Design, Synthesis and Evaluation of Bioreductively-Activated Fluoroquinolone Prodrugs

Abstract

During tuberculosis (TB) infection caused by Mycobacterium tuberculosis (Mtb), a subpopulation of mycobacteria enter into a metabolically inactive, non-replicating persistent state that are not susceptible to most frontline TB drugs. Persisters become a reservoir with conditions favouring the emergence of drug-resistant mutants and also contribute to extended duration of TB therapy. Recent clinical trial data indicated that inclusion of Moxifloxacin (MXF) led to a shortening of TB treatment from 6-months to 4-months. Although MXF demonstrated lethality against actively replicating Mtb, it has limited efficacy against mycobacterial persisters. One important reason for the lack of activity of MXF against nonreplicating Mtb is its poor accumulation within bacteria. Non-replicating Mtb is known to have a thick lipid-rich cell wall and being relatively hydrophilic (clogP = -0.49), the permeability of MXF may be limited. To address this problem, we utilized a prodrug approach to enhance the efficacy of MXF by increasing its permeation in non-replicating Mtb. Prodrugs are normally activated by enzymes and/or related metabolic conditions within the target cells. Non-replicating Mtb are associated with a reductive environment (hypoxia) and induce the expression of reductive enzymes such as nitroreducatases (NTRs). Since NTRs are prevalent in both replicating and non-replicating Mtb, we propose that a MXF prodrug can be cleaved by NTR to generate MXF and as a consequence may exhibit potent inhibitory activity against exponentially growing as well as dormant bacilli. Herein, we designed and developed a focussed library of nitroaryl and nitroheteroaryl conjugates of MXF with varying reduction potentials to modulate the propensity to undergo reduction, rate and efficiency of drug release. A systematic screening was then conducted to identify the optimal prodrug to generate MXF under reductive conditions, in the presence of NTRs as well as in bacterial lysates. Our data revealed that the 2-nitrothiazole-based prodrug was rapidly and efficiently cleaved under both chemo- and bio-reductive conditions to produce MXF. Notably, this prodrug was equipotent in its inhibitory activity in both cellular models and animal models, and had superior inhibitory activity against non-replicating Mtb when compared with MXF. LC-MS based studies further supported that the increased intracellular accumulation of MXF from the prodrug as compared to MXF alone, could contribute to enhanced potency in the non-replicating Mtb model. Hence, the newly developed 2-nitrothiazole-prodrug is rapidly, efficiently and selectively cleaved under reductive conditions to produce the active drug. Inspired by these differences in the sensitivity and specificity of 2-nitrothiazolyl group towards NTR, a dual colorimetric fluorescent probe sensitive to bacterial NTR was constructed. The probe exhibited high selectivity and sensitivity with an excellent ability for non-invasive real time detection of bacteria. The results outlined in this thesis lay the foundation for new prodrug approaches to developing interventions that are effective against both replicating as well as non-replicating Mtb and may have applications in imaging and diagnostics.