Synthesis and Stereochemical Assignment of Diastereomeric Steroidal Spiro-1,2,4-trioxolanes with Sitostanone Motif Using PLS2 Chemometric Analysis of DFT-Calculated and Experimental 13C NMR Data.

The synthesis and multi-step spectroscopic characterization of diastereomeric 3-spiro-1,2,4-trioxolanes obtained by Griesbaum co-ozonolysis of sitostanone O-methyl oxime with two types of fluorinated ketones are reported. The reaction furnished four possible diastereomers that differ in the α/β orientation of the peroxide bridge relative to the steroid A-ring and in the syn/anti orientation of the trifluoromethyl group at C5'. A chemometric PLS2 approach, linking experimental and DFT-calculated 13C chemical shifts, was used for the objective stereochemical assignment of each diastereomer in the inseparable mixtures. Single-crystal X-ray analysis of the isolated 3R,5'R and 3R,5'S stereoisomeric pair (α-anti and α-syn ozonides) provided unambiguous absolute configurations that validated the stereochemical assignments obtained by the combined NMR/DFT/PLS2 approach. This integrated synthetic, crystallographic, spectroscopic, and chemometric methodology provides reliable configurational assignment in complex spiro-peroxide mixtures and expands the analytical toolkit for such systems.