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

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Abstract

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.

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Data Availability

Crystallographic details for structures 2 (CCDC #2286286), 3 (CCDC #2286287), 4 (CCDC #2286288) and 5 (CCDC #2286289) have been deposited with the Cambridge Crystallographic Data Centre (https://www.ccdc.cam.ac.uk) and are available on request.

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  1. Department of Chemistry and Biochemistry, University of Portland, Portland, OR, 97203, USA

    Daniel A. Osborne, Edward Danielyan, Khoi Hoang & Edward J. Valente

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Contributions

National Science Foundation (MRI #0604188) for crystallographic equipment (EV), and University of Portland Butine and SURE funding for computational hardware and software (DO), and a research stipend (KH). DO, ED, and KH performed the computations, DO analyzed the computations and determined torsional, angle, and distance metrics. EJV performed the syntheses, spectroscopic and crystallographic experiments. We thank Dr. David H. Magers, Mississippi College, for discussions on computational strategies.

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Osborne, D.A., Danielyan, E., Hoang, K. et al. Warfarin Tautomers in Solution: A Structural, Computational and Thermodynamic Study. J Chem Crystallogr 54, 64–83 (2024). https://doi.org/10.1007/s10870-023-00999-9

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