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. 2019 Oct 16;9(57):32995-33006.
doi: 10.1039/c9ra03926k. eCollection 2019 Oct 15.

Sapitinib: reactive intermediates and bioactivation pathways characterized by LC-MS/MS

Mohamed W Attwa  1   2 , Adnan A Kadi  1
Affiliations

Sapitinib: reactive intermediates and bioactivation pathways characterized by LC-MS/MS

Mohamed W Attwa et al. RSC Adv. .

Abstract

Sapitinib (AZD8931, SAP) is an epidermal growth factor receptor (EGFR) family (pan-erbB) tyrosine kinase inhibitor. In multiple tumor cell lines, SAP has been shown to be a much more potent inhibitor of EGF-driven cellular proliferation than gefitinib. In this in vitro metabolic study, we tested the generation of reactive intermediates from SAP using human liver microsomes and a capturing agent (potassium cyanide) to trap the iminium reactive intermediates. The same metabolic reaction was further repeated in the presence of methoxyamine to trap aldehyde intermediates. The identification of SAP metabolites revealed that the hydroxylation metabolic reaction represents the major in vitro metabolic pathway occurring at the piperidine moiety. We characterized six in vitro phase I metabolites in addition to three reactive intermediates (i.e., two iminiums and one aldehyde), therefore suggesting two probable SAP-bioactivation pathways. We hypothesized that the piperidine ring nitrogen (cyclic tertiary amine) activated the two adjacent α-carbons within the ring. The oxidative dealkylation of the N-acetamide group led to an unstable aldehyde that was trapped using methoxyamine, generating an oxime adduct that was detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To the best of our knowledge, this is the first study presenting the structural characterization of SAP reactive intermediates.

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Conflict of interest statement

None.

Figures

Fig. 1
Fig. 1. Chemical structure of Sapitinib.
Scheme 1
Scheme 1. Fragments (DIs) observed after fragmentation of SAP.
Fig. 2
Fig. 2. SAP PI mass spectrum. SAP, sapitinib; PI, product ion.
Fig. 3
Fig. 3. M1 PI mass spectrum.
Scheme 2
Scheme 2. Fragments (DIs) observed after fragmentation of M1.
Fig. 4
Fig. 4. M2 PI mass spectrum.
Scheme 3
Scheme 3. Fragments (DIs) observed after fragmentation of M2.
Fig. 5
Fig. 5. M3 PI mass spectrum. SAP, sapitinib; PI, product ion.
Scheme 4
Scheme 4. Fragments (DIs) observed after fragmentation of M3.
Fig. 6
Fig. 6. PI mass spectrum of M4 (A), M5 (B), and M6 (C).
Scheme 5
Scheme 5. Fragments (DIs) observed after fragmentation of M4.
Scheme 6
Scheme 6. Fragments (DIs) observed after fragmentation of M5.
Scheme 7
Scheme 7. Fragments (DIs) observed after fragmentation of M6.
Fig. 7
Fig. 7. M7 PI mass spectrum.
Scheme 8
Scheme 8. Fragments (DIs) observed after fragmentation of M7.
Fig. 8
Fig. 8. M8 PI mass spectrum.
Scheme 9
Scheme 9. Fragments (DIs) observed after fragmentation of M8.
Fig. 9
Fig. 9. M9 PI mass spectrum.
Scheme 10
Scheme 10. Fragments (DIs) observed after fragmentation of M9.
Scheme 11
Scheme 11. Suggested metabolism for the generation of iminium and aldehyde intermediates of SAP.
Fig. 10
Fig. 10. Metabolic and bioactivation pathways of SAP.
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