The antagonism between 2-methyl-1,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1,25-dihydroxyvitamin D3 in non-genomic pathway-mediated biological responses induced by 1α,25-dihydroxyvitamin D3 assessed by NB4 cell differentiation

We synthesized all eight possible A-ring diastereomers of 2-methyl substituted analogs of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] and also all eight A-ring diastereomers of 2-methyl-20-epi-1α,25(OH)2D3. Their biological activities, especially the antagonistic effect on non-genomic pathway-mediated responses induced by 1α,25(OH)2D3 or its 6-s-cis-conformer analog, 1α,25(OH)2-lumisterol3, were assessed using an NB4 cell differentiation system. Antagonistic activity was observed for the 1β-hydroxyl diastereomers, including 2β-methyl-1β,25(OH)2D3 and 2β-methyl-3-epi-1β,25(OH)2D3. Very interestingly, 2β-methyl-3-epi-1α,25(OH)2D3 also antagonized the non-genomic pathway, despite its 1α-hydroxyl group. Other 1α-hydroxyl diastereomers did not show antagonistic activity. 20-epimerization diminished the antagonistic effect of all of these analogs on the non-genomic pathway. These findings suggested that the combination of the 2-methyl substitution of the A-ring and 20-epimerization of the side chain could alter the biological activities in terms of antagonism of non-genomic pathway-mediated biological response. Based on a previous report, 2-methyl substitution alters the equilibrium of the A-ring conformation between the α- and β-chair conformers. The 2β-methyl diastereomers, which exhibited antagonism on non-genomic pathway-mediated response, were considered to prefer the β-conformer. Further examination to elucidate the relationship between the altered ligand shape and receptors interaction will be important for molecular level understanding of the mechanism of antagonism of the non-genomic pathway.