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.