Background: Thiosemicarbazones of the di-2-pyridylketone thiosemicarbazone class possess potent anti-tumor activity via binding essential metal ions in cancer cells (e.g., iron (Fe(II)), copper (Cu(II)), and zinc (Zn(II)) to form metal complexes. These agents demonstrate activity at inhibiting the “triad-of-death” in cancer, namely (1) metastasis; (2) tumor growth; and (3) resistance (Richardson et al. PNAS 2006;103:14901-6; FASEB J. 2020;34:11511-28; FASEB J. 2021;35:e21347). However, clinical trials have indicated that some thiosemicarbazones induce deleterious oxidation of heme proteins such as oxyhemoglobin leading to hypoxia (Invest. New Drugs. 2010;28:91–97).

Objective/Aim: To design, synthesize, and characterize innovative thiosemicarbazone anti-cancer agents with marked and selective anti-tumor activity that do not induce problematic oxidation of oxy-myoglobin or oxyhemoglobin.

Hypothesis: That bespoke structural modifications of thiosemicarbazones derived from structure-activity relationships can lead to frontier agents with marked anti-cancer activity and selectivity.

Design/Approach/Methodology: Medicinal chemistry, cellular and molecular biology, biochemistry, molecular pharmacology, molecular modeling, and protein docking.

Results: A novel, potent, and selective anti-tumor thiosemicarbazone, PPP44mT, and its analogs were synthesized and characterized and displayed strikingly distinctive properties. This activity was mediated by the inclusion of a styrene moiety, which through steric and electrochemical mechanisms, prevented deleterious oxy-myoglobin or oxyhemoglobin oxidation relative to other thiosemicarbazones, namely di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC) or di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This prevention of heme oxidation was due to the ability of the styrene moiety to prevent the close association of PPP44mT to the heme plane of oxy-myoglobin or oxyhemoglobin. Furthermore, structure-activity relationship analysis demonstrated that specific tuning of PPP44mT electrochemistry further inhibited oxy-myoglobin or oxyhemoglobin oxidation. Both PPP44mT and its Cu(II) complexes showed conspicuous, almost immediate cytotoxicity against SK-N-MC tumor cells (within 3 h). In contrast, the Zn(II) complex of PPP44mT (i.e., [Zn(PPP44mT)2]) demonstrated a pronounced delay in activity, taking 48 h before marked anti-proliferative efficacy was apparent. As such, [Zn(PPP44mT)2] was designated as a “stealth Zn(II) complex” that overcomes the near immediate cytotoxicity of PPP44mT or its Cu(II) complexes. Examining suppression of oncogenic signaling, [Zn(PPP44mT)2] was superior to DpC or Dp44mT at inhibiting the expression of pro-oncogenic cyclin D1.

Significance: This research demonstrates that thiosemicarbazones can be specifically tailored to achieve bespoke activity that leads to marked anti-cancer activity and selectivity.

Support/Funding Information: National Health and Medical Research Council of Australia; Australian Research Council; Thrasher Foundation USA; Cancer Australia; National Breast Cancer Foundation of Australia.