Elucidation of a Novel Bioactivation Pathway of a 3,4-Unsubstituted Isoxazole in Human Liver Microsomes: Formation of a Glutathione Adduct of a Cyanoacrolein Derivative after Isoxazole Ring Opening

Abstract

ABSTRACT:Studies on the biotransformation of isoxazole rings have shown that molecules containing a C3-substituted isoxazole or a 1,2-benzisoxazole can undergo a two-electron reductive ring cleavage to form an imine. In the absence of a C3 substituent, the isoxazole ring opens via deprotonation of the C3 proton followed by N-O bond cleavage to yield an ␣-cyanoenol analog. We report the identification of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes. After the enzyme-catalyzed cleavage of the 3,4-unsubstituted isoxazole ring of N-((2-isopropyl-7-methyl-1-oxoisoindolin-5-yl)methyl)isoxazole-5-carboxamide (P) in human liver microsomes, the formed ␣-cyanoenol (M1) condenses with formaldehyde to generate an ␣,␤-unsaturated Michael acceptor intermediate (a cyanoacrolein derivative, VII), which further reacts with the cysteinyl thiol of glutathione to yield a GSH adduct of a cyanoacrolein derivative (M3). The same adduct also is formed when M1, generated in 0.1 N NaOH aqueous solution, reacts with formaldehyde and GSH. 13Clabeled methanol was used to confirm that methanol from the drug stock solution was oxidized by liver microsomal enzymes to formaldehyde and the carbon atom from methanol was finally incorporated in the corresponding GSH adduct. The formation of isoxazole ringopened products (M1 and M2) in human liver microsomes is NADPHdependent. M1 and M2 were found in human liver microsomes preincubated with 1-aminobenzotriazole (1 mM) and NADPH (5 mM) at ϳ10% of the levels found in the samples in the absence of 1-aminobenzotriazole, suggesting that this biotransformation pathway is primarily catalyzed by cytochrome P450. The formation of M3 also was inhibited by 1-aminobenzotriazole at a similar level.

A redox-mediated Kemp eliminase

“…Following the work with the model compound 1 , we turned to leflunomide as substrate, knowing that this compound is metabolized by human P450 (refs 22, 23, 24) (Fig. 4).…”

“…Based on our experimental and computational results using P450-BM3 as the catalyst in the reaction of the standard substrate 1 , the same redox mechanism may be postulated in human metabolism of leflunomide, but these mechanistic details still need to be explored. Relevant is also the reported human metabolism of a different isoxazole-based therapeutic drug, zonisamide23, active in the treatment of epilepsy, Parkinson's disease, dardive dyskinesia, migraine and obesity. The metabolite is a ring-opened compound, namely 2-(sulfamoylacetyl)phenol (Supplementary Fig.…”

“…We focused on cytochrome P450 monooxygenases (CYPs) for several reasons: Human cytochrome P450 (CYP) enzymes play a crucial role in the metabolism of therapeutic drugs192021, including the degradation of a number of prominent isoxazole-based pharmaceuticals222324. A prime example is the metabolism of leflunomide, an anti-inflammatory agent used in the treatment of rheumatoid arthritis23. The main metabolic outcome is the physiologically active teriflunomide (2-cyano-3-oxo-N-[(4-trifluormethyl)phenyl]butyramide, also called A771726), a formal Kemp elimination product.…”

“…The main metabolic outcome is the physiologically active teriflunomide (2-cyano-3-oxo-N-[(4-trifluormethyl)phenyl]butyramide, also called A771726), a formal Kemp elimination product. Two pathways have been postulated, an acid/base and an undefined redox-mediated process2223, but mechanistic studies to distinguish between the two possibilities have not been reported to date. The term Kemp elimination was not referred to in this connection.…”

Novel Bioactivation Mechanism of Reactive Metabolite Formation from Phenyl Methyl-Isoxazoles

“…However, certain drugs, such as trimetoprime and dasatinib, have been proposed to generate GSH adducts through enimine intermediates (Lai et al, 1999;Li et al, 2009). A metabolic pathway for the generation of reactive metabolites was recently described for 3,4-unsubstituted isoxazoles after an enzymecatalyzed cleavage of the ring to form an ␣-cyanoenol followed by a condensation with formaldehyde to yield a reactive cyanoacrolein derivative (Yu et al, 2011). However, Kalkutgar et al (2003 demonstrated using leflunomide and 3-methylleflunomide that the unsubstituted 3Ј-position was important for this ring opening to happen.…”