develop the active targeting folic acid(FA)-functionalized human serum albumin(HSA)nanoparticles(NPs)carrier for anticancer metal pro-drug designed by regulating the ligand(s)in metal pro-drug to be replaced by Lys199 and His242 of HSA.To this end,we first synthesized two(E)-N'-(5-chloro-2-hydroxybenzylidene)benzohydrazide Schiff-base(HL)Cu(II)compounds by designing a second ligand with a different coordination atom with Cu2+;namely,Cu(L)(QLXBr)[C1,QL=quinolone]and Cu(L)(DMF)(Br)[C2,DMF=N,N-dimethylformamide].The structures of the two HSA complexes revealed that Cu compounds bind to the hydrophobic cavity in the HSA ?A subdomain.The QL and Br-ligands of C1 are replaced by Lysl99 and His242,respectively,which coordinate with Cu2+,whereas the DMF ligand of C2 is kept intact and His242 is replaced with Br-of C2 and coordinates with Cu2+.Interestingly,the binding mode of Cu compounds to HSA has little influence on their release behavior from HSA at different pH levels.Compared with Cu compounds alone,the FA-HSA NPs enhance cytotoxicity in Bel-7402 cells approximately 2-4 fold,but do not raise cytotoxicity levels in normal cells in vitro.Importantly,in vivo data showed that FA-HSA-C2 NPs increased selectivity and capacity of inhibiting tumor growth of C2.Finally,a brief conclusion of the work is givea This work may guide the rational design and development of new metal prodrugs for future clinical applications.