Novel 2-Alkyl-1α,25-dihydroxy-19-norvitamin D₃: Efficient Synthesis with Julia Olefination, Evaluation of Biological Activity and Development of New Analyzing System for Co-activator Recruitment

Abstract

New 2-alkyl-1α,25-dihydroxy-19-norvitamin D₃ analogs were efficiently synthesized. The C2-alkyl-A-ring precursors were prepared as (3R,5R)-4-alkyl-3,5-dihydroxy-cyclohexanones from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The novel CD-ring coupling partner, with the elongated two carbon unit, was synthesized from 25-hydroxy Grundmann's ketone, and applied to modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were moderate (47-62%). After the separation of the diastereomers based on C2 stereochemistry using HPLC, the structure (2α or 2,) was determined by ¹H NMR studies including NOE (nuclear Overhauser effect) experiments. It was also found that ¹H NMR chemical shifts on the A-ring showed good correlation with DeLuca's 2-methyl- and 2-ethyl-1α,25-dihydroxy-19-norvitamin D₃ analogs. Among the synthesized 19-norvitamin D₃ analogs, 2α-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D₃ (8a) showed almost the same potency in binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone (1), while its β-isomer had only a 3% affinity. Both 2α-allyl- and 2α-propyl-1α,25-dihydroxy-19-norvitamin D₃ and their 2β-analogs possessed a weak affinity for the VDR. The strong VDR ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1 and, interestingly, even the weaker ligand, 2β-(3-hydroxypropyl)-1α,25-dihydroxy-19-norvitamin D₃ (8b), showed a 6.7-fold higher potency in cell differentiation activity than that of 1. Next, a novel rapid analyzing system of ligand-induced co-factor recruitment on VDR was developed using fluorophore labelled CoA and HCHO fixing of the complex. The efficiency of recruitment of co-activators would explain the discrepancy between the biological activity and the VDR affinity of the ligand.