2 alpha-(3-hydroxypropyl)- and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 accessible to vitamin D receptor mutant related to hereditary vitamin D-resistant rickets

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor, which lead to resistance to 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. We found that the A ring-modified analogues, 2alpha-(3-hydroxypropyl)- and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)(2)D(3), (O1C3 and O2C3) can bind better than the natural hormone to the mutant VDR (R274A), which similar to the HVDRR mutant, R274L, had lost the hydrogen bond to the 1alpha-hydroxyl group of 1alpha,25(OH)(2)D(3).     

Functionalization at C-12 of 1alpha,25-dihydroxyvitamin D(3) strongly modulates the affinity for the vitamin D receptor (VDR)

The first synthesis of analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1a) with substituents at C-12 is reported. The following are the relative affinities of the novel compounds for the vitamin D receptor (VDR) compared to that of 1a (100%): 1alpha,12alpha,25-(OH)(3)-D(3) (1b, 1%), 1alpha,25-(OH)(2)-12-methylene-D(3) (1c, 50%), and 1alpha,25-(OH)(2)-12beta-methyl-D(3) (1d, 440%). [structure: see text]     

Modulation effects of zinc on the formation of vitamin D receptor and retinoid X receptor alpha-DNA transcription complexes: analysis by microelectrospray mass spectrometry

The vitamin D receptor (VDR) binds zinc, and the activity of vitamin D dependent genes in cells is influenced by intracellular zinc concentrations. To determine whether zinc influences vitamin D action in cells by modulating the formation of VDR and retinoid x receptor alpha (RXR alpha) heterodimer-DNA complexes, we used microelectrospray ionization mass spectrometry (microESI-MS) to assess receptor-DNA interactions in the presence of varying amounts of zinc. In the absence of DNA, VDR and RXR alpha proteins were primarily monomeric with small amounts of protein homodimers also observed. Zn(2+) (up to 300 microM) did not change VDR or RXR alpha monomer/homodimer ratios. Mass spectra of VDR combined with RXR alpha were a sum of individual protein spectral data. Zn(2+) had no effect on the interactions of receptors. With increasing amounts of Zn(2+), additional Zn(2+) ions were detected bound to VDR and RXR alpha. microESI-MS analyses of RXR alpha in the presence of an osteopontin vitamin D DNA response element (OP-VDRE) showed RXR alpha homodimer/OP-VDRE complexes. DNA-protein complex formation increased on addition of Zn(2+) up to 200 microM; at 300 microM, Zn(2+) dissociation of the RXR alpha homodimer/OP-VDRE complexes occurred, coincident with the appearance of RXR alpha monomeric protein. When microESI-MS analyses were carried out with VDR and OP-VDRE, VDR homodimer/OP-VDRE complexes were not detected. Addition of Zn(2+) did not result in VDR/OP-VDRE complex formation. Heterodimeric VDR/RXR alpha complexes with OP-VDRE were detected by microESI-MS. Addition of 300 microM Zn(2+) resulted in dissociation of the heterodimeric VDR/RXR alpha/OP-VDRE complex. Addition of Mg(2+) in place of Zn(2+) did not alter protein/OP-VDRE complexes. Our results show that zinc modulates steroid hormone receptor-DNA interactions.     

A structural basis for the species-specific antagonism of 26,23-lactones on vitamin D signaling

The 26,23-lactone derivative of 1alpha,25-dihydroxyvitamin D3, TEI-9647, is a partial antagonist of the of human vitamin D receptor (VDR). However, we found that TEI-9647 in rat cells behaves as a weak VDR agonist. This behavior could be mimicked in human cells by the double mutagenesis of human VDR (specifically C403S and C410N). The increased agonistic action of TEI-9647 correlates to a gain in the interaction of the VDR with coactivator protein and a decreased stabilization of the antagonistic conformation of the receptor. Molecular dynamics simulations indicated that TEI-9647 acts as antagonist of human VDR by reducing the stability of helix 12 of the ligand binding domain. In contrast, N410 of the rat VDR stabilized, via backbone contacts, the interaction between helices 11 and 12. This results in TEI-9647 becoming a weak agonist in this organism.     

Catalytic asymmetric syntheses and biological activities of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs in cancer cell differentiation and apoptosis

BACKGROUND: 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation, but also apoptosis in malignant cells, indicating that it could be useful for treating cancer. Little information is available concerning the structural motifs of the 1alpha, 25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis, however. We set out to synthesize singly dehydroxylated A-ring analogs of 19-nor-1alpha,25(OH)(2)D(3) in a catalytic asymmetric fashion, and to investigate their biological activities in leukemia HL-60 cells. RESULTS: A series of singly dehydroxylated 19-nor-1alpha,25-dihydroxyvitamin D(3) A-ring analogs were synthesized using a combinatiorial sequence of regioselective propiolate-ene reaction and catalytic asymmetric carbonyl-ene cyclization. Surprisingly, the analogs could be clearly divided into two categories; one group, bearing 1alpha-hydroxy or 3beta-hydroxy groups in the A-ring, were potent differentiators and the second group, bearing 1beta-hydroxy or 3alpha-hydroxy groups, were potent stimulators of apoptosis. CONCLUSIONS: We have clearly identified the structural motifs of 19-nor-1alpha,25(OH)(2)D(3) analogs responsible for differentiation and apoptosis in HL-60 cells. These findings will provide useful information not only for development of therapeutic agents for treatment of leukemia and other cancers, but also for structure-function studies of 1alpha,25(OH)(2)D(3).     

A concise and efficient route to 2alpha-(omega-hydroxyalkoxy)-1alpha,25-dihydroxyvi tam in D3: remarkably high affinity to vitamin D receptor

[reaction: see text]A convenient and potentially valuable synthetic approach to the novel 2alpha-functionalized 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] derivatives (1a-c), which are the C2-epimer of ED-71 and its analogues, has been developed. The C2alpha-modified ring A precursors (1,7-enynes 16, n = 0, 1, and 2) were constructed stereoselectively starting from D-glucose in high yield. In the synthesized 2alpha-(omega-hydroxyalkoxy)-1alpha,25(OH)2D3 derivatives, 1a and 1b showed a greater binding affinity to vitamin D receptor (VDR), up to 1.8 times that of the native hormone.     

Remarkable effect of 2[small alpha]-modification on the VDR antagonistic activity of 1small alpha-hydroxyvitamin D3-26,23-lactones

Novel 2[small alpha]-methyl-, 2[small alpha]-(3-hydroxypropyl)- and 2[small alpha]-(3-hydroxypropoxy)-substituted 25-dehydro-1[small alpha]-hydroxyvitamin D-26,23-lactone derivatives were efficiently synthesized Reformatsky type allylation and palladium-catalyzed alkenylative cyclization processes, and their biological activities were evaluated. Introducing functional groups into the 2[small alpha]-position of the vitamin D-26,23-lactones resulted in remarkable enhancement of their antagonistic activity on vitamin D receptor (VDR).     

Superagonistic fluorinated vitamin D3 analogs stabilize helix 12 of the vitamin D receptor

Side chain fluorination is often used to make analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] resistant to degradation by 24-hydroxylase. The fluorinated nonsteroidal analogs CD578, WU515, and WY1113 have an increased prodifferentiating action on SW480-ADH colon cancer cells, which correlated with stronger induction of vitamin D receptor (VDR)-coactivator interactions and stronger repression of beta-catenin/TCF activity. Cocrystallization of analog CD578 with the zebrafish (z)VDR and an SRC-1 coactivator peptide showed that the fluorine atoms of CD578 make additional contacts with Val444 and Phe448 of activation helix 12 (H12) of the zVDR and with Leu440 of the H11-H12 loop. Consequently, the SRC-1 peptide makes more contacts with the VDR-CD578 complex than with the VDR-1,25(OH)2D3 complex. These data show that fluorination not only affects degradation of an analog but can also have direct effects on H12 stabilization.     

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.     

The effect of antagonists on the conformational exchange of the retinoid X receptor alpha ligand‐binding domain

The effect of retinoid X receptor (RXR) antagonists on the conformational exchange of the RXR ligand‐binding domain (LBD) remains poorly characterized. To address this question, we used nuclear magnetic resonance spectroscopy to compare the chemical shift perturbations induced by RXR antagonists and agonists on the RXRα LBD when partnered with itself as a homodimer and as the heterodimeric partner with the peroxisome proliferator‐activated receptor γ (PPARγ) LBD. Chemical shift mapping on the crystal structure showed that agonist binding abolished a line‐broadening effect caused by a conformational exchange on backbone amide signals for residues in helix H3 and other regions of either the homo‐ or hetero‐dimer, whereas binding of antagonists with similar binding affinities failed to do so. A lineshape analysis of a glucocorticoid receptor‐interacting protein 1 NR box 2 coactivator peptide showed that the antagonists enhanced peptide binding to the RXRα LBD homodimer, but to a lesser extent than that enhanced by the agonists. This was further supported by a lineshape analysis of the RXR C‐terminal residue, threonine 462 (T462) in the homodimer but not in the heterodimer. Contrary to the agonists, the antagonists failed to abolish a line‐broadening effect caused by a conformational exchange on the T462 signal corresponding to the RXRα LBD–antagonist–peptide ternary complex. These results suggest that the antagonists lack the ability of the agonists to shift the equilibrium of multiple RXRα LBD conformations in favor of a compact state, and that a PPARγ LBD‐agonist complex can prevent the antagonist from enhancing the RXRα LBD‐coactivator binding interaction. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect on carbon lengthening at the side chain terminal of 1 alpha,25-dihydroxyvitamin D3 for calcium regulating activity

A series of analogs of 1 alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3 (1)] with alkyl substitutions in 26- and 27-positions have been tested for their activity 1) in competing with 1,25-(OH)2D3 for binding to chick intestinal cytosol receptor, 2) in ability for formation of multinucleated cells (MNC) with various osteoclastic cell characteristics from blast cells, and 3) in stimulating bone calcium mobilization in vitamin D-deficient rats. The relative potencies of 1,25-(OH)2D3, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 (2), 1 alpha,25-dihydroxy-26,27-diethylvitamin D3 (3), and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 (4) in competing for intestinal cytosolic binding were 1:1.1:0.25:0.05. The similar order of the abilities on formation of the multinucleated cells in the same series was observed. In a bone calcium mobilization test with vitamin D-deficient rats, 1 alpha,25-dihydroxy-26,27-dimethylvitamin D3 showed slightly less activity than 1,25-(OH)2D3 at 12 h after administration, but long lasting activity was observed during time course experiments. 1 alpha,25-Dihydroxy-26,27-diethylvitamin D3, and 1 alpha,25-dihydroxy-26,27-dipropylvitamin D3 were found to be much less active than 1,25-(OH)2D3 in a bone calcium mobilization test.     

Efficient synthesis of 2-modified 1alpha,25-dihydroxy-19-norvitamin D3 with Julia olefination: high potency in induction of differentiation on HL-60 cells

Six novel 2-substituted analogues of 1alpha,25-dihydroxy-19-norvitamin D(3), 6a,b-8a,b, were efficiently synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared as 4-alkylated (3R,5R)-3,5-dihydroxycyclohexanones 12-15 from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The new type of the CD-ring coupling partner 23 was synthesized from 25-hydroxy Grundmann's ketone 19 to apply to the modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were 47-62%. After the separation of the diastereomers based on C2 stereochemistry, the structure (2alpha or 2beta) was determined by (1)H NMR experiments and compared to DeLuca's 2-methyl- and 2-ethyl-1alpha,25-dihydroxy-19-norvitamin D(3). Thus, the synthesized 2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D(3) (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 8b had only a 3% affinity. Both 2alpha-allyl- and 2alpha-propyl-1alpha,25-dihydroxy-19-norvitamin D(3) (6a and 7a) and their 2beta-analogues (6b and 7b) 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 8b showed a 6.7-fold higher potency in the cell differentiation activity than that of 1.     

Development of 14-epi-19-nortachysterol and its unprecedented binding configuration for the human vitamin D receptor

In the study of the synthesis of 14-epi-19-norprevitamin D(3), we found 14-epi-19-nortachysterol derivatives through C6,7-cis/trans isomerization. We also succeeded in their chemical synthesis and revealed their marked stability and potent VDR binding affinity. To the best of our knowledge, this is the first isolation of stable tachysterol analogues. Surprisingly, 14-epi-19-nortachysterol derivatives exhibited an unprecedented binding configurations for the ligand binding pocket in hVDR, C5,6-s-trans and C7,8-s-trans triene configurations, which were opposite the natural C7,8-ene-configuration of 1α,25(OH)(2)D(3).     

Synthesis and pharmacokinetics of 1 alpha-hydroxyvitamin D3 tritiated at 22 and 23 positions showing high specific radioactivity

A novel synthesis of a radioactive compound of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-(3)H4]1 alpha OHD3) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22,23-(3)H4]1 alpha OHD3 (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studies with rats. In the pharmacokinetics studies, the plasma concentration level of the active form of vitamin D3, 1 alpha,25-dihydroxy-vitamin D3 [1 alpha,25(OH)2D3], after oral or intravenous administration of [22,23-(3)H4]1 alpha OHD3 (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1 alpha,25(OH)2D3 tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1 alpha OHD3 (1) over the treatment of 1 alpha,25(OH)2D3 (2).     

Design and efficient synthesis of 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D3 Analogues, including 2-epi-ED-71 and their 20-epimers with HL-60 cell differentiation activity

A concise and efficient synthetic approach to 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D(3) (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2 alpha-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification"of 1 based on 4a-c with C20-epimerization, affording 2 alpha-(omega-hydroxyalkoxy)-20-epi-1 alpha,25-dihydroxyvitamin D(3) (20-epi-4a-c). All three 2 alpha-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.     

Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,25(OH)2D3 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml(-1) for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.