Synthesis, Characterization, Antimicrobial Activity and Molecular Modeling Studies of Novel Indazole-Benzimidazole Hybrids

<b>Background/Objectives</b>: In this work, a series of six new indazole-benzimidazole hybrids (<b>M1</b>-<b>M6</b>) were designed, synthesized, and fully characterized. The design of these compounds was based on the combination of two pharmacophoric units, indazole and benzimidazole, both known for their broad spectrum of biological activities. <b>Methods</b>: The molecular hybridization strategy was planned to combine these scaffolds through an effective synthetic pathway, using 6-nitroindazole, two 2-mercaptobenzimidazoles, and 1,3- or 1,5-dihaloalkanes as key precursors, affording the desired hybrids in good yields and with enhanced biological activity. Quantum chemical calculations were performed to investigate the structural, electronic, and electrostatic properties of <b>M1</b>-<b>M6</b> molecules using Density Functional Theory (DFT) at the B3LYP/6-311++G(d,p) level. The antimicrobial activity efficacy of these compounds was assessed in vitro against four Gram-positive bacteria (<i>Staphylococcus aureus</i>, <i>Enterococcus faecalis</i>, <i>Bacillus cereus</i>, and <i>Lactobacillus plantarum</i>), four Gram-negative bacteria (<i>Salmonella enteritidis</i>, <i>Escherichia coli</i>, <i>Campylobacter coli</i>, <i>Campylobacter jejuni</i>), and four fungal strains (<i>Saccharomyces cerevisiae</i>, <i>Candida albicans</i>, <i>Candida tropicalis</i>, and <i>Candida glabrata</i>) using ampicillin and tetracycline as reference standard drugs. <b>Results</b>: Among the series, compound <b>M6</b> exhibited remarkable antimicrobial activity, with minimum inhibitory concentrations (MIC) of 1.95 µg/mL against <i>S. cerevisiae</i> and <i>C. tropicalis</i>, and 3.90 µg/mL against <i>S. aureus</i>, <i>B. cereus</i>, and <i>S. enteritidis</i>, while the standards Ampicillin (AmB) (MIC ≥ 15.62 µg/mL) and Tetracycline (TET) (MIC ≥ 7.81 µg/mL) exhibited higher MIC values. To gain molecular insights into the compounds, an in silico docking study was performed to determine the interactions of <b>M1</b>-<b>M6</b> ligands against the antimicrobial target beta-ketoacyl-acyl carrier protein (ACP) synthase III complexed with malonyl-COA (PDB ID: 1HNJ). Molecular modeling data provided valuable information on the structure-activity relationship (SAR) and the binding modes influencing the candidate ligand-protein recognition. Amino acid residues, such as Arg249, located in the solvent-exposed region, were essential for hydrogen bonding with the nitro group of the 6-nitroindazole moiety. Furthermore, polar side chains such as Asn274, Asn247, and His244 participated in interactions mediated by hydrogen bonding with the 5-nitrobenzimidazole moiety of these compound series. <b>Conclusions</b>: The hybridization of indazole and benzimidazole scaffolds produced compounds with promising antimicrobial activity, particularly <b>M6</b>, which demonstrated superior potency compared to standard antibiotics. Computational and docking analyses provided insights into the structure-activity relationships, highlighting these hybrids as potential candidates for antimicrobial drug development.