Synthesis of New Pyrazolo[1,5‐ <i>a</i> ]Pyrazin‐4(5 <i>H</i> )‐One Derivatives and Effects on PI3K Protein Levels in A549 Lung Adenocarcinoma Cell Line

Molecular targeted therapies have significantly advanced non-small cell lung cancer (NSCLC) treatment; however, acquired resistance in advanced stages remains a critical challenge. To overcome this, novel therapeutic targets or combination strategies are urgently needed. In this study, a new series of pyrazolo[1,5-a]pyrazin-4(5H)-one derivatives (compounds 12-41) was designed and synthesized. Their antiproliferative activities were assessed in the A549 NSCLC cell line. Compounds 15, 16, 21, and 26-28 showed strong cytotoxicity, with IC₅₀ values of 8.19 µM (compound 27) and 7.01 µM (compound 28). Notably, compounds 21 and 26-28 significantly reduced phosphoinositide 3-kinase (PI3K) protein levels in A549 cells. To better understand their mode of action, molecular docking studies were performed using the crystal structure of PI3K (PDB ID: 4XE0), revealing favorable binding affinities and conserved interactions within the active site. Compounds 27 and 28, which showed the strongest in vitro activity, also exhibited the most stable binding modes and docking scores, consistent with the biological data. Structure-activity relationship (SAR) analysis indicated that a high electron-density benzene ring on the pyrazole moiety and a low electron-density ring on the pyrazine enhanced cytotoxic activity. Drug-likeness and pre-absorption, distribution, metabolism, excretion, and toxicity (ADMET) evaluations suggested favorable pharmacokinetic properties.