Design, docking, synthesis, and in vitro evaluation of potent anti-tubercular agents targeting DNA Gyrase

Tuberculosis caused by Mycobacterium tuberculosis has been reported to infect about two-third of the global population and to continuously develop multidrug resistance. DNA gyrase, a type II topoisomerase, is a promising target of the quinolone class of drugs in the treatment of tuberculosis. The present study is focused on the design and synthesis of newer nitrogen heterocyclics containing indole, n-methyl piperazine, piperidine, and pyrrolidine ring structures Initially designed compounds were evaluated for their affinity to the DNA gyrase target. The molecular docking performed using FlexX indicated compounds IIb5 (1-(R)-(4-hydroxyphenyl)(4-methylpiperazin-1-yl)methyl)-3-((S)-(4-hydroxyphenyl)(4-methylpiperazin-1-yl)methyl)urea and IIc5 ((1-(R)-(4-hydroxyphenyl)(4-methylpiperazin-1-yl)methyl)-3-((S)-(4-hydroxyphenyl)(4-methylpiperazin-1-yl)methyl) thiourea to exhibit promising binding affinity (dock score of -15.01 and -13.77) when compared to the reference MFX moxifloxacin (dock score -4.40) with the target 5BS8 (DNA gyrase). Further, the best 10 compounds were synthesized by one-pot synthesis employing the reaction of indole/N-methyl piperazine/piperidine/pyrrolidine with N-substituted benzaldehydes in the presence of acetamide/urea/thiourea to afford the compounds in 54.60% to 85.47% yield. The synthesized compounds were suitably characterized using chromatographic and spectroscopic tools. In the microplate Alamar Blue assay (MABA), compounds IIb1, IIIc2, IIIb1, and IIb5 exhibited good minimum inhibitory concentrations of 1.6µg/mL, 3.12µg/mL, and 12.5µg/mL, respectively, when compared to the standard rifampicin with 0.8µg/mL inhibitory concentration. The MTB gyrase supercoiling assay performed using Mycobacterium tuberculosis gyrase supercoiling assay kit demonstrated compound IIb5 at a concentration of 300µg/mL to show gyrase inhibition in comparison to MFX at 60 µg/mL. In the MTT assay performed using the human breast cancer cell line MCF-7, compounds IIc2, IIb5, and IIb1 showed IC50 values of 2.57µM, 12.54µM, and 12.75µM, respectively, compared to doxorubicin (1.10µM) at 7-48hrs and 72hrs of the study. Based on these observations, N-methyl piperazine class of compounds can serve as a lead/pharmacophore for the rational design of potent molecules against MTB gyrase to combat the growing issue of MDR-TB.