Biological Activity and Preliminary Safety Assessment of the Nitrobenzoate Derivative Methyl 3-hydroxy-4-methoxy-2,6-dinitrobenzoate (MMDB): Antimicrobial, Redox and Developmental Toxicity Profiles in vitro and in Zebrafish

Methyl 3-hydroxy-4-methoxy-2,6-dinitrobenzoate (MMDB), a derivative of Methyl 3-hydroxy-4-methoxybenzoate, was synthesized to assess its antimicrobial potential and preliminary safety profile against clinically relevant pathogens and human cell systems. The study systematically evaluated MMDB’s antimicrobial (antibacterial and antifungal), antioxidant (DPPH RSA), and anti-inflammatory (BSA denaturation assay) activity, as well as the toxicological properties in three human cell lines and the zebrafish model. MMDB showed remarkable antibacterial activity against multiple Gram-positive and Gram-negative bacterial strains, including multi-drug-resistant strains with MIC as low as 50 µg/ml and antifungal activity against Candida albicans , Penicillium notatum and Aspergillus niger at the concentrations ranging from 1000-1500 µg/ml. It also showed moderately antioxidant activity (59.74%) and weak anti-inflammatory activity (31.3%). Cytotoxicity investigations across multiple cell lines (HEK293T, 4T1, and MDA-MB-231) and zebrafish toxicity evaluations revealed a favorable safety profile, with minimal adverse effects observed at therapeutic concentrations while dose-dependent toxicity at higher concentrations, providing crucial insights into the MMDB's developmental and systemic toxicity safety profiling, supporting its potential for future clinical applications through careful lead optimization by SAR and subsequent dose optimization. These comprehensive findings position MMDB as a promising lead compound with potential antimicrobial, antioxidant, and anti-inflammatory activity, which can be further optimized to reduce the toxicity at higher doses. Future studies should focus on the lead optimization of MMDB, its mechanistic studies and advanced pre-clinical and clinical studies. • Potent antibacterial activity of MMDB was observed against both Gram-positive and Gram-negative bacteria at doses as low as 50 µg. • Anti-fungal activity was observed against Candida albicans, Penicillium notatum, and Aspergillus niger at higher doses. • MMDB showed weak anti-oxidant and anti-inflammatory properties. • The toxicity profiling of MMDB in human cell lines and zebrafish model showed a favorable safety profile upto 32.25 μg/ml dose.