Reactive intermediates in naquotinib metabolism identified by liquid chromatography-tandem mass spectrometry: phase I metabolic profiling

Abstract

Tyrosine kinase inhibitors (TKIs) are very efficient for the treatment of EGFR-mutated lung cancer and show improved therapeutic efficacy. However, treatment with both first- and second-generation TKIs results in acquired resistance and is related to various toxicities; the EGFR T790M mutation has been associated with this resistance. Naquotinib (ASP8273, NQT) is a novel third-generation epidermal growth factor receptor tyrosine kinase inhibitor that has been shown to be more potent than osimertinib in the management of L858R plus T790M mutations. However, its bioactivation may occur and promote the formation of reactive electrophiles that are toxic. We hypothesize that these reactive intermediates are potentially involved in the side effects of NQT. Reactive metabolites are often formed by phase I metabolic reactions and cannot be characterized directly as they are transient in nature. Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we screened for in vitro metabolites of NQT formed during incubation with human liver microsomes and evaluated the generation of reactive electrophiles using capturing agents, such as methoxyamine and potassium cyanide, as nucleophiles that form stable adducts for identification by LC-MS/MS. Eight NQT phase I metabolites were found that had been formed by N-demethylation, oxidation, hydroxylation, and reduction. In addition, three reactive electrophiles, two aldehydes, and one iminium ion were identified, and the corresponding bioactivation mechanisms were proposed. The reported side effects of NQT may be related to the generation of reactive metabolites. Based on a literature review, this may be the first study of in vitro phase I metabolites, detailed structural characterizations, and NQT reactive intermediates.

Fig. 1. Chemical structure of naquotinib (NQT).

Fig. 2. PI chromatogram at m/z 563 showing the NQT peak at 37.8 min (A). PI mass spectrum of NQT (B).

Scheme 1. Structural formulas of NQT and corresponding MS/MS fragments.

Fig. 3. PI chromatogram of the NQT549 peak at 36.9 min (A). PI mass spectrum of NQT549 (B).

Scheme 2. Structural formulas of NQT549 and corresponding MS/MS fragments.

Fig. 4. PI chromatogram of the NQT565 peak at 31.2 min (A). PI mass spectrum of NQT565 (B).

Scheme 3. Structural formulas of NQT565 and corresponding MS/MS fragments.

Fig. 5. PI chromatogram of NQT577a, NQT577b, and NQT577c peaks at 41.1, 41.4, and 42.4 min, respectively (A). PI mass spectra of NQT577a (B), NQT577b (C), and NQT577c (D).

Scheme 4. Structural formulas of NQT577a and corresponding MS/MS fragments.

Scheme 5. Structural formulas of NQT577b and corresponding MS/MS fragments.

Scheme 6. Structural formulas of NQT577c and corresponding MS/MS fragments.

Fig. 6. PI chromatogram of NQT579a, NQT579b, and NQT579c peaks at 38.0, 38.4, and 41.6 min, respectively (A). PI mass spectra of NQT579a (B), NQT579b (C), and NQT579c (D).

Scheme 7. Structural formulas of NQT579a and corresponding MS/MS fragments.

Scheme 8. Structural formulas of NQT579b and corresponding MS/MS fragments.

Scheme 9. Structural formulas of NQT579c and corresponding MS/MS fragments.

Fig. 7. PI mass spectrum of NQT588.

Scheme 10. Structural formulas of NQT588 and corresponding MS/MS fragments.

Fig. 8. PI mass spectrum of NQT566.

Scheme 11. Structural formulas of NQT566 and corresponding MS/MS fragments.

Fig. 9. PI mass spectrum of NQT580.

Scheme 12. Structural formulas of NQT580 and corresponding MS/MS fragments.

Scheme 13. Proposed mechanism of iminium intermediate formation during NQT metabolism and a potential trapping strategy.

Scheme 14. Proposed mechanism of aldehyde generation.

Fig. 10. NQT chemical structure showing different metabolic phase I reactions and bioactive centers.