COVALENT INHIBITION IN TRIPLE-NEGATIVE BREAST CANCER: DESIGN, SYNTHESIS AND BIOLOGICAL PROFILING OF ACRYLAMIDE-BASED QUINOLINES

In recent decades covalent inhibition has been rediscovered in medicinal chemistry as a powerful tool for the development of potent and selective drugs, particularly for cancer treatment.1 Covalent inhibitors consist of a recognition moiety (driver region) with a selective affinity for the biological target(s) and an electrophilic warhead capable of forming stable covalent bonds with nucleophilic amino acid residues within the binding site. Among the classes of warheads, the acrylamide group stands out due to its favourable reactivity profile (formation of irreversible adducts) and is currently featured in several approved targeted anticancer drugs (e.g. the EGFR inhibitors afatinib and dacomitinib).2 The present study focuses on the covalent-based lead optimization of a series of 4-piperazinylquinoline derivatives 1a–h (Figure 1), which were previously synthesized and evaluated for antiproliferative activity against a panel of 60 human cancer cell lines, as part of the NCI Developmental Therapeutics Program (DTP).3 In the newly developed compounds 2a–h, the nitro group, considered a toxicophore, was removed and replaced with an acrylamide warhead, to enable covalent inhibition of selected biological target(s). The piperazinylquinolines 2a–h were synthesized by multistep procedures and obtained in yields suitable for subsequent biological assays. A preliminary in vitro evaluation of cytotoxic activity was performed on the triple-negative breast cancer (TNBC) MDA-MB-231 cell line, well known for its aggressiveness and limited response to available therapies. Our data provided evidence that several tested compounds showed promising IC₅₀ values in the micromolar range as well as they were able to efficiently reduce breast cancer cell viability. Exploring their mode of action, we demonstrated that their antitumor activity was associated with the induction of oxidative injury and endoplasmic reticulum stress, as well as the activation of Nrf2 and its transcriptional targets. All these events promoted cancer cell commitment to apoptotic cell demise with the activation of caspase-3 and fragmentation of PARP-1. The ability of the compounds to form covalent bonds with reactive nucleophilic residues, such as cysteine or lysine, is currently being investigated by HPLC-MS experiments, to gain insight into the reactivity of the proposed warhead. In silico studies, including induced-fit docking and molecular dynamics simulations, have also been performed to analyse the interaction with key biological targets deregulated in TNBC. Quantitative proteomic analyses and binding assays will better elucidate the mechanism of action, and the potential targets involved. These studies could guide future efforts in the discovery of more effective quinoline-based anti-TNBC compounds.