A Highly Potent Class of Halogenated Phenazine Antibacterial and Biofilm-Eradicating Agents Accessed Through a Modular Wohl-Aue Synthesis

Unlike individual, free-floating planktonic bacteria, biofilms are surface-attached communities of slow- or non-replicating bacteria encased within a protective extracellular polymeric matrix enabling persistent bacterial populations to tolerate high concentrations of antimicrobials. Our current antibacterial arsenal is composed of growth-inhibiting agents that target rapidly-dividing planktonic bacteria but not metabolically dormant biofilm cells. We report the first modular synthesis of a library of 20 halogenated phenazines (HP), utilizing the Wohl-Aue reaction, that targets both planktonic and biofilm cells. New HPs, including 6-substituted analogues, demonstrate potent antibacterial activities against MRSA, MRSE and VRE (MIC = 0.003–0.78 µM). HPs bind metal(II) cations and demonstrate interesting activity profiles when co-treated in a panel of metal(II) cations in MIC assays. HP 1 inhibited RNA and protein biosynthesis while not inhibiting DNA biosynthesis using 3H-radiolabeled precursors in macromolecular synthesis inhibition assays against MRSA. New HPs reported here demonstrate potent eradication activities (MBEC = 0.59–9.38 µM) against MRSA, MRSE and VRE biofilms while showing minimal red blood cell lysis or cytotoxicity against HeLa cells. PEG-carbonate HPs 24 and 25 were found to have potent antibacterial activities with significantly improved water solubility. HP small molecules could have a dramatic impact on persistent, biofilm-associated bacterial infection treatments.

General procedure for demethylation of 1-methoxyphenazines: To a round bottom flask, 39 (376 mg, 1.68 mmol) was dissolved in anhydrous dichloromethane (50 mL) and cooled to -78 °C before dropwise addition of 1M boron tribromide solution in dichloromethane (10.0 mL, 10.0 mmol). The reaction was left to stir at -78 °C for 1 hour, and allowed to reach ambient temperature overnight. The reaction was then heated to reflux for 8 hours until complete (monitored by TLC). Upon completion of the reaction, brine (50 mL) was added to quench the reaction. The contents of the resulting biphasic mixture were then transferred to a separatory funnel and dichloromethane was used to extract the product. The resulting organic layers were dried with sodium sulfate, filtered through cotton, and removed in vacuo. The resulting solid was purified via column chromatography using dichloromethane to elute compound 60 as a yellow solid (100%, 350 mg). Note: Some S8 1-hydroxyphenazines were purified with the addition of 1% acetic acid to 99% dichloromethane via column chromatography.   General procedure for bromination of 1-hydroxyphenazines: 60 (156 mg, 0.742 mmol) and Nbromosuccinimide (277 mg, 1.56 mmol) were dissolved in dichloromethane (60.0 mL) and allowed to stir at room temperature for 4 hours. The reaction contents were washed with brine (60.0 mL) and extracted with dichloromethane. The extracts were dried with sodium sulfate, filtered, and concentrated in vacuo. The resulting solid was purified via column chromatography using 99:1 dichloromethane:acetic acid to elute 17 as a yellow solid. Note: When R = Me, 1.0 equivalent of N-bromosuccinimide was used for bromination.   Procedure for the synthesis of 58: To a round bottom flask was added 57 (160 mg, 0.40 mmol) dissolved in anhydrous dichloromethane (20 mL). The mixture was brought to -78 °C in a dry ice bath before dropwise addition of 1 M boron tribromide solution in dichloromethane (4.02 mL, 4.02 mmol). The reaction was left to stir at -78 °C for 1 hour, and then allowed to reach ambient temperature for reaction overnight. The reaction was heated to reflux for 8 hours until complete (monitored by TLC). Brine (20 mL) was then added to the mixture to quench the reaction. The mixture was then transferred to a separatory funnel, and then extracted with dichloromethane. Organic extracts were dried with sodium sulfate, filtered through cotton, and removed in vacuo. The resulting crude product was purified via column chromatography using dichloromethane to elute 58 as a red solid (100%, 149 mg). Procedure for the synthesis of 15: 58 (30.0 mg, 0.08 mmol) and N-bromosuccinimide (43.7 mg, 0.18 mmol) were suspended in 1 mL toluene and allowed to stir at room temperature for 1 hour. The reaction contents were filtered. The filtrate was washed with dichloromethane (6 mL), resulting product 15 as a dark red solid (60%, 26 mg).
General Procedure for the Synthesis of HP-Carbonate (24 and 25) Tetraethyleneglycol monomethyl ether (69 µL 0.33 mmol) was placed in an oven-dried round-bottomed flask and dissolved in anhydrous dichloromethane (1 mL). The solution was then cooled to 0 °C. Pyridine (37 µL, 0.47 mmol) and triethylamine (11 µL 0.73 mmol) was then added via syringe, followed by triphosgene (48.2 mg, 0.16 mmol) dissolved in dichloromethane (1mL). The resulting mixture was stirred from 0 °C to room temperature and continued to stir at room temperature for 5 hours. After that, then the reaction was cooled to 0 °C before the addition of solution of 17 (86 mg, 0.23 mmol) and triethylamine (49 µL 0.35 mmol) in anhydrous dichloromethane was added to the reaction in dropwise. The reaction solution was stirred for 5 min at 0 °C and then reach ambient temperature and stirred at room temperature overnight. After the reaction was complete, the reaction mixture was poured into a separatory funnel containing 1 M ammonium chloride (20 mL), and the biphasic mixture was shaken vigorously. Upon separation of layers, the aqueous layer was re-extracted with dichloromethane (2 × 30 mL). Organic extracts were collected, dried over Sodium Sulfate, filtered, and concentrated under vacuum. The resulting crude material was purified using flash column chromatography with 3:1 hexanes:ethyl acetate to 100% ethyl acetate as eluent yield 25 as a yellow oil (135 mg, 96%).  The minimum inhibitory concentration (MIC) for each test compound was determined by the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute (CLSI). In a 96-well plate, eleven two-fold serial dilutions of each compound were made in a final volume of 100 μL Luria Broth. Each well was inoculated with ~10 5 bacterial cells at the initial time of incubation, prepared from a fresh log phase culture (OD600 of 0.5 to 1.0 depending on bacterial strain). The MIC was defined as the lowest concentration of compound that prevented bacterial growth after incubating 16 to 18 hours at 37 °C (MIC values were supported by spectrophotometric readings at OD600). The concentration range tested for each test compound during this study was 0.10 to 100 μM. DMSO served as our vehicle and negative control in each microdilution MIC assay. DMSO was serially diluted with a top concentration of 1% v/v. All compounds were tested in a minimum of three independent experiments.

B.) MIC Assay with Metal(II) Cation Co-Treatment:
Metal(II) cation studies were performed in a similar setup to the standard MIC assay, with the addition of 200 µM of the metal(II) cation (i.e., copper(II) sulfate) to the media. 3 All data were obtained from three independent experiments.

C.) MIC Assay for Mycobacterium tuberculosis:
M. tuberculosis H37Ra (ATCC 25177) was inoculated in 10 mL Middlebrook 7H9 medium and allowed to grow for two weeks. The culture was then diluted with fresh medium until an OD600 of 0.01 was reached. Aliquots of 200 µL were then added to each well of a 96-well plate starting from the second column. Test compounds were dissolved in DMSO at final concentration of 10 mM. 7.5 µL of each compound along with DMSO (negative control) and streptomycin (positive control-40mg/ml stock solution) were added to 1.5 mL of the Mycobacterium diluted cultures, resulting in 50 µM final concentration of each halogenated phenazine analogues and 340 µM for streptomycin. The final DMSO concentration was maintained at 0.5%. Aliquots of 400 µl were added to wells of the first column of the 96-well plate and serially diluted two-fold (200 µl) per well across the plate to obtain final concentrations that ranges from 0.024 to 50 µM for the test compounds and 0.16 to 340 µM for streptomycin. Three rows were reserved for each compound. The plates were then incubated at 37C for seven days. Minimum inhibitory concentrations are reported as the lowest concentration at which no bacterial growth was observed. OD600 absorbance was recorded using SpectraMax M5 (Molecular Devices). Data obtained from three independent experiments were analyzed using Excel.

Minimum Bactericidal Concentrations (MBC) and Minimum Biofilm Eradication Concentrations (MBEC) Determination
Biofilm eradication experiments were performed using the Calgary Biofilm Device to determine MBC/MBEC values for various compounds of interest (Innovotech, product code: 19111). The Calgary device (96-well plate with lid containing pegs to establish biofilms on) was inoculated with 125 µL of a mid-log phase culture diluted 1,000-fold in tryptic soy broth with 0.5% glucose (TSBG) to establish bacterial biofilms after incubation at 37 °C for 24 hours. The lid of the Calgary device was then removed, washed and transferred to another 96-well plate containing 2-fold serial dilutions of the test compounds (the "challenge plate"). The total volume of media with compound in each well in the challenge plate is 150 µL. The Calgary device was then incubated at 37 °C for 24 hours. The lid was then removed from the challenge plate and MBC/MBEC values were determined using different experimental pathways. To determine MBC values, 20 µL of the challenge plate was transferred into a fresh 96-well plate containing 180 µL TSBG and incubated overnight at 37 °C. The MBC values were determined as the concentration giving a lack of visible bacterial growth (i.e., turbidity). For determination of MBEC values, the Calgary device lid (with attached pegs/treated biofilms) was transferred to a new 96-well plate containing 150 µL of fresh TSBG media in each well and incubated for 24 hours at 37 °C to allow viable biofilms to grow and disperse resulting in turbidity after the incubation period. MBEC values were determined as the lowest test concentration that resulted in eradicated biofilm (i.e., wells that had no turbidity after final incubation period). All data were obtained from a minimum of three independent experiments.
Pulse experiments followed a normal CBD assay protocol; however, the compound treatment phase (the "challenge plate") consisted of two sequential 24 hour compound treatment plates before the final recovery plate. Following this, CBD pegs were removed from the lid, sonicated for 30 minutes in PBS and plated out to determine biofilm cell killing in colony forming units per milliliter (CFU/mL).
Work flow for the determination of MBC and MBEC values using the Calgary Biofilm Device.

E.) Live / Dead staining (Fluorescence Microscopy) of MRSA BAA-1707 Biofilms:
A mid-log culture of MRSA BAA-1707 was diluted 1:1,000-fold and 500 µL was transferred to each compartment of a 4 compartment CELLview dish (Greiner Bio-One 627871). The dish was then incubated for 24 hours at 37 °C. After this time, the cultures were removed and the plate was washed with 0.9% saline. The dish was then treated with the compounds in fresh media at various concentrations. DMSO was used as our negative control in this assay. The dish was incubated with the compound for 24 hours at 37 °C. After this time, S21 the cultures were removed and the dish was washed with 0.9% saline for 2 minutes. Saline was then removed and 500 µL of the stain (Live/Dead BacLight Viability Kit, Invitrogen) were added for 15 minutes and left in the dark. After this time, the stain was removed and the dish was washed twice with 0.9% saline. Then the dish was fixed with 500 µL 4% paraformaldehyde in PBS for 30 minutes. Images of remaining MRSA biofilms were then taken with a fluorescence microscope. All data were analyzed using Image J software from three independent experiments.

F.) Hemolysis Assay with Red Blood Cells:
As previously described, freshly drawn human red blood cells (hRBC with ethylenediaminetetraacetic acid (EDTA) as an anticoagulant) were washed with Tris-buffered saline (0.01M Tris-base, 0.155 M sodium chloride (NaCl), pH 7.2) and centrifuged for 5 minutes at 3,500 rpm. The washing was repeated three times with the buffer. In 96-well plate, test compounds were added to the buffer from DMSO stocks. Then 2% hRBCs (50 µL) in buffer were added to test compounds to give a final concentration of 200 µM. The plate was then incubated for 1 hour at 37 °C. After incubation, the plate was centrifuged for 5 minutes at 3,500 rpm. Then 80 µL of the supernatant was transferred to another 96-well plate and the optical density (OD) was read at 405 nm. DMSO served as our negative control (0% hemolysis) while Triton X served as our positive control (100% hemolysis). The percent of hemolysis was calculated as (OD405 of the compound-OD405 DMSO) / (OD405 Triton X-OD405 buffer) from three independent experiments.

G.) LDH Release Assay for HeLa Cytotoxicity Assessment:
HeLa cytotoxicity was assessed using the LDH release assay described by CytoTox96 (Promega G1780). HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM; Gibco) supplemented with 10% Fetal Bovine Serum (FBS) at 37°C with 5% CO2. When the HeLa cultures exhibited 70-80% confluence, halogenated phenazines were then diluted by DMEM (10% FBS) at concentrations of 25, 50 and 100 µM and added to HeLa cells. Triton X-100 (at 2% v/v) was used as the positive control for maximum lactate dehydrogenate (LDH) activity in this assay (i.e., complete cell death) while "medium only" lanes served as negative control lanes (i.e., no cell death). DMSO was used as our vehicle control. HeLa cells were treated with compounds for 24 hours and then 50 µL of the supernatant was transferred into a fresh 96-well plate where 50 µL of the reaction mixture was added to the 96-well plate and incubated at room temperature for 30 minutes. Finally, Stop Solution (50 µL) was added to the incubating plates and the absorbance was measured at 490 nm. Results are on the next page and are from three independent experiments.

H.) Macromolecular Synthesis Inhibition Assay:
Macromolecular syntheses experiments were carried out in methicillin-resistant Staphylococcus aureus BAA-1707. An overnight culture (100 µL) of S. aureus BAA-1707 was sub-cultured into 10 mL of fresh TSBG media which was allowed to grow to exponential phase (OD600 = 0.2-0.3) before transferring 500 µL to each well in a 24 well-plate. The test compounds and vehicle control (DMSO) were added to achieve the desired concentrations relative to their MIC values against S. aureus BAA-1707. Treated cultures were then incubated at 37°C for 30 minutes before radioactive precursors for DNA ([ 3 H] thymidine (0.5 µCi)), RNA ([ 3 H] uridine (0.5 µCi)) and protein ([ 3 H] leucine (1 µCi)) were added. Antibiotics with known modes of action were used as positive controls in these experiments, these included: ciprofloxacin (DNA inhibition), rifampicin (RNA inhibition) and linezolid (protein inhibition). DMSO served as our negative control. DNA and RNA radiolabeled cultures were then incubated in 37°C for 15 minutes before being stopped by adding 60 µL of cold 5% trichloroacetic acid (TCA). The protein synthesis experiment was stopped after 40 minutes by adding 60 µL cold TCA. These mixtures were then incubated at 2°C for at least 30 minutes before the contents of the plates were transferred onto glass microfiber filters (24 mm) and washed 5 times with 1 mL of 5% TCA. The filters