Mechanistic Studies on a P450-Mediated Rearrangement of BMS-690514: Conversion of a Pyrrolotriazine to a Hydroxypyridotriazine

Abstract

BMS-690514 ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4] triazin-5-yl)methyl)-3-piperidinol) is an oral oncologic agent being developed for the treatment of patients with advanced nonsmall cell lung cancer and breast cancer. The compound is metabolized via multiple metabolic pathways, including P450-mediated oxidation at one of the carbons of its pyrrolotriazine group. Oxidation at this site results in the formation of two metabolites, M1 and M37. Mass spectrometric and NMR analysis revealed that M1 underwent an unusual structural change, where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group. In contrast, the structure of the pyrrolotriazine moiety remained intact in M37. In vitro experiments with liver microsomes and deuterated or tritiated BMS-690514 containing the isotopic label on the carbon that underwent oxidation indicated that during the formation of M1, the isotope label was retained at the site of hydroxylation, while the label was lost during the formation of M37. On the basis of these results, a mechanism for the formation of M1 was proposed as follows: BMS-690514 was first oxidized by P450 enzymes either via epoxidation or an iron-oxo addition pathway to form a zwitterionic intermediate. This was followed by opening of the pyrrolotriazine ring to form an aldehyde intermediate, which could be partially trapped with methoxyamine. The aldehyde intermediate then reacted with the secondary amine of the methoxyaniline group in the molecule to form the pyridotriazine moiety of M1. This mechanism is consistent with the observed retention of the isotope label in M1. Metabolite M37 may be formed either via a common zwitterionic intermediate, shared with M1, or through a direct insertion pathway. In in vitro human liver microsome incubations, the abundance of M1 was higher than M37, suggesting that breaking of the carbon-nitrogen bond to generate the aldehyde intermediate, a process similar to N-dealkylation, was a preferred pathway.

Unusual Cytochrome P450 Enzymes and Reactions

“…The reaction is rationalized by an electrophilic attack of a P450 (CYP3A4?) on a pyrrole ring to form a hydroxypyrrole, which rearranges to open the pyrrole ring and eventually involves reaction of a neighboring aniline ring to form a stable carbinolamine product (21).…”

In Vitro Characterization of the Metabolic Pathways and Cytochrome P450 Inhibition and Induction Potential of BMS-690514, an ErbB/Vascular Endothelial Growth Factor Receptor Inhibitor

“…M2, M37, and M3 were minor oxidative metabolites in the HLM incubation. Under the chromatographic conditions used in this study, M2 and M37 (hydroxylated metabolites) coeluted (Hong et al, 2011), and together they accounted for 0.4% of the sample radioactivity. Metabolite M3 (an O-demethylated metabolite) accounted for 1.9% of the radioactivity.…”

“…The flux through the M1 pathway could not be determined from the human absorption, distribution, metabolism, and excretion (ADME) study because M1 was thermally labile and decomposed into multiple unknown degradation products. It is likely that M1 did not survive in the gastrointestinal tract or was unstable during collection and processing of urine, bile, and fecal samples Hong et al, 2011). However, in vitro studies in human liver microsomes and in vivo human plasma profiles indicated that the flux through M1 could be significant Hong et al, 2011).…”

“…It is likely that M1 did not survive in the gastrointestinal tract or was unstable during collection and processing of urine, bile, and fecal samples Hong et al, 2011). However, in vitro studies in human liver microsomes and in vivo human plasma profiles indicated that the flux through M1 could be significant Hong et al, 2011). Thus, the 0.3% of dose detected as M1 in human excreta is likely to be an underestimate of this pathway.…”

Biocatalytic Synthesis and Structure Elucidation of Cyclized Metabolites of the Deacetylase Inhibitor Panobinostat (LBH589)

“…Other examples of cyclized metabolites are aminals, where the cyclized metabolite is formed from an aldehyde intermediate. A recent example is the inhibitor of the human epidermal growth factor BMS-690514 14, where a substituted pyrrolo[2,1-f][1,2,4]triazine ring is opened and the aldehyde formed binds to a different, more nucleophilic, nitrogen in the molecule with ring closure (Hong et al, 2011).…”