Synthesis of the CYP24A1 major metabolite of 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3.

Previously, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 (AH-1) showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect on ovariectomized (OVX) rats for enhancing bone mineral density than those of 1α,25(OH)2D3 without hypercalcemic side effects in vivo. Although CYP24A1 catalyzes multi-step oxidations toward the CD-ring side chain of the active vitamin D3 [1α,25(OH)2D3], the CYP24A1-dependent metabolism of AH-1 tended to stop at the first step hydroxylation at the C24-position of AH-1. Interestingly, the metabolite 24-hydroxy-AH-1 [24(OH)AH-1] showed potent VDR binding affinity, and the new chiral center of the 24-position might be the 24R configuration compared with the process of the natural 1α,25(OH)2D3 catabolism. This time, (24R)-2α-[2-(tetrazol-2-yl)ethyl]-1α,24,25-trihydroxyvitamin D3 [(24R-OH)AH-1] was synthesized as a candidate for the major metabolite of AH-1 using the Trost Pd-mediated coupling reaction between A-ring and CD-ring precursors to identify the metabolite and evaluate its biological activity. We confirmed that the CYP24A1-dependent major metabolite of AH-1 was (24R-OH)AH-1 by HPLC analyses.

Synthesis, metabolism, and biological activity of 2-[3-(tetrazolyl)propyl]-1α,25-dihydroxy-19-norvitamin D3.

Recently, we found that 2α-[2-(tetrazol-2-yl)ethyl]-1α,25-dihydroxyvitamin D3 showed higher osteocalcin promoter transactivation activity in human osteosarcoma (HOS) cells and a greater therapeutic effect in ovariectomized (OVX) rats in vivo than those of active vitamin D3, 1α,25(OH)2D3. We were interested in introducing a heterocyclic ring to the C2 position of the seco-steroidal structure via an alkyl linker, and four novel C2-(3-tetrazolylpropyl) substituted 1α,25-dihydroxy-19-norvitamin D3 analogs, 2α-[3-(tetrazol-1-yl)propyl]-, 2ß-[3-(tetrazol-1-yl)propyl]-, 2α-[3-(tetrazol-2-yl)propyl]-, and 2ß-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 were synthesized. Among them, 2α-[3-(tetrazol-1-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak binding affinity for human vitamin D receptor (hVDR) (2.6% of 1α,25(OH)2D3 and ca. 15% of 19-nor-1α,25(OH)2D3) and weak VDR transactivation activity in HOS cells (EC50 7.3nM, when 1α,25(OH)2D30.23nM). Although the other three compounds could not act as VDR binders by evaluation of the competition assays, 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 showed weak transactivation activity (EC50 12.5nM). Metabolic stability of the 2α-substituted compounds 2α-[3-(tetrazol-1-yl)propyl]- and 2α-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 was higher than that of the 2ß-substituted counterparts 2ß-[3-(tetrazol-1-yl)propyl]- and 2ß-[3-(tetrazol-2-yl)propyl]-19-nor-1α,25(OH)2D3 against human CYP24A1. Introduction of a tetrazole ring to the C2-position of the 19-norvitamin D3 skeleton with the propyl linker led to weak VDR agonistic activity with stability against CYP24A1 metabolism.