ML-162

In this study, we explored a dual-target strategy combining the inhibition of human carbonic anhydrase IX (hCA IX), a tumor-associated isoform, and glutathione peroxidase 4 (GPX4), a key regulator of ferroptosis. We demonstrated that the simultaneous inhibition of hCA IX and GPX4 disrupts redox and iron homeostasis, thereby enhancing cell death via ferroptosis. Three series of compounds were rationally designed and synthesized based on the ML162 scaffold using an integrated structural approach and their enzymatic inhibition was evaluated in vitro. Several dual-target compounds exhibited significant antitumor activity, with 18a-c, 22abab and 22abcb inducing dose-dependent cell death. In vivo, intratumoral administration of the lead active compound, 22abcb, significantly prevented the growth of CA IX-expressing human breast cancer xenografts, compared to inactive 22abbb. The effect on tumour growth was significantly reversed by the ferroptosis inhibitor, Fer-1, confirming ferroptosis as the underlying mechanism. These findings highlight the synergistic potential of dual-target inhibitors in disrupting tumor-specific metabolic pathways and position them as a promising therapeutic strategy for solid tumors.

Triple-negative breast cancer (TNBC) is limited in treatment options due to the absence of three receptors, and evidence suggests that TNBC is sensitive to ferroptosis. In this study, a series of genipin derivatives were synthesized through a hybridization strategy that integrated the structures of RSL3 and ML162. Among these derivatives, compound B23 demonstrated remarkable activity against the MDA-MB-231 cell line with an IC₅₀ value of 40 nM, significantly outperforming genipin, RSL3 and ML162. Furthermore, B23 exhibited high selectivity for ferroptosis with a selectivity ratio of up to 108-fold. Further studies revealed that B23 induces ferroptosis by affecting the expression of ferroptosis-related proteins ACSL4, GPX4, and FTH1, thereby disrupting intracellular iron homeostasis and the GSH/GPX4 antioxidant defense system, ultimately leading to the accumulation of lipid peroxidation (LPO). Animal model demonstrated that B23 exhibited potent tumor suppression in an MDA-MB-231 xenograft model, achieving a tumor inhibition rate of 78.46 % at a dose of 4 mg/kg, without observable toxic side effects. In conclusion, B23 represents a promising ferroptosis inducer for the treatment of TNBC and warrants further investigation.