Synthesis, in vitro exploration, and reactivity studies of 6-chloropyridin-2-yl benzoates as promising covalent fragments with antiviral and pro-apoptotic activities

Fragment-Based Drug Discovery (FBDD) focuses on identifying small molecular fragments that, despite their low molecular weight, can specifically bind to biological targets. [1-3] Due to their small size, ester-based inhibitors are particularly suitable as starting building blocks in covalent FBDD, aiding the development of potential bioactive compounds. [4] In this study, we synthesized a series of 6-chloropyridinyl ester compounds (Figure 1), which were screened for their dual anticancer and antiviral activity. These inhibitors take advantage of the electrophilic nature of the 6-chloropyridinyl ester warhead to form a covalent bond with reactive amino acid residues present in the targeted proteins. The 6-chloropyridinyl ester derivatives were first analyzed for their ADME properties and druglikeness using the SwissADME tool [5] and then synthesized following two different one-pot procedures. The ability of the compounds to form covalent bonds with reactive nucleophilic residues was confirmed by HPLCMS studies. Therefore, the antiviral effect of 6-chloropyridinyl esters was evaluated through enzymatic inhibition assays, proving a binding affinity in the micromolar range. In particular, compound 1g exhibited the lowest IC50 value of 30.0 ± 6.6 μM. The antiproliferative activity was investigated in several tumor cell lines (A-375, MCF-7, and HCT-116) and normal cells (16HBE). The ester derivatives showed remarkable selectivity against the melanoma cell line A-375, with IC50 values of 1.59 ± 0.03 μM and 1.08 ± 0.01 μM for compounds 1e and 1m, respectively. Furthermore, these two compounds were analyzed for their ability to induce cellular cycle arrest, apoptosis, evaluation of mitochondrial membrane potential, and ROS-production. These results emphasize the potential of the 6-chloropyridinyl ester derivatives as promising covalent reactive fragments for both antiviral and anticancer applications.