Due to their significant biological activity, thiosemicarbazones (TSCs) are promising candidates for anticancer therapy. In part, the efficacy of TSCs is linked to their ability to chelate essential metal ions such as copper and iron. Triapine, the best-studied anticancer TSC, has been tested clinically with promising results in hematological diseases. During the last years, a novel subclass of TSCs with improved anticancer activity was found to induce paraptosis, a recently characterized form of cell death. The aim of this study was to identify structural and chemical properties associated with anticancer activity and paraptosis induction of TSCs.Results: When testing a panel of structurally related TSCs, compounds with nanomolar anticancer activity and paraptosis-inducing properties showed higher copper(II) complex solution stability and a slower reduction rate, which resulted in reduced redox activity. In contrast, TSCs with lower anticancer activity induced higher levels of superoxide that rapidly stimulated superoxide dismutase expression in treated cells, effectively protecting the cells from drug-induced redox stress.Innovation: Consequently, we hypothesize that in case of close Triapine derivatives, intracellular reduction leads to rapid dissociation of intracellularly formed copper complexes. In contrast, TSCs characterized by highly stable, slowly reducible copper(II) complexes are able to reach new intracellular targets such as the ER-resident protein disulfide isomerase. Conclusions:The additional modes of actions observed with highly active TSC derivatives are based on intracellular formation of stable copper complexes, offering a new approach to combat (drug-resistant) cancer cells.

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COTI-2 is a novel anticancer thiosemicarbazone in phase I clinical trial. However, the effects of metal complexation (a main characteristic of thiosemicarbazones) and acquired resistance mechanisms are widely unknown. Therefore, in this study, the copper and iron complexes of COTI-2 were synthesized and evaluated for their anticancer activity and impact on drug resistance in comparison to metal-free thiosemicarbazones. Investigations using Triapine-resistant SW480/Tria and newly established COTI-2-resistant SW480/Coti cells revealed distinct structure–activity relationships. SW480/Coti cells were found to overexpress ABCC1, and COTI-2 being a substrate for this efflux pump. This was unexpected, as ABCC1 has strong selectivity for glutathione adducts. The recognition by ABCC1 could be explained by the reduction kinetics of a ternary Cu-COTI-2 complex with glutathione. Thus, only thiosemicarbazones forming stable, nonreducible copper(II)-glutathione adducts are recognized and, in turn, effluxed by ABCC1. This reveals a crucial connection between copper complex chemistry, glutathione interaction, and the resistance profile of clinically relevant thiosemicarbazones.

COTI-2 is currently being evaluated in a phase I clinical trial for the treatment of gynecological and other solid cancers. As a thiosemicarbazone, this compound contains an N,N,S-chelating moiety and is, therefore, expected to bind endogenous metal ions. However, besides zinc, the metal interaction properties of COTI-2 have not been investigated in detail so far. This is unexpected, as we have recently shown that COTI-2 forms stable ternary complexes with copper and glutathione, which renders this drug a substrate for the resistance efflux transporter ABCC1. Herein, the complex formation of COTI-2, two novel terminal N-disubstituted derivatives (COTI-NMe2 and COTI-NMeCy), and the non-substituted analogue (COTI-NH2) with iron, copper, and zinc ions was characterized in detail. Furthermore, their activities against drug-resistant cancer cells was investigated in comparison to COTI-2 and Triapine. These data revealed that, besides zinc, also iron and copper ions need to be considered to play a role in the mode of action and resistance development of these thiosemicarbazones. Moreover, we identified COTI-NMe2 as an interesting new drug candidate with improved anticancer activity and resistance profile.

Thiosemicarbazones continue to attract the interest of researchers as potential anticancer drugs. For example, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone, or triapine, is the most well-known representative of this class of compounds that has entered multiple phase I and II clinical trials. Two new triapine derivatives HL1 and HL2 were prepared by condensation reactions of 2-pyridinamidrazone and S-methylisothiosemicarbazidium chloride with 3-N-(tert-butyloxycarbonyl) amino-pyridine-2-carboxaldehyde, followed by a Boc-deprotection procedure. Subsequent reaction of HL1 and HL2 with CuCl2·2H2O in 1:1 molar ratio in methanol produced the complexes [CuII(HL1)Cl2]·H2O (1·H2O) and [CuII(HL2)Cl2] (2). The reaction of HL2 with Fe(NO3)3∙9H2O in 2:1 molar ratio in the presence of triethylamine afforded the complex [FeIII(L2)2]NO3∙0.75H2O (3∙0.75H2O), in which the isothiosemicarbazone acts as a tridentate monoanionic ligand. The crystal structures of HL1, HL2 and metal complexes 1 and 2 were determined by single crystal X-ray diffraction. The UV-Vis and EPR spectroelectrochemical measurements revealed that complexes 1 and 2 underwent irreversible reduction of Cu(II) with subsequent ligand release, while 3 showed an almost reversible electrochemical reduction in dimethyl sulfoxide (DMSO). Aqueous solution behaviour of HL1 and 1, as well as of HL2 and its complex 2, was monitored as well. Complexes 1−3 were tested against ovarian carcinoma cells, as well as noncancerous embryonic kidney cells, in comparison to respective free ligands, triapine and cisplatin. While the free ligands HL1 and HL2 were devoid of antiproliferative activity, their respective metal complexes showed remarkable antiproliferative activity in a micromolar concentration range. The activity was not related to the inhibition of ribonucleotide reductase (RNR) R2 protein, but rather to cancer cell homeostasis disturbance—leading to the disruption of cancer cell signalling.