Quinoline-6-Carboxylic Acid Derivatives: A New Class of 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 new and effective inhibitors, such as compound 4d , which showed good inhibition against h -NTPDase1 and -2, as reflected by IC ₅₀ value of 0.28±0.03 µM against h-NTPDase1 and 0.92±0.17 µM against h -NTPDase2. Similarly, compound 4g inhibited h- NTPDase3 with an IC ₅₀ value of 0.32±0.05 µM, and compound 4b inhibited h -NTPDase8 with an IC ₅₀ value of _{0.44±0.08 µM. Additionally, compound 4k demonstrated good inhibition against h -ENPP1 as reflected by IC 50 value 0.11±0.02 µM. Finally, compound 4a inhibited e5’NT enzyme with an IC 50 value of 0.092±0.02 µM. 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. These interactions involve residues such as Asp201, Asp213, Asp218, Asp326, Ala412, Tyr340, Tyr371, Thr90, Trp408, Trp450, Cys502, Gly178, Phe360, Phe257, Arg392, Ala347, Leu202, Leu290, His50, His360, His380, Ser49, Ser100, Gln148, Glu266, Lys50, Lys295, Gln523, Pro323, and Ile90. Density Functional Theory (DFT) analysis revealed that compound 4f exhibits the lowest energy gap, which correlates well with its strong ectonucleotidase inhibitory activity against NPP1 and NTPDase3. Finally, fluorescence microscopy was conducted to investigate the interaction of the compound 4d with plasma membrane in A549 lung cancer cells. Fluorescence imaging of compound 4d exhibited strong cellular emission, confirming its effective interaction with membrane-bound enzymes in these cancer cells. MTT and apoptosis studies also revealed compound 4i , 4j , and 4k to be potentially cytotoxic in the cancer cells, endorsing the potential of these quinoline derivatives to be used for drug development for cancer management.}