Vol. 30, Issue 1, 74-85, January 2002

CYP1A-Mediated Metabolism of the Janus Kinase-3 Inhibitor 4-(4'-Hydroxyphenyl)-amino-6,7-
dimethoxyquinazoline: Structural Basis for Inactivation by Regioselective O-Demethylation

Fatih M. Uckun, Jason Thoen, Hao Chen, Elise Sudbeck, Chen Mao, Ravi Malaviya, Xing-Ping Liu, and Chun-Lin Chen

Departments of Pharmaceutical Sciences (F.M.U., H.C., C.-L.C.), Chemistry (J.T., X.-P.L.), Structural Biology (E.S., C.M.), and Drug Discovery Program (F.M.U., R.M.), Parker Hughes Cancer Center, St. Paul, Minnesota

Here we report the phase I metabolism of the rationally designed Janus kinase-3 (JAK) inhibitor 4-(4'-hydroxyphenyl)-amino-6,7-dimethoxyquinazoline (WHI-P131; JANEX-1). JANEX-1 was metabolized by the cytochrome P450 enzymes CYP1A1 and CYP1A2 in a regioselective fashion to form the biologically inactive 7-O-demethylation product 4-(4'-hydroxyphenyl)-amino-6-methoxy-7-hydroxyquinazoline (JANEX-1-M). Our molecular modeling studies indicated that the CYP1A family enzymes bind and demethylate JANEX-1 at the C-7 position of the quinazoline ring since the alternative binding conformation with demethylation at the C-6 position would result in a severe steric clash with the binding site residues. The metabolism of JANEX-1 to JANEX-1-M in pooled human liver microsomes followed Michaelis-Menten kinetics with V_max and K_m values (mean ± S.D.) of 34.6 ± 9.8 pmol/min/mg and 107.3 ± 66.3 µM, respectively. -Naphthoflavone and furafylline, which both inhibit CYP1A2, significantly inhibited the formation of JANEX-1-M in human liver microsomes. There was a direct correlation between CYP1A activities and the magnitude of JANEX-1-M formation in the liver microsomes from different animal species. A significantly increased metabolic rate for JANEX-1 was observed in Aroclor 1254-, -naphthoflavone-, and 3-methylcholanthrene-induced microsomes but not in clofibrate-, dexamethasone-, isoniazid-, and phenobarbital-induced microsomes. The formation of JANEX-1-M in the presence of baculovirus-expressed CYP1A1 and 1A2 was consistent with Michaelis-Menten kinetics. The systemic clearance of JANEX-1-M was much faster than that of JANEX-1 (5525.1 ± 1926.2 ml/h/kg versus 1458.0 ± 258.6 ml/h/kg). Consequently, the area under the curve value for JANEX-1-M was much smaller than that for JANEX-1 (27.5 ± 8.0 versus 94.8 ± 18.4 µM · h; P < 0.001).