[Show abstract][Hide abstract] ABSTRACT: 1alpha,25(OH)(2)-16-ene-20-cyclopropyl-vitamin D(3) (13) is several fold more potent than the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1) as an anti-inflammatory agent. Here, we have further analyzed the anti-inflammatory properties of 13, confirming it as the most potent analogue tested within this family. We then determined the structures of all the natural metabolites of 13, including the 24-oxo metabolite 14, and carried out its synthesis. A comparison of 13 with 14 showed a similar induction of the primary VDR target genes CYP24A1 and CAMP and comparable anti-inflammatory properties as revealed by a similar inhibition of TNF-alpha, IL-12/23p40, IL-6, and IFN-gamma production. Interestingly, 14 displays a 3-fold lower calcemic activity in vivo compared to 13. Collectively, these findings indicate that the strong potency of 13 can be explained by the accumulation of its stable 24-oxo metabolite, which shows immunoregulatory and anti-inflammatory properties superimposable to those exerted by 13 itself.