Pentacyclic Nitrofurans with <i>In Vivo</i> Efficacy and Activity against Nonreplicating <i>Mycobacterium tuberculosis</i>

<div><p>The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as <b>9a</b>). In the current study, we report the synthesis and antimicrobial properties of <b>9a</b> and panel of <b>9a</b> analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of <i>Mycobacterium tuberculosis</i> with favorable selectivity indices (>150) and a narrow spectrum of activity. <i>In vivo</i>, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity <i>in vivo,</i><b>9a</b> remained the series lead. <b>9a</b> exerted a lengthy post antibiotic effect and was highly active against nonreplicating <i>M. tuberculosis</i> grown under hypoxia. <b>9a</b> showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that <b>9a</b> is a nanomolar inhibitor of actively growing as well as nonreplicating <i>M. tuberculosis</i>.</p></div

Murine model of acute tuberculosis infection.

<p>Log₁₀ reduction provided by compound <b>9a</b> in lungs (black bars) and spleen (grey bars) after 9 days of daily oral administration of 300 mg/kg was determined by calculating the difference between bacillary loads in organs from the untreated group and <b>9a</b> dissolved in (<b>1</b>) 0.5% methylcellulose in DI-H₂O (<b>2</b>) 30% captisol in DI-H₂O (<b>3</b>) 10% vitamin E TPGS in DI-H₂O (<b>4</b>) 0.5% Tween 80 in DI-H₂O (<b>5</b>) 20% cyclodextrin in DI-H₂O or (<b>6</b>) cold PEG (50∶35∶15 H₂O:PEG300:PG). Error bars indicate SEM within treatment groups of 5–7 mice per group.</p

Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.

<p>Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.</p

In vitro activity against NRP bacteria grown under hypoxic conditions.

<p>Viability of hypoxic <i>M. tuberculosis</i> cultures was assessed using the rapid anaerobic dormancy model.</p

MIC and MBCs for select nitrofurans and controls.

<p>Treatments were considered cidal when the MBC_99.9 for <i>M. tuberculosis</i> H37Rv was less than 4X the MIC.</p

Nutrient starvation model of nonreplicating persisters.

<p>The viability of mid-log phase (black bars) or nutrient-starved (gray bars) after exposure to DMSO carrier (1% v/v), 1 µg/mL of isoniazid (INH), or 1 µg/mL of <b>9a</b>. Averaged results and SEM from two biologically independent experiments are presented.</p

Cross Resistance Profiles for Compounds 9a Resistant Clones.

<p>Abbreviations: S, susceptible; <b>R</b>, resistant.</p

Synthesis of pentacyclic nitrofurans.

<p>Reagents and conditions: a) MsCl, Et₃N, CH₂Cl₂, RT, 2 h, 94%; b) substituted phenol, <i>n</i>Bu₄NCl, H₂O, 100°C, 12 h, 86–90%; c) TFA, CH₂Cl₂, RT, 1 h, 92–95%; d) aryl bromide, 2-(di-<i>tert</i>-butylphosphino)biphenyl, NaO<i>t</i>Bu, Pd(OAc)₂, toluene, 100°C, 3 h, 50–70%; e) <b>8</b>, Et₃N, CHCl₃, RT, 3 h, 50–68%.</p

<i>In vitro</i> Interactions with Antitubercular Drugs.

<p><i>In</i> vitro interactions determined by checkerboard assays.</p>a<p>Ex-vivo synergy assays were performed to determine if <b>9a</b> displayed synergy (FICI ≤0.5), indifference (FICI >0.5–4.0) or antagonism (FICI >4.0) with a panel of anti-tuberculosis agents.</p

<i>In vivo</i> pharmacokinetic parameters.

<p>Pharmacokinetic analysis of experimental compounds in rats.</p><p>Values represent means (% coefficient of variation).</p><p>Abbreviations: t_½: half life; CL: clearance; Vd: volume of distribution; fe: fraction excreted unchanged in urine; C_max: maximum plasma concentration; C_{min,24<b> </b>h}: minimum plasma concentration within 24 hours after dosing; AUC_0-∞: systematic exposure; F: oral bioavailability.</p