Phthalimide Derivatives as Anti-Inflammatory Agents: In Silico COX-2 Targeting and In Vitro Inhibition of PGE2 Production

<b>Background/Objectives</b>: The development of specific inhibitors for cyclooxygenase-2 (COX-2) is a challenge for public health. A series of 17 <i>N</i>-phthalimide hybrids was evaluated using a functional M06 meta-GGA hybrid in combination with a polarized 6-311G (d, p) basis set. The top three candidates (<b>6</b>, <b>10</b>, and <b>17</b>) were synthesized and evaluated as selective COX-2 inhibitors of PGE-2 using an integrated in silico-in vitro approach. <b>Methods</b>: Molecular docking against COX-2 (PDB 5KIR) and COX-1 (PDB 6Y3C), supported by homology modeling and DFT geometry optimization (B3LYP/6-31G*), revealed that the phthalimide carbonyl groups and the 3,4,5-trimethoxyphenyl or geranyl-derived moieties establish key hydrogen bonds and hydrophobic contacts with Arg120, Tyr355, Tyr385, and Ser530 in the COX-2 active site, conferring predicted selectivity ΔGCOX-2 vs. COX-1 = -1.4 to -2.8 kcal/mol. <b>Results</b>: The compounds complied with Lipinski's and Veber's rules and displayed favorable ADMET profiles. In vitro assessment in LPS-stimulated J774A.1 murine macrophages confirmed potent inhibition of PGE₂ production, 3.05 µg/mL, with compound <b>17</b> exhibiting the highest efficacy, 97.79 ± 5.02% inhibition at 50 µg/mL, and <b>10</b> showing 95.22 ± 6.03% inhibition at 50 µg/mL. Notably, all derivatives maintained >90% cell viability up to 250 µg/mL by resazurin assay and showed no evidence of cytotoxicity or mitosis potential in the tests at 24 h. <b>Conclusions</b>: These results demonstrate that strategic hybridization of phthalimide with natural and synthetic product-derived fragments yields highly potential PGE2 inhibitors. Therefore, compounds <b>6</b>, <b>10</b>, and <b>17</b> are promising lead candidates for the development of safer anti-inflammatory agents.