Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo IIα inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

As the di-2-pyridylketone thiosemicarbazone (DpT) and 2-acetylpyridine thiosemicarbazone (ApT) series show potent antitumor activity in vitro and in vivo, we synthesized their fluorescent zinc(II) complexes to assess their intracellular distribution. The Zn(II) complexes generally showed significantly greater cytotoxicity than the thiosemicarbazones alone in several tumor cell-types. Notably, specific structure-activity relationships demonstrated the importance of the di-2-pyridyl pharmacophore in their activity. Confocal fluorescence imaging and live cell microscopy showed that the Zn(II) complex of our lead compound, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which is scheduled to enter clinical trials, was localized to lysosomes. Under lysosomal conditions, the Zn(II) complexes were shown to transmetallate with copper ions, leading to redox-active copper complexes that induced lysosomal membrane permeabilization (LMP) and cytotoxicity. This is the first study to demonstrate direct lysosomal targeting of our novel Zn(II)-thiosemicarbazone complexes that mediate their activity via transmetalation with copper ions and LMP.

Multidrug resistance (MDR) mediated by P-glycoprotein (Pgp) represents a significant impediment to successful cancer treatment. The compound, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), has been shown to induce greater cytotoxicity against resistant cells than their nonresistant counterparts. Herein, the structure− activity relationships of selected thiosemicarbazones are explored and the novel mechanism underlying their ability to overcome resistance is further elucidated. Only thiosemicarbazones with electron-withdrawing substituents at the imine carbon mediated Pgp-dependent potentiated cytotoxicity, which was reversed by Pgp inhibition. Treatment of resistant cells with these thiosemicarbazones resulted in Pgp-dependent lysosomal membrane permeabilization (LMP) that relied on copper (Cu) chelation, reactive oxygen species generation, and increased relative lipophilicity. Hence, this study is the first to demonstrate the structural requirements of these thiosemicarbazones necessary to overcome MDR. We also demonstrate the mechanism that enables the targeting of resistant tumors, whereby thiosemicarbazones "hijack" lysosomal Pgp and form redox-active Cu complexes that mediate LMP and potentiate cytotoxicity.

A series of eight bis(thiosemicarbazone) ligands and 16 of their respective copper(II) and zinc(II) complexes containing a combination of hydrogen, methyl, pyridyl, phenyl, and/or ethyl substituents at the diimine position of the ligand backbone were synthesized and characterized. The objective of this study was to identify the structure−activity relationships within a series of analogues with different substituents at the diimine position of the backbone and at the terminal N atom. The Cu(II) complexes Cu(GTSM 2 ), Cu(GTSCM), Cu-(PyTSM 2 ), Cu(EMTSM 2 ) and Cu(PGTSM 2 ) demonstrated a distorted square planar geometry, while the Zn(II) complexes Zn(ATSM 2 )(DMSO), Zn(PyTSM 2 )(DMSO), and Zn-(PGTSM 2 )(H 2 O) formed a distorted square pyramidal geometry. Cyclic voltammetry showed that the Cu(II) complexes display quasi-reversible electrochemistry. Of the agents, Cu(II) glyoxal bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(GTSM 2 )] and Cu(II) diacetyl bis(4,4-dimethyl-3-thiosemicarbazone) [Cu(ATSM 2 )] demonstrated the greatest antiproliferative activity against tumor cells. Substitutions at the diimine position and at the terminal N atom with hydrophobic moieties markedly decreased their antiproliferative activity. Complexation of the bis(thiosemicarbazones) with Zn(II) generally decreased their antiproliferative activity, suggesting the Zn(II) complex did not act as a chaperone to deliver the ligand intracellularly, in contrast to similar bis(thiosemicarbazone) cobalt(III) complexes [King et al. Inorg. Chem. 2017, 56, 6609−6623]. However, five of the eight bis(thiosemicarbazone) Cu(II) complexes maintained or increased their antiproliferative activity, relative to the ligand alone, and a mechanism of Cu-induced oxidative stress is suggested. Surprisingly, relative to normoxic growth conditions, hypoxia that is found in the tumor microenvironment decreased the antiproliferative efficacy of most bis(thiosemicarbazones) and their copper complexes. This was independent of the potential hypoxia−selectivity mediated by Cu(II/I) redox potentials. These results provide structure−activity relationships useful for the rational design of bis(thiosemicarbazone) anticancer agents.

Copper is an essential trace metal required by organisms to perform a number of important biological processes. Copper readily cycles between its reduced Cu(i) and oxidised Cu(ii) states, which makes it redox active in biological systems. This redox-cycling propensity is vital for copper to act as a catalytic co-factor in enzymes. While copper is essential for normal physiology, enhanced copper levels in tumours leads to cancer progression. In particular, the stimulatory effect of copper on angiogenesis has been established in the last several decades. Additionally, it has been demonstrated that copper affects tumour growth and promotes metastasis. Based on the effects of copper on cancer progression, chelators that bind copper have been developed as anti-cancer agents. In fact, a novel class of thiosemicarbazone compounds, namely the di-2-pyridylketone thiosemicarbazones that bind copper, have shown great promise in terms of their anti-cancer activity. These agents have a unique mechanism of action, in which they form redox-active complexes with copper in the lysosomes of cancer cells. Furthermore, these agents are able to overcome P-glycoprotein (P-gp) mediated multi-drug resistance (MDR) and act as potent anti-oncogenic agents through their ability to up-regulate the metastasis suppressor protein, N-myc downstream regulated gene-1 (NDRG1). This review provides an overview of the metabolism and regulation of copper in normal physiology, followed by a discussion of the dysregulation of copper homeostasis in cancer and the effects of copper on cancer progression. Finally, recent advances in our understanding of the mechanisms of action of anti-cancer agents targeting copper are discussed.

Fluorescent zinc complexes have recently attracted a lot of interest owing to their vast applications in cellular imaging. We report the synthesis as well as physical, chemical and biological studies of a novel zinc glyoxalbis(4-methyl-4-phenyl-3-thiosemicarbazone), [Zn(GTSC)]₃, complex. As compared with the well-studied zinc biacetylbis(4-methyl-3-thiosemicarbazone), Zn(ATSM), complex, which was used as a reference, [Zn(GTSC)]₃ had 2.5-fold higher fluorescence. When cellular fluorescence was measured using flow cytometry, we observed that [Zn(GTSC)]₃ had 3.4-fold to 12-fold higher fluorescence than Zn(ATSM) in various cell lines (n = 9) of different tissue origin. Confocal fluorescence microscopy results showed that [Zn(GTSC)]₃ appeared to have a nuclear localization within 30 min of addition to MCF7 cells. Moreover, [Zn(GTSC)]₃ showed minimal cytotoxicity compared with Zn(ATSM), suggesting that [Zn(GTSC)]₃ may be less deleterious to cells when used as an imaging agent. Our data suggest that the novel [Zn(GTSC)]₃ complex can potentially serve as a biocompatible fluorescent imaging agent for live cells.

The synthesis and anticancer activity of a copper(II) diacetyl-bis(N4-methylthiosemicarbazone) complex and its nanoconjugates are reported. The copper(II) complex is connected to a carboxylic acid group through a cleavable disulfide link to enable smart delivery. The copper complex is tethered to highly water-soluble 20 nm gold nanoparticles (AuNPs), stabilized by amine terminated lipoic acid-polyethylene glycol (PEG). The gold nanoparticle carrier was further decorated with biotin to achieve targeted action. The copper complex and the conjugates with and without biotin, were tested against HeLa and HaCaT cells. They show very good anticancer activity against HeLa cells, a cell line derived from cervical cancer and are less active against HaCaT cells. Slow and sustained release of the complex from conjugates is demonstrated through cleavage of disulfide linker in the presence of glutathione (GSH), a reducing agent intrinsically present in high concentrations within cancer cells. Biotin appended conjugates do not show greater activity than conjugates without biotin against HeLa cells. This is consistent with drug uptake studies, which suggests similar uptake profiles for both conjugates in vitro. However, in vivo studies using a HeLa cell xenograft tumor model shows 3.8-fold reduction in tumor volume for the biotin conjugated nanoparticle compared to the control whereas the conjugate without biotin shows only 2.3-fold reduction in the tumor volume suggesting significant targeting.

Over 44 million people live with Alzheimer's disease (AD) worldwide. Currently, only symptomatic treatments are available for AD and no cure exists. Considering the lack of effective treatments for AD due to its multi-factorial pathology, development of novel multi-target-directed drugs are desirable. Herein, we report the development of a novel series of thiosemicarbazones derived from 1-benzylpiperidine, a pharmacophore within the acetylcholinesterase inhibitor, Donepezil. These thiosemicarbazones were designed to target five major AD hallmarks, including: low acetylcholine levels, dysfunctional autophagy, metal dys-homeostasis, protein aggregation and oxidative stress. Of these thiosemicarbazones, pyridoxal 4-N-(1-benzylpiperidin-4-yl)thiosemicarbazone (PBPT) emerged as the lead compound. This agent demonstrated the most promising multi-functional activity by exhibiting very low anti-proliferative activity, substantial iron chelation efficacy, inhibition of copper-mediated amyloid-β aggregation, inhibition of oxidative stress, moderate acetylcholinesterase inhibitory activity and autophagic induction. These diverse properties highlight the potential of the lead ligand, PBPT, as a promising multi-functional agent for AD treatment.