Indole-1,2,4-triazole mannich hybrids as dual anti-newcastle disease virus genotype VII D and antimicrobial agents: In ovo efficacy and computational profiling

Abstract Newcastle disease virus (NDV) remains a major threat to global poultry production, particularly when viral outbreaks are aggravated by secondary microbial infections. To address this challenge, a focused series of indole–1,2,4-triazole Mannich hybrids ( 4a–d , 5c–d , 6a–d , 7a–d , and 8a–b ) was designed and synthesized as potential dual antiviral–antimicrobial agents. The structures of the synthesized compounds were confirmed by IR, ¹H NMR, ¹³C NMR, and mass spectrometry. In ovo antiviral evaluation using specific pathogen-free embryonated chicken eggs revealed pronounced inhibition of NDV genotype VII D replication. The untreated virus exhibited a high hemagglutination (HA) titer (10⁸·⁶), whereas treatment with the most active compounds reduced titers to approximately 10⁴·¹–10⁴·⁸, with compound 7c demonstrating activity comparable to the reference drug ribavirin (10⁴·¹). Antiviral potency was further supported by favorable inhibitory and selectivity parameters, with the most active derivatives displaying IC₅₀ values in the low-micromolar range and therapeutic indices approaching or exceeding 100, indicating strong antiviral efficacy with limited cytotoxicity. Beyond antiviral activity, several compounds exhibited broad-spectrum antimicrobial effects against Gram-positive, Gram-negative, and fungal pathogens. Notably, strong inhibition of Staphylococcus aureus was observed (up to 38 mm inhibition zone), alongside meaningful activity against Haemophilus paragallinarum and Salmonella typhimurium (up to 25 mm and 20 mm, respectively). Selected derivatives also demonstrated antifungal activity against Candida albicans , in some cases approaching the efficacy of the reference antifungal agent. Mechanistic investigation targeting the neuraminidase domain of recombinant NDV hemagglutinin–neuraminidase (HN) protein identified compounds 5d and 7c as potent inhibitors, with IC₅₀ values of 1.375 ± 0.03 and 3.343 ± 0.09 µM, respectively, supporting interference with HN-mediated viral entry as a plausible antiviral mechanism. Molecular docking against NDV HN (PDB: 1USX), complemented by in silico ADMET profiling, further rationalized ligand–protein interactions and highlighted favorable pharmacokinetic characteristics for the leading compounds. Collectively, these findings identify indole–triazole Mannich hybrids as promising dual-acting scaffolds capable of suppressing NDV genotype VII D replication while simultaneously targeting opportunistic microbial coinfections, providing a rational platform for further optimization toward integrated antiviral and antimicrobial therapies in poultry health management.