Quinoline carboxylic acid derivatives as potent ectonucleotidase inhibitors

Ectonucleotidases, including h-NTPDases, h-ENPP, and h-e5′NT, play a crucial role in regulating extracellular nucleotide levels by converting ATP into immunosuppressive adenosine, thereby facilitating tumor immune evasion. Inhibiting these enzymes can restore antitumor immunity by preventing adenosine accumulation within the tumor microenvironment. Herein, we report the design and synthesis of quinoline-6-carboxylic acid derivatives (4a–4l), a biologically relevant scaffold, and evaluate their potential to inhibit recombinant h-ENPP1, h-e5′NT, and h-NTPDases. This study led to the identification of a new and effective inhibitor, compound 4d, which exhibited strong inhibitory activity against both h-NTPDase1 and h-NTPDase2, with IC50 values of 0.28 ± 0.03 µM and 0.92 ± 0.17 µM, respectively. Molecular docking studies were performed to complement the in vitro analysis, revealing that the tested compounds show favorable interaction with the amino acid of the target enzymes h-NTPDase1, -2, -3, and -8, h-NPP1, and h-e5′NT enzymes. The geometry of the selected compounds was optimized using Density Functional Theory (DFT) at the B3LYP/3-21G level to obtain energy-minimized structures for subsequent analysis, fluorescence microscopy was conducted to investigate the interaction of the compound 4d with plasma membrane in A549 lung cancer cells. Fluorescence microscopy of compound 4d confirmed its membrane-localized interaction in A549 cells, supporting its potential engagement with ectonucleotidase targets. MTT and SRB proliferation assays indicated that compounds 4j, 4k, and 4h exhibited moderate cytotoxic activity against the tested cancer cells, suggesting their potential as preliminary leads for further development in anticancer drug discovery. In-contrast, to MTT, compound 4a have shown an increase in percentage cytotoxicity with time.