Novel ring A stereoisomers of 2-methyl-1α,25-dihydroxyvitamin D3 and 2-methyl-20-epi-1α,25-dihydroxyvitamin D3:: Transactivation of target genes and modulation of differentiation in human promyelocytic leukemia (HL-60) cells

We evaluated the biological activity of two sets of ring A stereoisomers of 2-methyl-1 alpha,25-dihydroxyvitamin D-3 (2-methyl-1 alpha,25(OH)(2)D-3) and 2-methyl-20-epi-1 alpha,25-dihydroxyvitamin D-3 (2-methyl-20-epi-1 alpha,25 (OH)(2)D-3) in terms of the following: transactivation of a rat 25-hydroxyvitamin D-3-24-hydroxylase gene promoter including two vitamin D response elements (VDREs) and a human osteocalcin gene promoter including a VDRE in transfected human osteosarcoma (MG-63) cells; a Vitamin D receptor (VDR)-mediated response using a VDR-GAL4 one-hybrid luciferase reporter system and a retinoid X receptor alpha (RXR alpha)-mediated response using an expressed VDR/RXR alpha-GAL4 modified two hybrid luciferase reporter system in transfected human epitheloid carcinoma, cervix (HeLa) cells; and modulation of cell surface CD11b antigen expression in human leukemia (HL-60) cells. All the diastereomers of both analogues exhibited unique biological activity profiles depending upon the configurations of the C-1 and C-3 hydroxyl groups, the C-2 methyl group in ring A, and the C-20 methyl group in the side chain. Of the eight possible diastereomers of the 2-methyl analogues, 2 alpha-methyl-1 alpha,25(OH)(2)D-3 was the most potent and exhibited comparable or even greater biological potency than 1 alpha,25(OH)(2)D-3. Of the eight possible diastereomers of the 2-methyl-20-epi analogues, 2 alpha-methyl-20-epi-1 alpha,25(OH)(2)D-3 was the most potent and exhibited 100- to 200-fold higher transcriptional potencies than 1 alpha,25(OH)(2)D-3 and exceptionally high cell regulatory activities. 2 beta-Methyl-20-epi-1 alpha,25(OH)(2)D-3 was nearly as potent as its 2-epimer, 2 alpha-methyl-20-epi-1 alpha,25(OH)(2)D-3, whereas its 20-epimer, 2 beta-methyl-1 alpha,25(OH)(2)D-3, was almost completely biologically inactive. In these respects, it can be postulated that the double modification of 2-methyl substitution and 20-epimerization to 1 alpha,25(OH)(2)D-3 induces remarkable changes in a VDR/RXR alpha/VDRE-mediated signaling response and greatly enhances biological activity. The other striking finding was that 2 beta-methyl-20-epi-3-epi-1 beta,25(OH)(2)D-3 is transcriptionally more active than 1 alpha,25(OH)(2)D-3 despite lacking the 1 alpha-hydroxyl group, which was believed to be essential for expressing VDR-mediated gene transcription. Since the C-20 natural counterpart, 2 beta-methyl-3-epi-1 beta,25(OH)(2)D-3, was almost completely biologically inactive, 20-epimerization is probably responsible for activation of gene expression. Although earlier extensive structure-activity studies of vitamin D analogues showed stereochemistry at the C-1, C-3, and C-20 of 1 alpha,25(OH),D, to be the key structural motif for Vitamin D action, our results clearly demonstrated that stereochemistry at the C-2 is also an important structural motif for Vitamin D action and imply that 2-methyl substitution possibly induces conformational changes in ring A depending upon the combinations of configurations of the C-1 and C-3 hydroxyl groups with C-20 stereochemistry. Consequently, several of these analogues exhibit exceptionally high or unexpected biological activities at the molecular and cellular levels. These results suggest that 2-methyl substitution together with alterations of stereochemistry in both ring A and the side chain of 1 alpha,25(OH)(2)D-3 will provide useful analogues for structure-activity studies and development of therapeutic agents with unique biological activity profiles. (C) 2000 Elsevier Science Inc.