Manabu Chokki

A novel and practical route to A-ring enyne synthon for 1α,25-dihydroxyvitamin D3 analogs: Synthesis of A-ring diastereomers of 1α,25-dihydroxyvitamin D3 and 2-methyl-1,25-dihydroxyvitamin D3

A novel and practical route to the A-ring enyne synthon (2), which can be versatile for a variety of A-ring analogs of 1α,25-dihydroxyvitamin D3 (1), was developed. This novel method led to an improved synthesis of the A-ring diastereomers of 1, the compounds 13–15, and synthesis of the new analogs, 2-methyl-1,25-dihydroxyvitamin D3 (4) with its all possible diastereomers. The biological evaluation of the 2-methyl analogs showed the ααβ-isomer to be more potent than 1.

Human airway trypsin-like protease induces amphiregulin release through a mechanism involving protease-activated receptor-2-mediated ERK activation and TNF alpha-converting enzyme activity in airway epithelial cells

Human airway trypsin‐like protease (HAT), a serine protease found in the sputum of patients with chronic airway diseases, is an agonist of protease‐activated receptor‐2 (PAR‐2). Previous results have shown that HAT enhances the release of amphiregulin (AR); further, it causes MUC5AC gene expression through the AR‐epidermal growth factor receptor pathway in the airway epithelial cell line NCI‐H292. In this study, the mechanisms by which HAT‐induced AR release can occur were investigated. HAT‐induced AR gene expression was mediated by extracellular signal‐regulated kinase (ERK) pathway, as pretreatment of cells with ERK pathway inhibitor eliminated the effect of HAT on AR mRNA. Both HAT and PAR‐2 agonist peptide (PAR‐2 AP) induced ERK phosphorylation; further, desensitization of PAR‐2 with a brief exposure of cells to PAR‐2 AP resulted in inhibition of HAT‐induced ERK phosphorylation, suggesting that HAT activates ERK through PAR‐2. Moreover, PAR‐2 AP induced AR gene expression subsequent to protein production in the cellular fraction through the ERK pathway indicating that PAR‐2‐mediated activation of ERK is essential for HAT‐induced AR production. However, in contrast to HAT, PAR‐2 AP could not cause AR release into extracellular space; it appears that activation of PAR‐2 is not sufficient for HAT‐induced AR release. Finally, HAT‐induced AR release was eliminated by blockade of tumour necrosis factor α‐converting enzyme (TACE) by the TAPI‐1 and RNA interference, suggesting that TACE activity is necessary for HAT‐induced AR release. These observations show that HAT induces AR production through the PAR‐2 mediated ERK pathway, and then causes AR release by a TACE‐dependent mechanism.

Synthesis and biological activity of 2-methyl-20-epi analogues of 1α,25-dihydroxyvitamin D3

Abstract Synthesis and biological evaluation of all eight possible A-ring diastereomers of 2-methyl-20-epi-1,25-dihydroxyvitamin D3 are described. Among the analogues synthesized, 2α-methyl-20-epi-1α,25-dihydroxyvitamin D3 exhibited exceptionally high potency. The double modification of 2-methyl substitution and 20-epimerization yielded analogues with unique activity profiles.

Antagonistic Actions in Vivo of (23S)-25-Dehydro-1α-Hydroxyvitamin D3-26,23-Lactone on Calcium Metabolism Induced by 1α,25-Dihydroxyvitamin D3

The vitamin D analog, (23S)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone (TEI-9647), is an antagonist of the 1α,25-dihydroxyvitamin D3[ 1α,25(OH)2D3] nuclear receptor (VDR)-mediated differentiation of human leukemia (HL-60) cells. To clarify whether TEI-9647 could function as an antagonist of 1α,25(OH)2D3 in vivo, we investigated in vitamin d-deficient (-D) rats the effects of single doses of TEI-9647 on several parameters of calcium metabolism modulated by 1α,25(OH)2D3. TEI-9647 (50μ g/kg iv) acting alone slightly, but significantly, stimulated intestinal calcium transport (ICA) and bone calcium mobilization (BCM) only at 8 h, but not at 24 h. In contrast, TEI-9647 dose-dependently inhibited ICA and BCM stimulated by an iv dose of 0.25 μg/kg 1α,25(OH)2D3 after 24 h, but not after 8 h. With respect to serum PTH levels, the administration of either TEI-9647, 50 μg/kg, or 1α,25(OH)2D3, 0.25 μg/kg, began to decrease the circulating levels by 4 h, which reached a nadir 24 h after administration. But, when TE...

(23S)- and (23R)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone function as antagonists of vitamin D receptor-mediated genomic actions of 1α,25-dihydroxyvitamin D3

Abstract We synthesized various analogues of 1α,25-(OH)2D3-26,23-lactone and examined the effects of them on HL-60 cell differentiation using the evaluation system of the genomic action of 1α,25-(OH)2D3. We found that (23S)- and (23R)-25-dehydro-1α-OH-D3-26,23-lactone (TEI-9647 and TEI-9648) strongly bound to the VDR, but did not induce HL-60 cell differentiation. Intriguingly, TEI-9647 and TEI-9648 did inhibit that induced by 1α,25-(OH)2D3, whereas they did not suppress that caused by retinoic acid or TPA. On the contrary, the similar 25-dehydrated 24-dehydro analogues, TEI-D1807 and TEI-D1808, weakly but significantly induced HL-60 cell differentiation, never showing inhibitory effect on HL-60 cell differentiation induced by 1α,25-(OH)2D3. In other experiments, TEI-9647 and TEI-9648 markedly suppressed 25-OH-D3-24-hydroxylase gene expression induced by 1α,25-(OH)2D3 in HL-60 cells. TEI-9647 also inhibited the heterodimer formation between VDR and RXRα, and the VDR interaction with co-activator SRC-1 according to the results obtained from the mammalian two-hybrid system in Saos-2 cells. Taking all these results into consideration, we reached a manifest conclusion that TEI-9647 and TEI-9648 are the specific and first antagonists of 1α,25-(OH)2D3 action, specifically VDR-VDRE mediated genomic action.

Systematic studies on synthesis, structural elucidation, and biological evaluation of A-ring diastereomers of 2-methyl-1α, 25-dihydroxyvitamin D3 and 20-epi-2-methyl-1α, 25-dihydroxyvitamin D3

Abstract All possible A-ring diastereomers of 2-methyl-1α,25-dihydroxyvitamin D 3 ( 2 ) and 20- epi -2-methyl-1α,25-dihydroxyvitamin D 3 ( 3 ) were synthesized by palladium-catalyzed coupling reaction of A-ring ‘enyne’ synthons with CD-ring portions. The A-ring synthons were rationally synthesized via a novel and practical route, starting with methyl ( R )-(+)- and ( S )-(-)-3-hydroxy-2-methyl-propionate, in good yields. X-ray crystallographic analysis of 2α-methyl-1α,25-dihydroxyvitamin D 3 ( 2b ) and conformational analysis of the A-ring of 2α-methyl-( 2b ) and 2β-methyl-1α,25-dihydroxyvitamin D 3 ( 2f ) were carried out, and the results are described. All A-ring diastereomers ( 2 and 3 ), thus synthesized, were biologically evaluated both in vitro and in vivo. The biologic potency was highly dependent on the stereochemistry of the A-ring substituents. In particular, 2b showed 4-fold higher vitamin D receptor [VDR] binding activity than the natural hormone, and its 20-epimer ( 3b ) exhibited exceptionally high activity, 12-fold more potent in VDR binding, 7-fold in calcium mobilization, and 590-fold in induction of human promyelocytic leukemia (HL-60) cell differentiation as compared with the natural hormone. Further, the 20- epi –2β-Me-1β, 3α(OH) 2 isomer ( 3g ) had significant biologic potencies compared to the natural hormone despite having 1β-OH configuration. The transcriptional activities on human osteocalcin gene promoter, including VDRE in transfected mammalian cells, were also evaluated. Finally, there was a clear contrast between the effects of the 2-methyl group on the HL-60 cell differentiation- and apoptosis-inducing activities of 2 and 3 .

TEI-A00114: A new vitamin D3 analogue that inhibits neutrophil recruitment in an acute lung injury hamster model while showing reduced hypercalcemic activity

While searching for vitamin D(3) analogues which inhibit neutrophil recruitment in the lung without elevating plasma calcium level, we found that (5Z,7E)-(1S,3R)-20(R)-[(5E)-(2S)-2-hydroxy-2-methyl-cyclopentanone-5-ylidene]methyl-9,10-secopregna-5,7,10(19)-triene-1,3-diol (TEI-A00114) had the best efficacy and calcemic action. TEI-A00114 has a vitamin D receptor affinity 2.5-fold weaker and a vitamin D binding protein affinity 330.9-fold weaker than those of 1α,25(OH)(2)D(3). The estimated effective doses for 40% inhibition (ED(40)) via peroral and intratracheal administration are 7.6 and 0.4 μg/kg, respectively. TEI-A00114 was also tested by inhaled administration, and its ED(40) was calculated as 0.2 μg/kg. The estimated 40% inhibitory concentration (IC(40)) of TEI-A00114 on interleukin (IL)-8 production induced by lipopolysaccharide and on IL-1β in human whole blood cells in vitro were 9.8 × 10(-8) or 1.8 × 10(-9)M, respectively. These levels of TEI-A00114s activities are equal to those of 1α,25(OH)(2)D(3). On the other hand, the calcemic action of TEI-A00114, which was evaluated at day 14 after sequential peroral quaque die administration, was 89-fold weaker (molar ratio) than that of 1α,25(OH)(2)D(3). These results indicate that TEI-A00114 has a dissociated profile between inhibition of neutrophil recruitment in the lung and calcemic action, suggesting its suitability over 1α,25(OH)(2)D(3) as a candidate for the treatment of acute lung injury.