General Methods Chemistry

Commercially available reagents and dry solvents were purchased from commercial sources and used without further purification. Anhydrous reactions were carried out using oven-dried glassware and under an argon atmosphere. Reaction progress was monitored through thin layer chromatography (TLC) using aluminium plates pre-coated with Silica Gel 60 F254 (E. Merck). TLC plates were observed under UV light at 254 nm and were then visualized by application of a solution of H₂SO₄ in EtOH (5% v/v) followed by heating. Flash chromatography was carried out using Silica Gel 60 (0.040–0.063 mm) and distilled solvents. ¹H and ¹³C NMR spectra were recorded at 400 and 100 MHz, respectively on a BrukerAvance 400 MHz spectrometer, while ¹⁹F NMR spectra were recorded at 376 MHz. Chemical shifts (δ) are reported in parts per million, relative to the internal reference residual solvent peak [CDCl₃: 7.26 (s) for ¹H, 77.0 (t) for ¹³C; D₂O: 4.79 (s) for ¹H]. 2D COSY and HSQC experiments were run when necessary to support assignments. Low-resolution mass spectra (LRMS) were recorded using positive mode, in electrospray ionization mode, on a BrukerDaltonics Esquire 3000 ESI spectrometer. High-resolution mass spectra (HRMS) were recorded for the final derivatives using Griffith University SmartWater Research Centre facility on Agilent 6530 QTOF, with an Agilent 1290 HPLC. Final deprotected derivatives were purified by reversed phase chromatography, using GracePure^TM SPE C18-Aq 5000 mg/20 mL and 2% acetonitrile/H₂O. The purities of all synthetic intermediates after chromatographic purification were > 90% as determined by ¹H and ¹³C NMR, while the purity of reference compounds synthesized for screening purposes 5 and 6, and of the new final products 7 and 8 was judged to be ≥ 95%. The synthesis of intermediate 9 (ref. ⁹) and reference inhibitors 5 and 6 (ref. ¹⁰) followed typical literature procedures. The details of synthetic methods used and full characterization data of key intermediates and novel final products are reported here in the supplementary material.