Abstract
Small-molecule inhibitors targeting the B7-CD28 pathway represent a promising strategy for cancer immunotherapy. This study presents a comprehensive in silico characterization of 2-(4-fluorophenyl)-6-methyl-4-(3-(trifluoromethyl)-phenyl)-1,2-dihydropyrazolo-[3,4-b:3',4'-d]-pyridin-3-(6H)-one to evaluate its potential as a dual-target inhibitor within the B7-CD28 pathway. Using density functional theory (DFT) at the B3LYP/6- 311G-(d) level, the molecule's structural and electronic properties were thoroughly characterized. Simulated spectroscopic profiles (Fourier transform infrared spectroscopy (FT-IR), Raman, and ultraviolet-visible (UV-Vis)) were generated, with vibrational modes validated through potential energy distribution (PED) analysis. Electronic characterizationincluding the highest occupied molecular orbital-lowest unoccupied molecular orbital (HOMO-LUMO) energy gap, density of states (DOS), and topological descriptors such as the electron localization function (ELF), localized orbital locator (LOL), and reduced density gradient (RDG)highlighted features governing the molecule's reactivity and stability. Natural bond orbital (NBO) analysis quantified key hyperconjugative interactions (E (2)) contributing to intramolecular stability, while local reactivity was further examined using Fukui functions. The 1H and 13C NMR chemical shifts were predicted via the gauge-independent atomic orbital (GIAO) method. Molecular docking with AutoDock Vina revealed strong binding affinities toward CD80 (-7.54 kcal·mol-1) and CTLA-4 (-7.97 kcal·mol-1), suggesting effective inhibition of the B7-CD28/CTLA-4 signaling axis. Additionally, in silico ADMET analysis indicated favorable pharmacokinetic and absorption properties, supporting the compound's potential as a promising immunotherapeutic candidate.