Binding pose analysis and scaffold optimization of petasis products for selective COX-2 inhibition

Selective COX-2 inhibitors are associated with cardiovascular safety concerns, motivating continued exploration of alternative chemotypes. In this study, we investigated the COX-2 inhibitor activity of a library of alkylaminophenols. This was achieved through single-concentration screens used to prioritize compounds with COX-2 inhibition and acceptable cellular tolerability in THP-1 cells, generation of analogs via positional analog scanning (PAS) and molecular docking studies to guide scaffold optimization. Novel compounds were then synthesized by a green Petasis borono-Mannich protocol. Preliminary structure activity relationships (SAR) and molecular descriptor analysis of our one-shot assays allowed us to observe that indoline and para substituted phenols derivatives showed the most promising inhibitory activity. Compound 35e demonstrated a 23-fold increase in COX-2 potency over its parent compound 35, while simultaneously increasing COX-1/COX-2 selectivity by a factor of 25. In LPS-stimulated THP-1 macrophages, 35e reduced IL-6 and TNF-α release in a concentration-dependent manner, with estimated IC₅₀ values of approximately 35 μM and 42 μM, respectively. These concentrations remained below the cytotoxic IC₅₀ in THP-1 cells (67.9 μM), indicating cytokine modulation under non-cytotoxic conditions. Although cellular activity required higher concentrations than direct enzymatic inhibition (COX-2 IC₅₀ = 8.43 μM), these results support a measurable functional anti-inflammatory phenotype. This work establishes alkylaminophenols as an early-stage COX-2 inhibitory chemotype and identifies 35e as a starting point for further optimization.