Development and Evaluation of Bis-benzothiazoles as a New Class of Benzothiazoles Targeting DprE1 as Antitubercular Agents

Benzothiazole-bearing compounds have emerged as potential noncovalent DprE1 (decaprenylphosphoryl-β-d-ribose-2'-epimerase) inhibitors active against <i>Mycobacterium tuberculosis</i>. Based on structure-based virtual screening (PDB ID: 4KW5), a focused library of thirty-one skeletally diverse benzothiazole amides was prepared, and the compounds were assessed for their antitubercular activity against <i>M.tb</i> H37Ra. Most potent compounds <b>3b</b> and <b>3n</b> were further evaluated against the <i>M.tb</i> H37Rv strain by the microdilution assay method. Among the compounds evaluated, bis<i>-</i>benzothiazole amide <b>3n</b> emerged as a hit molecule and demonstrated promising antitubercular activity with minimum inhibitory concentration (MIC) values of 0.45 μg/mL and 8.0 μg/mL against H₃₇Ra and H₃₇Rv, respectively. Based on the preliminary hit molecule (<b>3n)</b>, a focused library of 12 more bis-benzothiazole amide derivatives was further prepared by varying the substituents on either side to obtain new leads and generate a structure-activity relationship (SAR). Among these compounds, <b>6a, 6c</b>, and <b>6d</b> demonstrated remarkable antitubercular activity with MIC values of 0.5 μg/mL against H37Ra and 1.0, 2.0, and 8.0 μg/mL against H₃₇Rv, respectively. The most active compound, <b>6a</b>, also displayed significant efficacy against four drug-resistant tuberculosis strains. Compound <b>6a</b> was assessed for <i>in vitro</i> cytotoxicity against the HepG2 cell line, and it displayed insignificant cytotoxicity. Furthermore, time-kill kinetic studies demonstrated time- and dose-dependent bactericidal activity of this compound. The GFP release assay revealed that compound <b>6a</b> targets the inhibition of a cell wall component. SNPs in <i>dprE-1</i> gene assessment revealed that compound <b>6a</b> binds to tyrosine at position 314 of DprE1 and replaces it with histidine, causing resistance similar to that of standard TCA1. <i>In silico</i> docking studies further suggest that the strong noncovalent interactions of these compounds may lead to the development of potent noncovalent DprE1 inhibitors.