Fluorinated vitamin D analogs to probe the conformation of vitamin D in its receptor complex: 19F-NMR studies and biological activity.

To investigate vitamin D conformation, specifically the A-ring and seco-B ring parts, in its complex with the vitamin D receptor (VDR), we have previously suggested the use of 19F-NMR spectroscopy and have synthesized three fluorovitamin D derivatives to be used for the study, 4,4-difluoro-1alpha,25-dihydroxyvitamin D3 [4,4-F2-1,25-(OH)2D3, 2a], and (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 [19-F-1,25-(OH)2D3, 3a, 4a]. In this paper, we examined the 19F-NMR spectra of these and related fluorovitamin D compounds in detail. In the low temperature 19F-NMR spectra, we observed two well-separated rigid conformations of 2a (51:49) and 4a (84:16), while only one conformation was detected with 3a. The two observed conformers of 2a and 4a were respectively assigned to the known alpha- and beta-conformers formed by the flipping of the A-ring where the C(19) exocyclic methylene points to the alpha- and beta-faces. The single conformation observed for 3a was assigned to the alpha-conformer. We detected no other conformation in the 19F-NMR of all vitamin D compounds examined. The effect of solvents on the 19F chemical shifts of fluorovitamin D compounds was found to be small (less than 6.3 ppm). This was much smaller than the difference between the two A-ring conformers (13-30 ppm). Using the dynamic 1H-NMR studies of fluorovitamin D compounds, we determined the free energy of activation for the interconversion between the two conformations of 2a (9.9 kcal/mol) and 4a (10.8, 11.5 kcal/mol). Introduction of the 1alpha-hydroxyl group raised the activation energy about 1 kcal/mol. The affinity for VDR was evaluated, and the relative potency of 2a, 3a and 4a was found to be 1%, 8% and 9%, respectively, of that of 1,25-(OH)2D3 (1). Though the affinity for VDR was considerably reduced in these compounds, the ability to activate gene transcription was similar and remained in the range 30-50% of the effect of 1. This biological information in combination with the NMR properties indicates that 2a and 4a are promising probes for studying the VDR-bound A-ring conformation of vitamin D.