Vitamin D3 can be metabolized by cytochrome P450 side chain cleavage enzyme (P450scc/CYP11A1) to give 20S-hydroxyvitamin D3 [20S(OH)D3] as a major product. Further hydroxylation of 20S(OH)D3 by CYP11A1 produces one 23-hydroxlated product (20S,23-dihydroxyvitamin D3, 23S or 23R). However, the limited amount of this enzymatic product has hindered the determination of its C23 configuratio, and its biological evaluation. In this study, we chemically synthesized both 20(S),23(S)-dihydroxyvitamin D3 [20S,23S(OH)2D3] and 20(S),23(R)-dihydroxyvitamin D3 [20S,23R(OH)2D3] in 13 steps with an overall yield of 1.5%. They were separated by preparative HPLC and characterized by NMR. The C23 configurations were determined by Mosher’s method and further validated by 2D-NMR spectra. We confirmed that 20S,23S(OH)2D3 is identical to the enzymatically generated product. Kinetic measurements showed that 20S,23S(OH)2D3 is a better substrate for CYP27B1 than 20S,23R(OH)2D3, for production of their 1α-hydroxyl derivatives [1,20S,23S(OH)3D3 and 1,20S,23R(OH)3D3]. We tested their vitamin D receptor (VDR) activation activity in three cell lines (Jurkat, HaCaT and Caco2), and their anti-proliferation activities against cell growth using 1α,25-dihydroxyvitamin D3 [1,25(OH)D3] as a positive control. In addition, their anti-inflammatory activities have been studied by investigating their inhibitory effects against INFγ production and their stimulatory effects on leukocyte-associated immunoglobulin-like receptor 1 (LAIR1). This study provides an efficient synthetic route for production of the 20-hydroxyvitamin D analogs. The comparisons of their biological activities are of great importance for understanding the structural basis for the differential actions of vitamin D analogs/metabolites.

Synthesis and biological evaluation of 20S,23S- and 20S,23R-dihydroxyvitamin D3 isomers together with their 1α-hydroxyl derivatives

251st National Meeting (2016)

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