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Mechanistic Studies on a P450-Mediated Rearrangement of BMS-690514: Conversion of a Pyrrolotriazine to a Hydroxypyridotriazine† Haizheng Hong,‡ Janet Caceres-Cortes,‡ Hong Su,‡ Xiaohua Huang,‡ Vikram Roongta,‡ Samuel Bonacorsi, Jr.,§ Yang Hong,§ Yuan Tian,§ Ramaswamy A. Iyer,‡ W. Griffith Humphreys,‡ and Lisa J. Christopher*,‡ Departments of Pharmaceutical Candidate Optimization and Radiochemistry, Bristol-Myers Squibb Research, Princeton, New Jersey 08543, United States ReceiVed September 28, 2010
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 BMS690514 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. Introduction 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 a potent inhibitor of human epidermal growth factor receptors HER 1 (EGFR), HER2, and HER41 and vascular endothelial growth factor receptors (VEGFR, 1-3) (1). It is under development as an oral agent for the treatment of advanced nonsmall cell lung cancer (NSCLC) and other solid tumors, such as breast cancer (2-4). BMS-690514 contains a pyrrolo[1,2,4]triazine † A portion of this work was presented at the 240th National American Chemical Society Meeting, Boston MA, August 22-26, 2010. * To whom correspondence should be addressed. Department of Biotransformation, Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Research, Route 206 & Province Line Road, Mail Stop F13-01, Princeton, NJ 08543. Tel: 609-252-6371. Fax: 609-252-6802. E-mail:
[email protected]. ‡ Department of Pharmaceutical Candidate Optimization. § Department of Radiochemistry. 1 Abbreviations: ADME, absorption, distribution, metabolism, and excretion; P450, cytochrome P450 enzyme; DMF, dimethylformaldehyde; DLM, dog liver microsomes; HMBC, heteronuclear multiple-bond correlation spectroscopy; HSQC, heteronuclear single quantum coherence; HLM, human liver microsomes; HER1 (EGFR), HER2 and HER4, human epidermal growth factor receptors; LC/MS/MS, liquid chromatography with tandem mass spectrometry; ROESY, rotating frame Overhauser enhancement spectroscopy;UDPGA,uridine5′-diphosphoglucuronicacid;VEGFR1-VEGFR3, vascular endothelial growth factor receptors; XIC, extracted ion chromatogram.
moiety (Figure 1), which effectively mimics the well-known quinazoline kinase inhibitors (5). It was found that the attachment of substituents, e.g., (3-hydroxyphenyl)amino or (3methoxyphenyl)amino, to the pyrrolo[1,2,4]triazine nucleus provided potent inhibition of EGFR and VEGFR tyrosine kinase activities (5, 6). In vitro experiments suggested that direct glucuronidation and CYP2D6 and CYP3A4-mediated oxidation were likely to be important pathways in the metabolism of BMS-690514 (7). In vivo, BMS-690514 was well absorbed and highly metabolized in rats, dogs, and humans (8, 9). The primary metabolic pathways of this compound included hydroxylation, O-demethylation, and glucuronidation (8, 9) (Figure 1). The structural elucidation and detailed analytical characterization for important metabolites has been reported previously (7, 9). Although the structures of M1 and M37 were also disclosed (9), detailed experiments to characterize these two metabolites have not been published. Metabolites M1 and M37 were each hydroxylated on the pyrrolotriazine moiety at the C-16 position (Figure 1). M1 underwent an unusual structural rearrangement where the pyrrolotriazine moiety rearranged to yield a hydroxypyridotriazine group, while the pyrrolotriazine moiety of M37 remained intact. M1 was a prominent circulating metabolite found in the plasma of rats, rabbits, dogs, and humans after the administration
10.1021/tx100337s 2011 American Chemical Society Published on Web 11/16/2010
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Figure 1. Major metabolic pathways of BMS-690514. For the parent compound and metabolites M1, M37, and M7, the carbon at position 16 is indicated. Atom numbers are for illustrative purposes and do not conform to International Union of Pure and Applied Chemistry nomenclature.
Scheme 1. Mechanism of CYP-Mediated Aromatic Hydroxylation of Deuterium Labeled Benzenea
a Pathway a: initial formation of an arene oxide species, which subsequently rearranges to the corresponding phenol following an NIH shift. Pathway b: addition of high valent iron(IV)-oxo species to the π-system of the aromatic ring to produce a tetrahedral intermediate or cationic σ-complex, which subsequently rearranges to form an epoxide or a zwitterionic intermediate. These intermediates then proceed to yield the phenol via the NIH shift. Pathway c: direct oxygen insertion in which the carbon-hydrogen bond is broken, leading to the loss of the isotope label at the site of hydroxylation.
of BMS-690514 (8, 9). M37, also present in these four species, was the precursor of a prominent O-glucuronide conjugate, M7 (9). The mechanism of enzymatic hydroxylation on aromatic hydrocarbons has been extensively studied, and currently three pathways have been proposed (Scheme 1): (a) an epoxidation pathway, (b) an iron-oxo addition pathway, and (c) a direct insertion pathway. However, little has been reported on the hydroxylation of heteroaromatic compounds, e.g., pyrrole or pyrrolotriazine rings. The formation of metabolites M1 and M37 from BMS-690514 presents a unique opportunity to study the mechanism of hydroxylation on the pyrrolotriazine template. In this article, we describe the detailed experiments that were
conducted to characterize the structures of metabolites M1 and M37. In addition, the mechanism for the formation of M1 and M37 was explored through a series of studies, including the use of isotopic tritium and deuterium labels at the site of hydroxylation.
Materials and Methods Materials. BMS-690514 (10) (Figure 1) and a piperidine N-oxide of BMS-690514, ((3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)-3-piperidinol 1-oxide (structure shown in Figure S4, Supporting Information) were supplied by Department of Chemical Synthesis, Bristol-Myers Squibb (BMS), New Brunswick, NJ, USA. [14C]BMS-690514
OxidatiVe Biotransformation of BMS-690514 (radiochemical purity of 98.96%), [2H]BMS-690514, and [3H]BMS690514 were supplied by Radiochemistry Group of the Department of Chemical Synthesis, BMS, Princeton, NJ, USA. The C-14 label of [14C]BMS-690514 was evenly distributed among the six carbons of the methoxyaniline ring (9). [2H]BMS-690514 and [3H]BMS690514, each isotopically labeled on position 16, were synthesized by exchange with deuterium or tritum gas using their corresponding bromo analogue. The percent of deuterium or tritium label incorporation was determined to be about 60% for both [2H]BMS690514 and [3H]BMS-690514 (Supporting Information). It should be noted that the atom numbers of BMS-690514 and its metabolites shown in this article are for illustrative purposes and do not conform to International Union of Pure and Applied Chemistry nomenclature. Pooled human liver microsomes (HLM, pooled from 19 subjects) and dog liver microsomes (DLM, pooled from 4 dogs) were purchased from BD Bioscience (Woburn, MA). Deuterium oxide (D, 99.9%) was purchased from Cambridge Isotope Laboratory (Andover, MA). All other reagents were purchased from SigmaAldrich Chemical Co. (St. Louis, MO) except where indicated. Liver Microsome Incubations. BMS-690514, [14C]BMS690514, [3H]BMS-690514, or [2H]BMS-690514 (50 µM) was incubated with pooled HLM (1 mg/mL) fortified with 1 mM NADPH in 100 mM phosphate buffer (pH 7.4) to generate M1 and M37. [3H]BMS-690514 (50 µM) was incubated with pooled HLM or DLM (1 mg/mL) fortified with 1 mM NADPH, 2 mM UDPGA (uridine 5′-diphosphoglucuronic acid), and 25 µg/mL alamethicin in 100 mM phosphate buffer (pH 7.4) to generate M7. All incubations were conducted at 37 °C in a shaking water bath for 60 min. At the end of the incubation period, an equal volume of ice-cold acetonitrile was added to each mixture to quench the reaction. The samples were vortex mixed and centrifuged at 13,000 rpm for 10 min, and the supernatant was frozen at -80 °C until analysis. HPLC Purification of M1 for NMR Analysis. HPLC purification was performed on a Shimadzu LC-8A preparative HPLC system with a photodiode array detector, set at a wavelength of 264 nm. Separation of drug-related components was achieved with a 150 × 21.2 mm Phenomenex Synergi Fusion-RP column (4 µm in particle size and 80 Å in pore size). The HPLC mobile phase consisted of two solvents: mobile phase A (10 mM ammonium acetate in water, pH 5.0, and acetonitrile, 95:5 v/v) and mobile phase B (100% acetonitrile). The gradient program used for separation was as follows: hold isocratic at 0% B (0-5 min); linear gradient from 0 to 35% B (5-40 min); linear gradient from 35 to 90% B (40-45 min); hold isocratic at 90% B (45-50 min); and re-equilibrate at 0% B for 10 min. The mobile phase flow rate was 10 mL/min. The HPLC fractions containing M1 were collected on ice and dried by lyophilization under low temperature (shelf temperature 1 µM (8). Moreover, structure-activity studies identified that the substitution of a methyl group at the 7-position of the pyrrolotriazine group (position 16 of BMS-690514) led to substantial loss of inhibitory activity (5), implicating the importance of this position for the retention of activity. Since M37 had a hydroxyl group located at this position, it is likely that M37 is less potent than the parent drug. Since M37 was not a circulating metabolite in humans, the pharmacological activity of M37 was not further explored in receptor binding assays. Although M1 was relatively stable at low temperature (
