Oxidative ipso Substitution of 2,4-Difluoro-benzylphthalazines: Identification of a Rare Stable Quinone Methide and Subsequent GSH Conjugate

ABSTRACT: In vitro metabolite identification and GSH trapping studies in human liver microsomes were conducted to understand the bioactivation potential of compound 1 [2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol], an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a GSH adduct (M2). The stability of this metabolite arises from extensive resonance-stabilized conjugation of the substituted benzylphthalazine moiety. Cytochrome P450 (P450) phenotyping studies revealed that the formation of M1 and M2 were NADPH-dependent and primarily catalyzed by CYP3A4 among the studied P450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified, and a mechanism was proposed for its formation via an oxidative ipso substitution

Oxidative ipso substitution of 2,4-difluoro-benzylphthalazines -Identification of a rare stable quinone methide and subsequent glutathione conjugate DMD #46268 2 Running Title: Oxidative ipso substitution2,4-difluoro-benzylphthalazines

Abstract In vitro metabolite identification and glutathione trapping studies in human liver microsomes were conducted in order to understand the bioactivation potential of compound 1, 2-(6-(4-(4-(2,4-difluorobenzyl)phthalazin-1-yl)piperazin-1-yl)pyridin-3-yl)propan-2-ol), an inhibitor of the Hedgehog pathway. The results revealed the formation of a unique, stable quinone methide metabolite (M1) via ipso substitution of a fluorine atom and subsequent formation of a glutathione adduct (M2). The stability of this metabolite arises from extensive resonance stabilized conjugation of the substituted benzylphthalazine moiety. CYP450 phenotyping studies revealed that the formation of M1 and M2 were NADPH dependent and primarily catalyzed by CYP3A4 among the studied CYP450 isoforms. In summary, an unusual and stable quinone methide metabolite of compound 1 was identified and a mechanism was proposed for its formation via an oxidative ipso substitution. DMD #46268