Phlms

2-Oxo-PCcP hydrochloride, 2-oxo-PCE hydrochloride, 2-oxo-PCiP hydrochloride, 2-oxo-PCMe hydrochloride, and 2-oxo-PCPr were synthesized by the standard methods and analytically characterized by ¹H and ¹³C nuclear magnetic resonance (NMR), high-performance liquid chromatography (HPLC) (>95% pure on HPLC and NMR), and high-resolution mass spectrometry (HRMS.) Acetonitrile (ACN), ammonium formate, dichloromethane (DCM), ethyl acetate, hydrochloric acid (HCl), isopropanol, methanol (MeOH), sodium hydroxide solution (NaOH), and all other chemicals were obtained from VWR International GmbH (Darmstadt, Germany). Diazepam-d5 was obtained from LGC Standards GmbH (Wesel, Germany). NADP⁺ was obtained from Biomol (Hamburg, Germany), superoxide dismutase, magnesium chloride (MgCl₂), isocitrate, and isocitrate dehydrogenase were from Merck (Darmstadt, Germany). pHLMs (20 mg microsomal protein/mL, 330 pmol total CYP/mg protein) were obtained from Corning (Amsterdam, the Netherlands). All enzyme-containing preparations were thawed at 37 °C after delivery, aliquoted, snap frozen in liquid nitrogen and stored at −80 °C until use. Water was purified with a Millipore (Merck, Darmstadt, Germany) filtration unit, which purifies water to a resistance of 18.2 Ω × cm. Isolute HCX solid-phase extraction (SPE) cartridges (130 mg, 3 mL) were obtained from Biotage (Uppsala, Sweden).

PCYP hydrochloride was provided by the State Bureau of Criminal Investigation Schleswig-Holstein (E.U. project ADEBAR plus, Kiel, Germany) for research purposes. The chemical purity of >93% and the identity of the compound was verified by MS and nuclear magnetic resonance analysis. Ammonium formate, ammonium acetate, creatinine-d₃, dipotassium phosphate, formic acid, D-glucose-1,2,3,4,5,6,6-d₇, isocitrate dehydrogenase, isocitrate, magnesium chloride, palmitic acid-d₃₁, superoxide dismutase, and tripotassium phosphate were obtained from Merck (Darmstadt, Germany). Acetonitrile, ethanol, methanol (all LC-MS grade), and NADP-Na₂ were from VWR (Darmstadt, Germany). L-Tryptophan-d₅ was obtained from Alsachim (Illkirch-Graffenstaden, France). 1-Palmitoyl-d₉-2-palmitoyl-sn-glycero-3-PC and prostaglandin-E₃-d₉ were from Cayman Chemical (Ann Arbor, MI, USA). Water was purified with a millipore filtration unit (18.2 W × cm water resistance). pHLMs (20 mg microsomal protein × mL⁻¹, 360 pmol total CYP/mg, 26 donors) were obtained from Corning (Amsterdam, The Netherlands). After delivery, pHLMs were thawed at 37 °C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 °C until use.