STRUCTURAL ELUCIDATION OF WARFARIN, APPLICATION OF THE DP4+ METHOD TO EQUILIBRIUM SYSTEMS

Warfarin is an oral anticoagulant widely used in the treatment of cardiovascular diseases and remains a relevant model in pharmacological and structural studies. Its behavior in solution, particularly the equilibrium between different neutral species, is crucial for understanding its bioactivity and affinity for its molecular target, the VKOR enzyme. Nuclear magnetic resonance (NMR) studies and theoretical calculations based on density functional theory (DFT) have confirmed the existence in solution of keto and enol forms in dynamic equilibrium, in addition to cyclic hemiacetal species.

In this context, the DP4+ method, which statistically compares experimental and calculated chemical shifts, allows for the validation of structures with high precision, even in the presence of equilibrium species. Its Bayesian approach improves reliability in complex assignments, integrating ¹H and ¹³C data and solvent effects.

For the structural study, a commercial formula (sodium salt) was used. After its isolation by dissolution in water (filtering out insoluble residues under vacuum) and subsequent controlled acidification of the aqueous medium, the neutral compound was obtained and characterized by ¹HNMR in DMSO at different temperatures. One-dimensional and two-dimensional experiments (¹³CNMR, COSY, HSQC, HMBC, and NOESY) were also performed, and theoretical shifts were calculated with Gaussian 09 (B3LYP/6-31+G(d,p)).

The analysis revealed the presence of two neutral species in equilibrium (Keq = 1.8). The application of DP4+ allowed confirming that these species were two diastereomeric hemiacetals. The integration of NMR, DFT calculations, and DP4+ enabled a precise structural assignment of warfarin in solution, validating its complex chemical behavior.