Selective membrane disrupting dispiro-oxindole Dehydrocostuslactone hybrids with potent anti-MRSA, antibiofilm, and synergistic activity

A structurally diverse library of dispiro-oxindole dehydrocostuslactone (DHC) hybrids was synthesized via a modular, catalyst-free, regio- and stereoselective three-component 1,3-dipolar cycloaddition involving natural DHC, isatin derivatives, and amino acid-based dipoles including sarcosine, L-thioproline, and L-proline. This green approach yielded 21 novel spirocyclic architectures featuring densely functionalized, sp³-rich frameworks with multiple stereocenters and rigid quaternary spiro junctions. All compounds were fully characterized by 1D/2D NMR and HRMS, and their stereochemistry was confirmed through single-crystal X-ray diffraction of selected analogues. The library was evaluated for antimicrobial activity against Gram-positive and Gram-negative bacterial strains. Notably, L-proline based analogues, especially LP-03, displayed potent and selective activity against methicillin-resistant Staphylococcus aureus (MRSA), with MIC values as low as 6.2 μM. LP-03 retained strong efficacy across six multidrug-resistant clinical isolates, matching vancomycin in four cases. Selectivity indices derived from cytotoxicity and hemolysis assays confirmed low mammalian toxicity. Mechanistic studies, including membrane depolarization, SEM, and fluorescence imaging, revealed bacterial membrane disruption as a likely mode of action. LP-03 also showed synergistic interactions with vancomycin, daptomycin, tetracycline, and moxifloxacin, along with rapid bactericidal action in time-kill assays. In silico ADMET profiling supported favourable pharmacokinetic properties, including high predicted oral bioavailability and low CYP inhibition for selected compounds (LP-01, LP-03, and LP-05). Taken together, this study establishes spirocyclic DHC hybrids as a promising class of selective, membrane active anti-MRSA agents with validated efficacy against clinical isolates and favourable drug-like profiles.