Design, Synthesis and Biological Evaluation of C3‐Indolyl/(3‐chloro‐indolyl)‐C4‐aryl/heteroaryl‐azetidin‐2‐ones

Herein, trans‐ and cis‐ azetidin‐2‐ones 3–6 were strategically synthesized, capitalizing on the bioactivity of azetidin‐2‐ones and indole pharmacophore, followed by a comprehensive characterization using a diverse array of spectroscopic techniques. The sixteen azetidin‐2‐ones were examined for antimicrobial activities against both Gram‐negative ( P. aeruginosa , E. coli , A. baumannii ) and Gram‐positive bacteria (S . aureus, E. faecium, B. cereus ), as well as against C. albicans and C. tropicalis fungal strains. The highly potent compounds ( 5 a , 6 b , 6 d ) demonstrated maximum inhibition against all multidrug‐resistant strains, with minimum inhibitory concentrations ranging from 0.97–3.9 μg/mL, surpassing the potency of standard ampicillin (MIC: 3.12–50 μg/mL). Moreover, 6 b and 6 d exhibited significant inhibitory effects on C. albicans (MIC: 0.97 μg/mL), comparable to fluconazole. The presence of C3 ‐(3‐chloro‐indolyl) scaffold, combined with diverse electronic effects at N1/C4 ‐centers, particularly the inclusion of thiophen‐2‐yl motif, greatly influenced the activity of target compounds. Assessment of 4 d , 4 i–k and 6 d on THLE‐2 cell lines revealed their preferential safety. Molecular docking studies revealed seven compounds with active dual targeting of DNA GyrB and PBP2a proteins, demonstrating a potent broad‐spectrum antibacterial effect. In silico ADME analysis affirms positive drug‐likeness and favorable pharmacokinetic characteristics of indole‐derived hybrids, indicating a promising potential for addressing challenges in evolving multidrug resistance.