Development of A Narrow Spectrum Anti-Tubercular Agent

Abstract
Complex and lengthy treatment regimens coupled with rising drug resistance underscore the urgent need for
new and better drugs to treat tuberculosis (TB) caused by the bacterium Mycobacterium tuberculosis (Mtb).
Current treatment regimens for drug resistant TB involve the use of costly, less effective, and toxic drugs that
must be continued for up to 24 months. The work proposed here focuses on a proprietary triazolothiadiazole
series of narrow spectrum anti-tubercular agent, which demonstrates exclusive selectivity against Mtb with little
or no activity against other eubacteria. Due to such high Mtb-selectivity, the triazolothiadiazoles are expected to
minimally impact the gut microbiome without promoting selection of cross resistance in non-targeted species
during protracted treatment regimens; therefore, this series represent an exciting starting point for the
development of an entirely novel class of anti-tubercular agent to specifically treat drug-resistant TB. The unique
anti-microbial profile of the triazolothiadiazoles was initially confirmed with three de novo derivatives of the same
scaffold, providing robust verification of the Mtb selectivity. The minimal inhibitory concentration (MIC) values of
44 additional de novo derivatives range from 0.05 to 6.25 µg/ml against drug-susceptible and multi-drug resistant
(MDR) Mtb clinical isolates. Activity against intracellular Mtb replicating within macrophages shows bacteriostatic
and bacteriocidal effects at 0.1 µg/ml and 10 µg/ml. This series has excellent spontaneous resistance
frequencies of andlt;10-9 and minimal cytotoxicity in mammalian cell lines with therapeutic index of ≥50. However,
metabolite profiling in liver microsomes revealed rapid metabolism via N-oxidation and demethylation of an
amine moiety in the triazolothiadiazole core. The immediate goal of this Phase I project is to obtain analogs
with improved metabolic stability while retaining potent Mtb activity for in vivo efficacy evaluation in the
TB mouse model. For the current project, we have developed a medicinal chemistry plan highly focused on
resolving metabolic instability. Approximately 50 analogs containing stable substitutions at the metabolically
vulnerable spot will be synthesized and profiled in microbiological assays against extracellular and intracellular
Mtb along with stability assessment in human and mouse liver microsomes. Our goal is to obtain 3 to 4 potent
(MIC andlt;0.5 µg/ml) analogs with acceptable microsomal stability in human and mouse liver microsomes (extraction
ratio ≤30%) for pharmacokinetic experiments and efficacy evaluations in the mouse TB model. A successful
outcome for the current Phase I project is a quantitative evidence of treatment-induced suppression of
bacterial replication within the lungs of Mtb-infected mice to support the continued advancement of this
series in a future Phase II project.Public Health Significance
The goal of this project is to advance a novel anti-bacterial agent to treat tuberculosis (TB), a disease that kills
1.5 million people annually and affects 2.4 billion people worldwide. The effectiveness of current TB therapy is
compromised by drug resistance; therefore, new anti-tubercular agents to treat drug resistant forms of TB are
urgently needed. We are developing an agent that can selectively kill the drug-resistant forms of the TB
bacterium without affecting the growth of beneficial gut microflora.