Synthesis and biological evaluation of thiosemicarbazone-based antibody–drug conjugates

Metal chelators belonging to the di-pyridyl-thiosemicarbazone (DpTs) class have shown great promise as adjuvant therapeutics for treating cancer, with DpC and Dp44mT emerging as the lead candidates. Despite their efficacy, these molecules also induce various undesirable side effects due to insufficient cancer cell targeting, highlighting the need to improve their selectivity. Here, we present a first generation of DpT-antibody conjugates. To this end, we developed a facile synthesis to functionalize DpTs strategically with click-able azido linkers. Moreover, selective side-chain modification of the clinical antibody trastuzumab (Tras) with a complementary bis-alkyne moiety is described. Using this new chemistry, we conjugated four different azido DpTs to trastuzumab <i>via</i> a combination of oxidation-controlled quinone (SPOCQ) and strain-promoted alkyne-azide click (SPAAC) chemistry. We evaluated the antiproliferative activity of the resulting novel antibody-drug conjugates (ADCs) against MCF-7 and SK-BR-3 cell lines. Linker positioning on the DpT scaffold significantly influences the cytotoxicity of the conjugates. For instance, conjugating Tras at the <i>ortho</i> position on the Dp44mT scaffold is more efficacious than conjugating at the <i>para</i> position with IC₅₀ values of 25.7 ± 5.5 nM and 103.5 ± 2.0 nM, respectively, against MCF-7 cells. Furthermore, we observe intriguing cell line-dependent activity of the ADCs with increased selectivity towards MCF-7 cells, providing novel insights into the cytotoxic activity of DpTs and their antibody conjugates.