Novel 2-alkyl-1alpha,25-dihydroxy-19-norvitamin D3: efficient synthesis with Julia olefination, evaluation of biological activity and development of new analyzing system for co-activator recruitment.
New 2-alkyl-1alpha,25-dihydroxy-19-norvitamin D3 analogs were efficiently synthesized. The C2-alkyl-A-ring precursors were prepared as (3R,5R)-4-alkyl-3,5-dihydroxycyclohexanones 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 (2alpha or 2beta) 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-1alpha,25-dihydroxy-19-norvitamin D3 analogs. Among the synthesized 19-norvitamin D3 analogs, 2alpha-(3-hydroxypropyl)-1alpha,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 beta-isomer had only a 3% affinity. Both 2alpha-allyl- and 2alpha-propyl-1alpha,25-dihydroxy-19-norvitamin D3 and their 2beta-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, 2beta-(3-hydroxypropyl)-1alpha,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.
Arai
Midori A
MA
Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan.
Kittaka
Atsushi
A
D000478:Alkylation; D000818:Animals; D002117:Calcitriol / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D002417:Cattle; D002454:Cell Differentiation / Q000187:drug effects; D003512:Cyclohexanones / Q000737:chemistry; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011801:Quinic Acid / Q000737:chemistry; D018167:Receptors, Calcitriol / Q000378:metabolism; D013552:Swine
D003512:Cyclohexanones; D018167:Receptors, Calcitriol; D011801:Quinic Acid; C083610:1,25-dihydroxy-19-norvitamin D3; D002117:Calcitriol