Green and scalable supercritical fluid chromatographic resolution of 5-nitroindoline derivatives: Enantiomer-specific biological evaluation and COX-2 inhibition insights.

Abstract

We describe an integrated, environmentally conscious workflow that combines synthesis, enantioselective separation, and biological evaluation of 5-nitroindoline-based derivatives. Ten racemic compounds were synthesized via a Petasis multicomponent reaction involving 5-nitroindoline, glyoxylic acid, and various (hetero)aryl boronic acids under trifluoroacetic acid (TFA) catalysis. Enantiomeric resolution was achieved using packed-column supercritical fluid chromatography (SFC) on immobilised and coated polysaccharide chiral stationary phases (Chiralpak IG, Chiral Art Amylose SA, and Chiralpak AS-H), employing CO2 with 30-40% isopropanol containing 0.5% isopropylamine (IPAm). Rapid screening delivered baseline resolution within 4-7 min under analytical conditions, which translated directly to 21 and 30 mm i. d. preparative columns without loss of selectivity, enabling high-throughput isolation of both enantiomers. The racemic compounds and isolated enantiomers were comprehensively characterized by UPLC-QTOF, LC-MS, 1H and 13C NMR, FT-IR, CHNS analysis, HPLC purity, and specific optical rotation, followed by biological evaluation to assess enantiomer-dependent activity trends. Antibacterial screening against Staphylococcus aureus and Escherichia coli using the disk diffusion assay revealed moderate to significant antibacterial activity for several derivatives, with compounds 4a and 4b exhibiting the most pronounced inhibitory effects and enantiomer-dependent antibacterial behaviour. Furthermore, these derivatives demonstrated strong antibiofilm activity by significantly reducing biofilm formation and biomass in both bacterial strains. A series of enantiomerically enriched derivatives was further evaluated against COX-1 and COX-2 using molecular docking, MM/GBSA binding free-energy calculations, and molecular dynamics simulations. Compound 4a (R) showed selective preference for COX-2, with favourable docking affinity and thermodynamic stabilization. FMO analysis revealed that reduced HOMO-LUMO energy gap and enhanced electrophilicity contribute significantly to biological activity and intermolecular interactions. These findings identify 4a (+) as a promising scaffold for selective COX-2 inhibition and provide important structural and electronic insights for the development of novel anti-inflammatory and antibacterial agents. Overall, the study demonstrates a generalizable, mass-efficient, and scalable chiral separation strategy that minimizes organic solvent consumption while enabling access to stereo-chemically defined molecules for downstream biological investigations. The primary objective of this study was to develop a rapid, scalable, and environmentally sustainable supercritical fluid chromatographic (SFC) platform for the enantiomeric resolution of novel 5-nitroindoline derivatives. The isolated stereoisomers were subsequently evaluated for antibacterial, antibiofilm, and COX-2 inhibitory activities to investigate the influence of stereochemistry on biological activity and demonstrate the utility of the developed SFC methodology.

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