A Multi-Target Nitrogen-Fused Azole Drug Platform Derived from a Pyrazoline-Thiadiazole Moiety: In Vivo Antimicrobial...

Background: Cancer patients are highly susceptible to microbial infections due to immune suppression, necessitating therapeutic strategies that integrate anticancer efficacy with effective antimicrobial intervention. Chalcone-derived nitrogen-fused heterocycles represent a promising platform for developing multi-target agents with relevance to antimicrobial drug delivery, particularly for localized infections. Methods: A series of chalcone-based pyrazoline-thiadiazole nitrogen-fused azole hybrids was synthesized via thiosemicarbohydrazide-functionalized intermediates and fully characterized. Antiproliferative activity was evaluated against MCF-7, HepG-2, HeLa, and HCT-116 cell lines, alongside selectivity toward WI-38 normal fibroblasts. Antibacterial, antibiofilm, and in vivo efficacy were assessed against methicillin-resistant Staphylococcus aureus (MRSA USA300) and Acinetobacter baumannii AB5057. Mechanistic investigations included cell-cycle analysis, apoptosis assays, ERK2, RIPK3, p53, BAX/Bcl-2 quantification, DNA gyrase inhibition, molecular docking, molecular dynamics simulations, and density functional theory calculations. Results: Compound 13 exhibited potent cytotoxicity, particularly against MCF-7 (IC₅₀ = 3.87 ± 0.2 µM), outperforming doxorubicin (IC₅₀ = 4.17 ± 0.2 µM), with high selectivity indices (SI = 10.7 for MCF-7). Mechanistically, compound 13 induced G₂/M arrest (40.16% vs. 14.15% control), increased apoptosis to 32.89%, up-regulated ERK2 (3.17-fold), RIPK3 (11.97-fold), and p53 (3.54-fold), and markedly increased the BAX/Bcl-2 ratio (~42-fold). Compounds 7 and 13 displayed bactericidal activity against MRSA and A. baumannii (MIC/MBC = 10 mg/mL), potent antibiofilm effects, and significant in vivo efficacy in an MRSA skin infection model. Compound 13 reduced bacterial load by ~5 log units, outperforming vancomycin. DNA gyrase inhibition (IC₅₀ = 17.10 ± 0.17 µM) and computational studies supported target engagement. Conclusions: Pyrazoline-thiadiazole-based nitrogen-fused azole hybrids, particularly compound 13, demonstrated quantifiable anticancer and antimicrobial efficacy with strong in vivo validation, supporting their potential as multi-target candidates relevant to antimicrobial drug delivery in infection-prone cancer patients.