Anisotropic Nuclear Magnetic Resonance Spectroscopy and Density Functional Theory Methodologies Combine with CASE‐3D Analysis for Unambiguous Diastereomeric Differentiation of AMG 176 Macrocycles

As the molecular complexity of active pharmaceutical ingredients (APIs) continues to rise, it is necessary for analytical chemists to evolve and develop robust analytical techniques to provide unambiguous structure identification and ensure drug product quality. The use of orthogonal analytical methods, combined with stereospecific and/or stereoselective synthetic steps, ensures that the chemical integrity of APIs in drug products delivered to patients is maintained. However, molecular characteristics such as multiple distal stereocenters and macrocyclic rings, which often require numerous synthetic steps, make structure analysis particularly challenging. These complexities place significant demands on traditional analytical methods used for elucidating structures. Herein, the synergistic utilization of nuclear magnetic resonance, density functional theory (DFT), and CASE‐3D methodologies for unambiguous structure analysis of the complex macrocyclic AMG 176 diastereomers is showcased. The large macrocyclic rings confer high flexibility to the molecules, making the generation of accurate diastereomeric conformational pools the bottleneck for CASE‐3D structure elucidation. A robust computational workflow for conformational space sampling, along with a stepwise DFT conformer filtering and optimization protocol, is utilized to reduce the high computational demand. The low‐energy conformers produced for AMG 176 are evaluated against its crystal structure to verify the accuracy of the conformer pool and the efficacy of the method. The conformers show a root‐mean‐square deviation of atomic positions in the range of 0.655–2.338 Å when compared to the crystal structure, validating the method. Furthermore, unambiguous diastereomeric CASE‐3D analyses for all four diastereomeric AMG 176 molecules, including AMG 176, 20 R ‐AMG176, 15 S ‐AMG176, and 15 S ,16 R ‐AMG176, are achieved.