In-Source Fragmentation of Pyrrolidine-Containing KRAS Scaffolds Leads to Enhanced Structure Elucidation and Tandem...

Area of Science:

• Analytical Chemistry
• Mass Spectrometry
• Medicinal Chemistry

Background:

• Small-molecule therapeutics with basic moieties, like pyrrolidine groups in KRAS G12C inhibitors, pose challenges for structure elucidation using tandem mass spectrometry (MS/MS).
• Proton sequestration by the pyrrolidine during fragmentation leads to uninformative ions, hindering impurity and metabolite identification.

Purpose of the Study:

• To develop an analytical workflow to overcome MS/MS limitations in characterizing basic small-molecule therapeutics.
• To enhance structure elucidation of KRAS inhibitors and related compounds for pharmaceutical process development.

Main Methods:

• Development and application of an analytical workflow utilizing In-Source Fragmentation (ISF).
• Optimization of source parameters to promote selective neutral loss of the pyrrolidine moiety before MS/MS.
• Characterization of GDC-6036, its intermediate, and other KRAS inhibitors (Adagrasib, MRTX1133) using the ISF-MS/MS workflow.

Main Results:

• ISF enables selective neutral loss of the pyrrolidine, generating informative core fragment ions.
• MS/MS analysis of ISF-generated precursors provides superior fragmentation and structural coverage compared to traditional methods.
• Successful characterization of GDC-6036, its synthetic intermediate, and other KRAS inhibitors was demonstrated.

Conclusions:

• The developed ISF workflow is a transferable and universal tool for enhanced structure elucidation of challenging basic compounds.
• This approach significantly supports pharmaceutical process development by improving the identification of impurities and metabolites.