Alkyltriphenylphosphonium turns naphthoquinoneimidazoles into potent membrane depolarizers against mycobacteria

Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure–activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.


Synthesis and characterization of intermediates
The synthetic route and procedures by Li et al. was modified and employed. [S1] General procedure for alkylation of the naphthoimidazolediones 22 and 35 (Procedure A) Compound 22 or 35 was reacted with NaH (60% dispersion in mineral oil, 2 eq.) in anhydrous DMF (3 mL) at 0°C for 30 minutes under N2. The corresponding bromochloroalkane (1.3 eq.) was added to the reaction mixture and allowed to warm to room temperature and stirred overnight under N2. Upon completion, the mixture was quenched with cold water at 0°C. The crude product was extracted with DCM (4 ´ 20 mL), washed with water (4 ´ 20 mL) and brine (1 ´ 20 mL). The organic layers were combined and dried over anhydrous MgSO4, filtered and concentrated under reduced pressure to obtain crude residues, which were purified by column chromatography using DCM:EtOAc (16:1 -1:1) to obtain the desired products as a yellow solid. This procedure is referred to as Procedure A.

N-(5-Chloropentyl)-2-methylnaphtho
General procedure for synthesis of bromochloroalkanes To a mixture of the corresponding bromoalcohol (2 mmol) and pyridine (5 µL), SOCl2 (2.2 mmol) was added slowly over 30 mins at 0°C. The mixture was then heated under reflux at 90°C for 5 h. The mixture was quenched with water (10 mL) and extracted with DCM (3 ´ 20 mL). The combined organic layer was washed with NaHCO3 (10 mL), brine (10 mL) and dried with MgSO4. The solution was concentrated and purified through column chromatography using hexane. The product was then dried in vacuo to yield a colourless oil.

Synthesis of 5 without sequential addition of reagents
Compound 25 (64 mg, 0.19 mmol), KI (93 mg, 0.56 mmol) and triphenylphosphine (146 mg, 0.56 mmol) were dissolved in ACN (5 mL) and heated under reflux for 3 days. Upon completion, the reaction mixture was filtered and purification was carried out as in Procedure B to afford the desired product as yellow solid (113.4 mg, 87%).

Minimum Inhibitory Concentrations (MICs) Determination
Minimum inhibitory concentrations (MICs) of test compounds were determined on M. bovis BCG and Mtb H37Rv following a previously described broth dilution method. [S1] MIC50 and MIC90 are the concentrations required to inhibit 50% and 90% of bacterial growth respectively, as compared to untreated drug free controls.

Minimum Bactericidal Concentrations (MBCs) Determination
Minimum bactericidal concentrations (MBCs) of test compounds were determined by CFU (colony forming unit) enumeration on complete Middlebrook 7H10 agar as described previously. [S14] MBC90, MBC99, and MBC99.9 are the concentrations required to reduce CFUs by 10-, 100-, and 1000-fold, respectively, as compared to the untreated inoculum at time point zero.

Time-kill Kinetic Determination
The 21-day time-kill kinetic profiles of 6 was determined as follows. Briefly, pellets of mid-log-phase M. bovis BCG (OD600 0.4 -0.8) were spun down (3200 ´ g, 10 min) and resuspended in fresh complete Middlebrook 7H9 broth at OD600 0.

Membrane Potential and Permeability Determinations
Reported methods were followed to determine the membrane depolarizing and permeabilizing activities of test compounds on M. bovis BCG cultures. [S1], [S14] Briefly, M. bovis BCG cultures at mid-log phase were diluted at OD600 0.1 in complete 7H9 broth. Diluted cultures were treated with test compounds at 4´ MIC90 for 24 h. At selected time points, aliquots were removed from culture media and tested for changes in membrane potential using BacLight TM Bacterial Membrane Potential kit (Life Technologies, CA) and membrane permeability using fluorescent probes SYTOâ9 and propidium iodide (PI) (Molecular Probes, Invitrogen, MA). RIF at 4´ MIC90 (0.08 µM) was used the negative control for both assays. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) at 5 µM and SDS at 5% (v/v) were used as the positive controls in membrane potential and permeability tests, respectively. [S14] In the membrane depolarization assay, the dye DiOC2(3) accumulates within polarized membrane and exhibits red fluorescence; upon dissociation due to loss of membrane potential, the fluorescence emission shifts to green. Therefore, the extent of membrane depolarization is shown by the extent to which the red/green fluorescence ratio decreases. Meanwhile, in the membrane permeabilization assay, SYTOâ9 exhibits green fluorescence and stains all bacteria regardless of membrane integrity, while propidium iodide (PI), which exhibits red fluorescence, only stains bacteria with damaged membrane. Therefore, a decrease in green/red fluorescence ratio indicates membrane permeabilization.

piniBAC Cell Membrane Stress Reporter System
A previously reported method was followed. [S14] Briefly, test compounds and the positive control INH (0.1 − 50 μM) were incubated with the recombinant M. bovis BCG-piniBAC-RFP strain at OD600 0.2 for 24 h. Thereafter, red fluorescence (RFU, λEx 587 nm/λEm 630 nm) and OD600 readings of cultures were recorded on a Tecan Infinite M200 PRO plate reader to assess induction of promoter activity. INH was employed as positive control. Normalized fluorescence readings (RFU/OD600) were recorded to account for changes in cell number during the incubation period. Details of the plasmid constructs are provided in Section 11.

Reactive Oxygen Species (ROS) Detection by CellROX TM Green Reagent
The generation of reactive oxygen species ( To each well in a 96-well plate was seeded 30,000 Vero-E6 cells in 100 μL media. The plate was incubated at 37°C, 24 h, 5% CO2 for cell adherence. After this time, media was removed from the well by aspiration and replaced with 100 μL aliquot of fresh media (99 μL) and test compound (1 μL from a DMSO stock solution that was 200fold more concentrated than the final concentration in the well). DMSO content in each well was 0.5% v/v. Treated plates were incubated for 48 h (5% CO2, 37°C) after which 10 μL of Celltitre 96 ®Aqueous One Solution was added to each well (without removal of media) and incubated for 3 h. Thereafter, absorbance readings were taken at 490 nm on a Tecan Infinite M200 Pro Microplate reader. Cell viability was determined from the expression: Cell viability (%) = ( + ) − ( ) ( + ) − ( ) × 100% where Absorbance (cells + cpd) = absorbance of wells containing cells and test compound in vehicle (media + 0.5% DMSO); Absorbance (cpd) = absorbance of wells containing test compound in vehicle without cells (compound control); Absorbance (cells + vc) = absorbance of wells containing cells in vehicle (vc) only (untreated cells); Absorbance (vc) = absorbance of wells containing vehicle without cells (vehicle control).The percentage viability readings were plotted against log concentration on GraphPad Prism (Version 5.0, San Diego, CA) to give a sigmoidal curve from which IC50 (concentration required to reduce viability by 50% compared to control/untreated cells) was obtained. The plot was constrained to ≥ 0 and ≤ 100%. At least three separate determinations were carried out.

Determination of aqueous solubilities of compounds 6 and 7
Determination of aqueous solubility was carried out on Multiscreen Ò Solubility filter plates (Millipore-MSSLBPC10) from Millipore Corporation (MA, USA). The protocol (PC2445EN00, Millipore Corporation) was followed. Briefly, various concentrations of the test compound (stock solution in DMSO) were prepared in Universal buffer (pH 7.4)/acetonitrile (80:20). DMSO content was kept at 1% in final solutions. The UV absorbance of these solutions were read at pre-determined wavelengths and used to construct calibration curves for the test compounds. Next, aliquots of the stock solution of test compound (100 µM in Universal Buffer with 1% DMSO) were dispensed into wells in the Multiscreen Solubility filter plate, and agitated for 24 h at room temperature (25°C). The suspension was filtered, the filtrate diluted with acetonitrile to give the same solvent composition used to prepare the calibration solutions. The absorbance of the diluted filtrate was read at the predetermined wavelength and the concentration of the filtrate (equivalent to the solubility of the test compound) was determined from the calibration curve. The solubility determinations were carried out on at least 3 separate occasions using different stock solutions.