Synthesis and structure-activity relationships of 16-ene-22-thia-1alpha,25-dihydroxy-26,27-dimethyl-19-norvitamin D3 analogs having side chains of different sizes.

We have synthesized eight novel 16-ene-22-thia-26,27-dimethyl-19-norvitamin D3 analogs 1-5 bearing side chains of different sizes, in combination with 20S- and 20R-isomers. The target compounds were prepared by Wittig-Horner reaction of A-ring phosphine oxide with 16-ene-22-thia-25-hydroxy Grundmann's ketones having different sized side chains, which were derived from the S-phenyloxycarbonyl derivative 13 as key intermediates. The binding affinity to the vitamin D receptor (VDR), VDR-mediated transcriptional activity, and osteoclast-inducing activity of synthetic 22-thia-19-norvitamin D analogs 1-5 were investigated. The (20S)-22-thia-19,24-dinorvitamin D analog 1a is as active as the natural hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3) in terms of biological activities tested in vitro. The analogs 2a and 3a exhibited almost the same potency as 1alpha,25-(OH)2D3 in binding to the VDR, were about 20 times more potent than 1alpha,25-(OH)2D3 in terms of transcriptional activity, and 3a was approximately 100 times as potent as 1alpha,25-(OH)2D3 in eliciting osteoclast formation. The biological activities of (20S)-22-thia compounds were more potent (by more than 10-fold) than those of the corresponding 20R-counterparts, but the activity of (20R)-compounds 1b, 2b, and 3b in stimulating the formation of osteoclasts was similar to that of 1alpha,25-(OH)2D3, and the 24-dihomo- and trihomo-analogs 4a and 5a showed low transcriptional activity. These results suggest that elongation of the side chain in 22-thia analogs by up to one carbon can be stably accommodated in the VDR ligand binding pocket.

2-Substituted-16-ene-22-thia-1alpha,25-dihydroxy-26,27-dimethyl-19-norvitamin D3 analogs: Synthesis, biological evaluation, and crystal structure.

Recently, we have found that 16-ene-22-thia-26,27-dimethyl-19-norvitamin D(3) analogs 1a (n=2, 3) are 20 times more active than the natural hormone 1alpha,25-dihydroxyvitamin D(3) in terms of transcriptional activity. To further investigate the effects of the A-ring modification of 1a, b on the biological activity profile, novel 22-thia-19-norvitamin D analogs 2-11 bearing a hydroxyethoxy-, hydroxyethylidene- or methyl group at C-2 in combination with 20S- and 20R-isomers were prepared and tested for their in vitro biological activities. All of the synthesized analogs showed 0.5-140% of the activity of the natural hormone in binding to the vitamin D receptor (VDR). When compared with the transcriptional activity of C-2 or C-20 isomeric pairs of the 22-thia analogs, the 20S-isomers 2-11a were more potent than the 20R-isomers 2, 3, 8-11b, and the 2beta-hydroxyethoxy, 2E-hydroxyethylidene, and 2alpha-methyl-2beta-hydroxy-22-thia isomers showed higher potency than their corresponding counterparts. In particular, 3a exhibited an extremely higher level of potency (210-fold) than the natural hormone. To elucidate the action mode of superagonist 3a at the molecular level, we determined the crystal structures of the rat VDR-ligand-binding domain complexed with 3a or 3b in the presence of peptide containing a nuclear box motif (LxxLL) at 1.9-2.0A resolution. The crystal structures demonstrated that the 1alpha-OH, 3beta-OH, and 25-OH groups of the natural hormone and 3a were anchored by the same amino acid residues in the ligand-binding pocket, and the terminal OH moiety of the substituent at C-2 formed hydrogen bonds with Arg270 and a water molecule to create a tight water molecule network. Moreover, the methyl groups at C-26a and C-27a make additional contact with hydrophobic residues such as Leu223, Ala227, Val230, and Ala299. These hydrophilic and hydrophobic interactions in 3a may underlie the induction of superagonistic activity.