Development of benzo[1,4]oxazines as biofilm inhibitors and dispersal agents against Vibrio cholerae † †Electronic supplementary information (ESI) available: Experimental procedures and analytical data along with protocols for all biological experiments. See DOI: 10.1039/c4cc07003h Click here for additional data file.

Synthesis of a library of natural product-inspired biofilm inhibitors has revealed a compound with selective and potent anti-biofilm activity against V. cholerae.

BIC50 curves for active compounds S6 -S7 EC50 curves of the antibiotics used in the co-dosing experiments S8 -S10 Flow cell experiments of oxazine 25 S11 BioMAP antibacterial profiling of oxazine 25 S12 CFU analysis of oxazine 25 S13 HeLa cell line toxicity study of oxazine 25 S14 Stability study of oxazine 25 in culture media S15 Experimental procedures S16 -S32 1 H and 13 C NMR spectra S33 -S73

References S74
Electronic Supplementary Material (ESI) for ChemComm. This journal is © The Royal Society of Chemistry 2014

General remarks
All reactions were performed in an open flask using acetone washed, oven dried glassware with magnetic stirring and if required heated through the use of Dry Syn TM blocks. All reagents used were acquired from chemical supply companies or, as indicated in the individual experimental details, prepared within the laboratory. Reactions that were performed at 0 °C were done so using water/ice baths. All solvents used in the course of the project were obtained from the departmental Grubbs solvent system.
Analytical thin layer chromatography (TLC) was carried out utilizing aluminium backed Merck TLC plates (silica gel 60 F254) and visualized with UV light (254 nm) or basic KMnO4 solution. Flash column chromatography was performed using Alfa Aesar, silica gel 60, 0.032 -0.063 mm (230 -450 mesh) as the stationary phase. Columns were typically packed as a slurry, with the eluent used for a particular purification noted within the individual experimental details for each reaction.
All 1 H and 13 C NMR spectra were obtained on either a Varian Unity 500+ or a Varian Inova 600 MHz spectrometer equipped with a 5 mm HCN triple resonance cryoprobe. Chemical shifts are expressed in parts per million (ppm) downfield from tetramethylsilane (TMS). All coupling constants given are in Hz. High resolution mass spectrometry was performed on an Agilent 6230 electrospray ionization (ESI) accurate-mass time-of-flight (TOF) liquid chromatograph/ mass spectrometer.
All procedures for determination of the biofilm inhibitory concentration (BIC50) occurred as previously described. 1 See individual experimental details for antibiotic and co-dosing procedure. All BIC50 and BDC50 reported are the result of three biological replicates each consisting of two technical replicates. The preformed biofilm screen followed the general experimental procedure developed in P. aeruginosa. 1 For the V.
cholerae biofilm dispersal assay (BDC50), compound, antibiotic or DMSO control were pinned into the screening plate following two hours of incubation and incubated for a further 4 hours at 32°C. An identical washing and analytical procedure to that reported in the literature was performed. 1 For the antibiotic co-dosing experiments, both oxazine 25 and the relevant antibiotic were added after 2 hours of incubation and OD600 readings immediately taken to determine initial OD600 values. After incubation, OD600 readings were acquired, and the change in OD600 values used as a measure of cell growth for each well. Immediately following OD600 readings, the plates were washed, PBS buffer added, and each well imaged using our standard protocol to determine biofilm coverage.

Data interpretation
In both the biofilm inhibition and dispersal assays, four outcomes are possible for any assay well. In the case of strong antibiotic activity, both planktonic and attached cells are eliminated, and the resulting screening images are blank, with low OD600 readings (Image A). For compounds capable of eradicating only the planktonic cells without impacting attached cells a lower OD600 reading would be expected, but with retention of large biofilm colonies in the image (Image B). If the compound has no effect on planktonic or biofilm-associated cells, then both the OD600 and biofilm coverage are high (Image C). Finally if the compound is capable of only inducing detachment of the bacteria with no bactericidal effects then

Flow cell experiments of oxazine 25
Overnight culture of rugose wild type V. cholerae (A1552 harboring a Tn7GFP insertion) was diluted 200-fold into 2% LB medium containing either the indicated concentration of test compound or an equal volume of DMSO as a vehicle control and inoculated into an Ibidi µ 0.4 6-well flow cell. After 1 hour of static incubation at room temperature, flow of 2% LB containing test compound or DMSO was initiated at 7.5 mL/minute at room temperature for 6 hours. Flow cells were imaged on a Zeiss LSM5 confocal microscope. Z-projections of Z-stacks were created with the FIJI build of Image J.
Quantitative analysis of images was performed with COMSTAT. 2 BioMAP antibacterial profiling of oxazine 25 The antibacterial profiling of oxazine 25 followed that previously reported in the literature. 3  Samples were harvested at specific time points and plated to enumerate CFU/ml. A negative control of doxycycline at 10 µM was also utilized. In all instances growth of V. cholerae in the presence of the oxazine 25 was comparable to that of the DMSO control vehicle. It should be noted that at the 24 hour time point a depreciation in CFU is observed. This is typical for such experiments.

HeLa cell line toxicity study of oxazine 25
Cytological profiling was performed as previously described. 4 Plates were imaged using an ImageXpress Micro epifluorescent microscope (Molecular Devices, LLC) with a 10× Nikon objective lens. Images were analysed using MetaXpress (Molecular Devices, LLC). In all instances up to 200 µM, oxazine 25 exerted no toxicity toward HeLa cells, with comparable cellular counts compared to the DMSO control vehicle (see below). White bar indicates a distance of 100 µm.

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Stability study of oxazine 25 in culture media Oxazine 25 (5 mg, 0.02 mmol) was dissolved in DMSO (100 µl) and added in a single portion to the appropriate culture media and, if appropriate, heated to 37 °C. In all instances agitation of the mixture was obtained by mechanical stirring.
Following overnight incubation, the solution was diluted with methanol (5 mL) and subjected to reverse-phase HPLC using a Phenomenex synergi-A 10µ fusion C18 column. An isocratic gradient of 6:4 Methanol/H2O (acidified with 0.02% of formic acid) was used as the solvent system. A wavelength of λ = 254 nm was used in all instances. The oxazine 25 was identified to have a retention time of 7.0 minutes.
The nitrophenol 5 (0.2 mmol) was added in a single portion to a suspension of platinum(IV) oxide (10% wt) in ethanol (5 cm³). The system was evacuated and backfilled with hydrogen gas five times. Following completion of the final cycle, the mixture was stirred for 4 hours. The suspension was filtered and the solvent removed in vacuo to afford the title compound.

General Procedure B for the sulphuric acid catalysed addition of an alcohol to the oxazine 1
The alcohol (1 mmol) was added to a solution of oxazine 1 (62 mg, 0.3 mmol) and sulphuric acid (3 drops) in THF (1 cm³) at 0 ºC. The resulting solution was stirred for 12 hours and the volatiles removed in vacuo to afford the crude product.
Purification of the crude material occurred as described in the individual experimental details.

General procedure C for the formation of α-ketoamides 37 -41 from the aniline 6
The α-ketoacid chloride (2 mmol) was added in a single portion to a solution of aniline (1 mmol) and pyridine (237 mg, 0.3 cm³, 3 mmol) in DCM (10 cm³) at 0 ºC. The resulting mixture was stirred for 1 hour before being quenched through addition of a saturated aqueous solution of NaHCO3 (10 cm³). The organic layer was separated and the aqueous phase extracted with dichloromethane (3 × 10 cm³). The organic layers were combined, washed with brine (10 cm³) and dried over magnesium sulfate. Removal of the solvent in vacuo afforded the crude α-ketoamide. Purification of the crude material occurred as described in the individual experimental details.
nOe spectra of oxazine 13 Key nOe interactions of irradiated proton (δH = 2.91, highlighted in blue) with protons highlighted in red.