Differential Metabolic Stability of 4α,25- and 4β,25-Dihydroxyvitamin D(3) and Identification of Their Metabolites

Vitamin D(3) (1) is metabolized by various cytochrome P450 (CYP) enzymes, resulting in the formation of diverse metabolites. Among them, 4α,25-dihydroxyvitamin D(3) (6a) and 4β,25-dihydroxyvitamin D(3) (6b) are both produced from 25-hydroxyvitamin D(3) (2) by CYP3A4. However, 6b is detectable in serum, whereas 6a is not. We hypothesized that the reason for this is a difference in the susceptibility of 6a and 6b to CYP24A1-mediated metabolism. Here, we synthesized 6a and 6b, and confirmed that 6b has greater metabolic stability than 6a. We also identified 4α,24R,25- and 4β,24R,25-trihydroxyvitamin D(3) (16a and 16b) as metabolites of 6a and 6b, respectively, by HPLC comparison with synthesized authentic samples. Docking studies suggest that the β-hydroxy group at C4 contributes to the greater metabolic stability of 6b by blocking a crucial hydrogen-bonding interaction between the C25 hydroxy group and Leu325 of CYP24A1.