EFFECT OF IRON-NITROGEN COORDINATION (TYPE II BINDING) ON DRUG METABOLISM BY CYTOCHROME P450: STUDIES TO UNDERSTAND THE KINETICS, METABOLIC STABILITY AND REGIOSELECTIVITY

Thesis (Ph.D.), Department of Chemistry, Washington State UniversityCytochrome P450 (CYP) enzymes metabolize more than 80% of clinically used drugs. Given a large role of CYP enzymes in drug metabolism, one of the goals in pharmaceutical industry is to understand metabolic rate and regioselectivity of a drug candidate in the early phase of drug discovery. A common practice in pharmaceutical industry to increase metabolic stability of a drug is to replace a carbon in an aromatic ring by a nitrogen so that the inserted nitrogen can coordinate with heme iron to form type II binding. This minor change in structure of a drug is believed to increase metabolic stability without altering the geometry and size of the compound. It has also been reported that type II binding compounds get metabolized significantly. But kinetics of metabolism of type II binding compounds is unknown. This work is focused to understand the metabolism, metabolic stability and regioselectivity of type II binding compounds. To explore the metabolism of type II binding compounds, we used saturation kinetics, surface plasmon resonance and electrochemical studies. Three possible mechanisms were described and analyzed to identify the kinetic of metabolism. Furthermore, to determine the effect of the type II binding on the metabolic stability of the drugs we developed a small library of the quinoline carboxamide compounds to systematically compare the metabolic stability and regioselectivity between structurally similar type I and type II binding compounds. The library contained same core structure but number and position of the nitrogen ware varied. On the basis of kinetic parameters variation in metabolic stability and regioselectivity was explained for the two binding modes.To measure the metabolic rate one should quantify the metabolites from a drug. A common practice for quantification is to use liquid chromatography mass spectrometry. Given the difficulty in obtaining pure metabolites, usually signals from the metabolite and the substrate is considered equivalent. Herein we describe the error associated with this assumption by comparing the signals from twenty substrate/metabolite pairs using both conventional electrospray ionization and captive flow ionization in LCMS.Department of Chemistry, Washington State Universit