New highly active isomers of the natural hormone 1alpha, 25-dihydroxyvitamin D3 possessing an exomethylene group at the 2-position were prepared in a convergent manner, starting with (-)-quinic acid and the corresponding (20R)- and (20S)-25-hydroxy Grundmann ketones. These 2-methylene-19-norvitamins were efficiently converted to the 2-methyl and 2-hydroxymethyl derivatives, some of which exhibited pronounced in vivo biological activity. Configurations of the A-ring substituents were determined by 1H NOE difference spectroscopy as well as by spin decoupling experiments. It was established that the bulky methyl and hydroxymethyl substituents at C-2, due to their large conformational free energies, occupy mainly equatorial positions. Additionally, hydroxylation of the C(10)-C(19) double bond in 1alpha,25-(OH)2D3 was performed, resulting in 1alpha,19,25-trihydroxy-10,19-dihydrovitamin D3 derivatives in which the hydroxymethyl substituent at C-10, for steric reasons, is forced to occupy an axial position. In consequence, the vitamin D3 analogues were synthesized in which the 1alpha-hydroxy group, required for biological activity, is almost exclusively axially or equatorially oriented because of stabilization of the single A-ring chair conformations. The relative ability of the synthesized analogues to bind the porcine intestinal vitamin D receptor was assessed and compared with that of the natural hormone. It was established that vitamins possessing the axial orientation of the 1alpha-hydroxy substituent exhibit a significantly increased receptor binding affinity. Compounds with a 2-methylene substituent showed selective calcemic activity profiles, being extremely effective on bone calcium mobilization. 2alpha-Methyl-substituted vitamins proved to be much more active in vivo than the corresponding epimers with 2beta-configuration. All of the 2-substituted vitamins exhibited pronounced HL-60 differentiating activity, those 2alpha-substituted in the 20S-series being especially potent. The present studies imply that the axial orientation of the 1alpha-hydroxy group is necessary for biological activity of vitamin D compounds.

All eight possible A-ring diastereomers of 2-methyl-1, 25-dihydroxyvitamin D(3) (2) and 2-methyl-20-epi-1, 25-dihydroxyvitamin D(3) (3) were convergently synthesized. The A-ring enyne synthons 19 were synthesized starting with methyl (S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8). This was converted to the alcohol 14 as a 1:1 epimeric mixture in several steps. After having been separated by column chromatography, each isomer led to the requisite A-ring enyne synthons 19 again as 1:1 mixtures at C-1. Coupling of the resulting A-ring enynes 20a-h with the CD-ring portions 5a,b in the presence of a Pd catalyst afforded the 2-methyl analogues 2a-h and 3a-h in good yield. In this way, all possible A-ring diastereomers were synthesized. The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency was highly dependent on the stereochemistry of each isomer. In particular, the alpha alpha beta-isomer 2g exhibited 4-fold higher potency than 1 alpha,25-dihydroxyvitamin D(3) (1) both in bovine thymus VDR binding and in elevation of rat serum calcium concentration and was twice as potent as the parent compound in HL-60 cell differentiation. Furthermore, its 20-epimer, that is, 20-epi-alpha alpha beta 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity, 7-fold higher in calcium mobilization, and 590-fold higher in HL-60 cell differentiation, as compared to 1 alpha,25-dihydroxyvitamin D(3) (1). Accordingly, the double modification of 2-methyl substitution and 20-epimerization resulted in unique activity profiles. Conformational analysis of the A-ring by (1)H NMR and an X-ray crystallographic analysis of the alpha alpha beta-isomer 2g are also described.