Warfarin Tautomers in Solution: A Structural, Computational and Thermodynamic Study

Variable temperature NMR spectroscopic measurements on (S)-warfarin [open-form: 3-(1′-phenyl-3′-oxobut-1′-yl)-4-hydroxycoumarin] in CDCl₃, CD₃OD and d₆-DMSO generally showed tautomeric compositions in the order trans (2S,4S) coumarin hemiketal > cis (2R,4S) coumarin hemiketal > open (S) coumarin enol in slow dynamic equilibrium over temperature ranges rising modestly from ambient. A computational (DFT-M06-2X) examination of the lower energy tautomers including coumarin and chromone open and cyclic forms (gas phase, chloroform or DMSO fields) was consistent with the general solution compositions. The crystal and molecular structures for model compounds of the major solution tautomers are reported: (2S,4S)-warfarin methyl ketal [orthorhombic, P2₁2₁2₁], (2R,4S)-warfarin methyl ketal [orthorhombic, P2₁2₁2₁], (rac)-warfarin-4-methyl ether [monoclinic, P2₁/n], and the open chromone (S)-warfarin-2-methyl ether [monoclinic, P2₁, Z = 8]. A combination of direct integration and line-fitting methods were used to determine solution (S)-warfarin tautomer compositions. As temperatures were increased, the concentrations of the open coumarin form increased at the expense of the cyclic hemiketals. Equilibrium constants were used to determine the standard free-energy differences for the two open-cyclic equilibria (trans hemiketal \(\rightleftharpoons\) open, open \(\rightleftharpoons\) cis hemiketal, respectively) in three solvents: CDCl₃ [+ 3.7(4), − 2.8(6) kJ/mol], CD₃OD [+ 7.6(16), − 4.7(9) k/mol], d₆-DMSO [+ 3.5(7), − 1.1(2) kJ/mol]. Standard enthalpy and entropy differences were also determined from van’t Hoff analysis. Rates of the respective reactions were estimated from line-widths for the cyclic hemiketals and solution equilibrium compositions for each species. Eyring analysis gave ΔG^‡, ΔH^‡, and ΔS^‡, respectively, for the forward and reverse reactions of coumarin trans hemiketal \(\rightleftharpoons\) open-form and for the open-form \(\rightleftharpoons\) cis hemiketal. Negative entropic contributions to the observed transition state energies were consistent with solvent or solute ordering in the prototropic reactions. Open-form NMR signals were broader than could be accounted for by the open-cyclic equilibria alone, increasingly so in polar and protic solvents and with rising temperatures. While a conformational equilibrium may operate, an increasingly faster intermediate dynamic equilibrium between open coumarin-chromone tautomers may be a more likely explanation.

Graphical Abstract

Structures of methylated warfarin tautomers and computational models enabled assignment of overlapping warfarin tautomeric NMR spectra and through variable temperature analysis, provided the thermodynamics of the tautomeric equilibria in three solvents.