A trisubstituted benzimidazole cell division inhibitor with efficacy against Mycobacterium tuberculosis

Abstract

Trisubstituted benzimidazoles have demonstrated potency against Gram-positive and Gram-negative bacterial pathogens. Previously, a library of novel trisubstituted benzimidazoles was constructed for high throughput screening, and compounds were identified that exhibited potency against M. tuberculosis H37Rv and clinical isolates, and were not toxic to Vero cells. A new series of 2-cyclohexyl-5-acylamino-6-N, N-dimethylaminobenzimidazoles derivatives has been developed based on SAR studies. Screening identified compounds with potency against M. tuberculosis. A lead compound from this series, SB-P17G-A20, was discovered to have an MIC of 0.16 µg/mL and demonstrated efficacy in the TB murine acute model of infection based on the reduction of bacterial load in the lungs and spleen by 1.73 ± 0.24 Log10 CFU and 2.68 ± Log10 CFU, respectively, when delivered at 50 mg/kg by intraperitoneal injection (IP) twice daily (bid). The activity of SB-P17G-A20 was determined to be concentration dependent and to have excellent stability in mouse and human plasma, and liver microsomes. Together, these studies demonstrate that SB-P17G-A20 has potency against M. tuberculosis clinical strains with varying susceptibility and efficacy in animal models of infection, and that trisubstituted benzimidazoles continue to be a platform for the development of novel inhibitors with efficacy.

Figure 1. Structure of lead benzimidazoles.

Figure 2. Activity of SB-P17G-A20 against M. tuberculosis clinical isolates.

Dose response curves were generated from MABA data for M. tuberculosis strains (H37Rv, TN587, NHN382, W210 and NHN20) treated with SB-P17G-A20. The curves were generated by graphing the log₁₀ drug concentrations against the difference in growth between the drug treated wells and control wells using GraphPad Prism Version 5.0d for Mac OS X (GraphPad Software, San Diego CA., USA, www.graphpad.com).

Figure 3. Transmission Electron Microscopy of FtsZ.

FtsZ (5 µM) was polymerized by GTP (25 µM) in the absence (A) and presence of SB-P17G-A20 at 40 µM (B) and 80 µM (C). Images are at 49,000x magnification (scale bar 500 nm).

Figure 4. Killing characteristics of SB-P17G

-A20 against whole bacteria. The time dose curves were generated from OD_{600 nm} (a) and from CFU enumeration (b) data. Different concentrations of the compound were tested in triplicate and the mean and standard deviation of the OD_{600 nm} values or the CFU counts from Day 0, 2, 4, and 6 were plotted against time using GraphPad Prism Version 5.0d for Mac OS X (GraphPad Software, San Diego CA., USA, www.graphpad.com).

Figure 5. Efficacy of SB-P17G-A20 in a tuberculosis murine model of infection.

Scatter plot of the CFU counts from the lung and spleens of infected mice after drug therapy with SB-P17G-A20 delivered IP at 50 mg/kg bid. The colony counts were converted to logarithms. The lower level of detection was 1 log₁₀ CFU. Outliers were identified by the Grubbs’ Test using an online calculator. (GraphPad Software, San Diego CA., USA www.graphpad.com). A scatter plot of the CFU data from the lung and spleen of individual mice from the treatment and control groups were plotted with the mean and SE from each group using GraphPad Prism Version 5.0d for Mac OS X (GraphPad Software, San Diego CA., USA www.graphpad.com).