Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D-3 and their 20-epimers: Unique activity profiles depending on the stereochemistry of the A-ring and atC-20

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 m ethyl(S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8). This was convert ed to the alcohol 14 as a 1:1 epimeric mixture in several steps. After havi ng been separated by column chromatography, each isomer led to the requisit e A-ring enyne synthons 19 again as 1:1 mixtures at C-1. Coupling of the re sulting 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 yiel d. In this way, all possible A-ring diastereomers were synthesized. The syn thesized analogues were biologically evaluated both in vitro and in vivo. T he potency was highly dependent on the stereochemistry of each isomer. In p articular, 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 poten t, as the parent compound in HL-60 cell differentiation. Furthermore, its 2 0-epimer, that is, 20epi-alpha alpha beta 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity, 7-fold higher in calciu m mobilization, and 590-fold higher in HL-60 cell differentiation, as compa red to 1 alpha ,25-dihydroxyvitamin D3 (1). Accordingly, the double modific ation of 2-methyl substitution and 20-epimerization resulted in unique acti vity profiles. Conformational analysis of the A-ring by H-1 NMR and an X-ra y crystallographic analysis of the alpha alpha beta -isomer 2g are also des cribed.