title: A mass spectrometry study of tirapazamine and its metabolites. insights into the mechanism of metabolic transformations and the characterization of reaction intermediates.
abstract: Tandem mass spectrometry methods were used to study the sites of protonation and for identification of 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine), and its metabolites (3-amino-1,2,4-benzotriazine 1-oxide (3), 3-amino-1,2,4-benzotriazine 4-oxide (4), 3-amino-1,2,4-benzotriazine (5), and a related isomer 3-amino-1,2,4-benzotriazine 2-oxide (6). Fragmentation pathways of 3 and 5 indicated the 4-N-atom as the most likely site of protonation. Among the N-oxides studied, the 4-oxide (4) showed the highest degree of protonation at the oxygen atom. The differences in collision-induced dissociation of isomeric protonated 1-, 2- and 4-oxides allowed for their identification by LC/MS/MS. Gas phase and liquid phase protonation of tirapazamine occurred exclusively at the oxygen in the 4-position. A loss of OH radical from these ions (2(+)) resulted in ionized 3. Neutralization-reionization mass spectrometry (NR MS) experiments demonstrated the stability of the neutral analogue of protonated tirapazamine in the gas phase in the micro s time-frame. A significant portion of the neutral tirapazamine radicals (2) dissociated by loss of hydroxyl radical during the NR MS event, which indicates that previously proposed mechanisms for redox-activated DNA damage are reasonable. The activation energy for loss of hydroxyl radical from activated tirapazamine (2) was estimated to be approximately 14 kcal mol(-1). Stable neutral analogues of [3 + H](+) and [5 + H](+) ions were also generated in the course of NR MS experiments. Structures of these radicals were assigned to the molecules having an extra hydrogen atom at one of the ring N-atoms. Quantum chemical calculations of protonated 1, 3, 4 and 5 and the corresponding neutrals were performed to assist in the interpretation of experimental results and to help identify their structures.
authors.affiliation: Department of Chemistry, Rensselaer Polytechnic Institute, Troy, New York, USA. zagord@rpi.edu
authors.forename: Dmitri
authors.initials: D
authors.lastname: Zagorevskii
authors.forename: Minghu
authors.initials: M
authors.lastname: Song
authors.forename: Curt
authors.initials: C
authors.lastname: Breneman
authors.forename: Yang
authors.initials: Y
authors.lastname: Yuan
authors.forename: Tarra
authors.initials: T
authors.lastname: Fuchs
authors.forename: Kent S
authors.initials: KS
authors.lastname: Gates
authors.forename: C Michael
authors.initials: CM
authors.lastname: Greenlief
mesh_terms: D000970:Antineoplastic Agents / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D005609:Free Radicals / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D000077704:Tirapazamine; D014227:Triazines / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D005609:Free Radicals; D011522:Protons; D014227:Triazines; D000077704:Tirapazamine