DMD#43505 Supplemental material Drug Metabolism and Disposition Identifying a Selective Substrate and Inhibitor Pair for the Evaluation of CYP2J2 Activity Caroline A. Lee, JP Jones III, Jonathan Katayama, Rüdiger Kaspera, Ying Jiang, Sascha Freiwald, Evan Smith, Greg Walker, and Rheem A. Totah Department of Medicinal Chemistry, University of Washington, 1959 Pacific Ave NE, Seattle, WA 98195. (JPJ, JK, RK, RAT) and Department of Drug Metabolism, Pfizer Global Research, La Jolla, CA. (CAL, YJ, SF, ES) and Groton, CT(GW) 1 DMD#43505 Tables Legend: Table1: List of drugs used for to screen for CYP2J2 inhibition. Figures Legend: Figure 1: 1H NMR of 4-hydroxy amiodarone compared to amiodarone Figure 2: 4-HydroxyAmiodarone 1H-1H COSY Figure 3: 4-HydroxyAmiodarone1H-13C multiplicity edited HSQC Figure 4: 1H NMR of 3-hydroxy amiodarone compared to amiodarone. Figure 5: 3-Hydroxy amiodarone 1H-1H COSY Figure 6: 3-HydroxyAmiodarone 1H-13C multiplicity edited HSQC Figure 7: Michaelis-Menten Kinetics for 4-OH amiodarone formation with recombinant CYP2J2 2 DMD#43505 Table 1: List of drugs tested for CYP2J2 inhibition Diphen- Hydrochloro- Acebutolol Captopril hydramine thiazide Miconazole Probenecid Diphenyl- Acetaminophen Carbamazepine hydantoin Ibuprofen Milrinone Procainamide Acetazolamide Cefazolin Disopyramide Imipramine Minocycline Progesterone AcycloguanosineChloroquine Dobutamine Indapamide Nabumetone Promazine Albendazole Chlorpheniramine Domperidone Indomethacin Nadolol Promethazine Amantadine Chlorpromazine Doxepin Isoniazid Naproxen Propranolol Amethopterin Chlorthalidone Droperidol Isradipine Nicardipine Quinapril Amiloride Chlorzoxazone Enalapril Ketoconazole Norfloxacin Quinidine Amiodarone Cimetidine Erythromycin Ketoprofen Nortriptyline Ramipril Amitriptyline Cisapride Etodolac Ketorolac Ofloxacin Ranolazine Amodiaquin Clomipramine Etoposide Labetalol Omeprazole Rifamycin Amoxicillin Clotrimazole Famotidine Lansoprazole Ondansetron Roxithromycin Amphotericin Clozapine Felbamate Levofloxacin OrphenadrineSotalol Antipyrine Colchicine Flecainide Meloxicam Paclitaxel Sparfloxacin Atenolol Cyclobenzaprine Fluconazole Mesoridazine Pentamidine Spironolactone Azathioprine CyclophosphamideFluoxetine Methazolamide PentoxifyllineSulindac Azithromycin Cyclosporin A Flurbiprofen Methimazole Perphenazine Tamoxifen Benazepril Danazol Fluvoxamine Methotrexate Phenacetin Terfenadine Bepridil Desipramine Furosemide MethoxypsoralenPimozide Testosterone 3 DMD#43505 Bisoprolol Dexamethasone Gemfibrozil MetoclopramidePiroxicam Thioridazine Budesonide Diclofenac Glipizide Metolazone Pravastatin Trazodone Bumetanide Dicloxacillin Griseofulvin Metoprolol Prazosin Vinblastine Bupropion Digoxin Haloperidol Mevinolin Proadifen Warfarin 4 DMD#43505 Figure 1: 1H NMR of 4-hydroxy amiodarone compared to amiodarone 4‐Hydroxy amiodarone 4 Butyl 1 Butyl 4 Butyl OH 2 Butyl 3 Butyl 8 7 6 5 4 3 2 1 ppm 4 Butyl CH3 4 butyl 3 butyl 2 butyl O 2' I 1 butyl O 6' O 2 DEAE Amiodarone 4 1 DEAE I 1 Butyl 2 Butyl N 4 DEAE 3 Butyl 7 5 H3C 6 CH3 5 DEAE 8 7 6 5 4 3 2 1 ppm 5 DMD#43505 Figure 2: 4-HydroxyAmiodarone 1H-1H COSY 3 Butyl 4 Butyl 1 Butyl 2 Butyl 4 Butyl OH ppm 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 6 DMD#43505 Figure 3: 4-HydroxyAmiodarone1H-13C multiplicity edited HSQC 4 Butyl OH ppm 10 20 2 Butyl 1 Butyl 3 Butyl 30 40 50 4 Butyl 60 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 7 DMD#43505 Figure 4: 1H NMR of 3-hydroxy amiodarone compared to amiodarone. 3‐HydroxyAmiodarone 4 Butyl 1 3 Butyl OH Butyl 3 Butyl 2 Butyl 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm CH3 4 butyl 3 butyl 4 Butyl 2 butyl O 2' I 1 butyl O 6' O 2 DEAE 4 I 1 DEAE N 4 DEAE 1 Butyl 7 5 H3C 2 Butyl 3 Butyl 6 CH3 5 DEAE Amiodarone 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 8 DMD#43505 Figure 5: 3-Hydroxy amiodarone 1H-1H COSY 1 Butyl 4 Butyl 3 Butyl 4 Butyl OH 2 Butyl ppm 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm 9 DMD#43505 Figure 6: 3-HydroxyAmiodarone 1H-13C multiplicity edited HSQC 4 Butyl OH ppm 1 Butyl weak 10 4 Butyl 20 30 2 Butyl 40 50 60 3 Butyl 70 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm 10 DMD#43505 Figure 7: Michaelis-Menten Kinetics for 4-OH amiodarone formation with recombinant CYP2J2 4 3 Vmax = 4.6 pmol/min/pmol CYP2J2 2 Km = 5.0 M 1 4-Hydroxyamiodarone (pmol/min/pmol CYP2J2) (pmol/min/pmol 0 0 10 20 30 40 50 60 Amiodarone (M) 11 .