Lcms 2020

All reactions were performed in oven-dried round-bottom flasks with magnetic stir bars or overhead mechanical stirrers under an argon atmosphere unless the reaction conditions were supposed to contain water. Organic solvents were purified when necessary by standard methods⁵⁵ or purchased from Sigma-Aldrich.™ Chemicals were purchased from either Sigma Aldrich™, Oakwood Chemical, Alfa Aesar, Matrix Scientific, or Acros Organic. The progress of the reactions was monitored by TLC on a silica gel plate (25% EtOAc in hexanes or 10% MeOH in DCM). The ¹H and ¹³C NMR data were obtained on Bruker Spectrospin 300 MHz and GE 500 MHz instruments with the chemical shifts in δ (ppm) reported relative to TMS. The HRMS spectral data was obtained on a LCMS-IT-TOF by Shimadzu Scientific. Purity of all final compounds was 98% or higher and was determined by HPLC on a LC-MS with Shimadzu LCMS 2020, (Shimadzu Scientific Instruments, Columbia, MD) using a PDA detector at 254 nm. The column was a Shimadzu C18 3 μm 50 × 4.6mm reversed phase LC column. LC mobile phase: 90% acetonitrile (w. 0.1% TFA) and 10% H₂O (w/ 0.1% TFA) with a flow rate of 1 mL min⁻¹, column temperature: 25 °C, injection size: 1.0μL.

Metabolite analysis by LCMS confirmed OCH₃ rate > OCD₃ rate and that the primary metabolites for the PQs was generated via O-demethylation of the methoxy groups to form hydroxypyrazoloquinolinones. The 60 min samples from the metabolism study above were injected on a LC-MS with Shimadzu LCMS 2020, (Shimadzu Scientific Instruments, Columbia, MD) using a SIM mode (Dual Ion Source (DUIS) ionization, simultaneous ESI and APCI) method calibrated to the respective retention times and mass units of each component and the respective peak intensities were measured. Compound peaks were resolved using a Shimadzu C18 3 μm 50 × 4.6mm reversed phase LC column. LC mobile phase: 90% acetonitrile (w. 0.1% TFA) and 10% H₂O (w/ 0.1% TFA). Results shown in Scheme 4.