WY 1048, a 17-methyl 19-nor D-ring analog of vitamin D3, in combination with risedronate restores bone mass in a mouse model of postmenopausal osteoporosis.

Bisphosphonates like risedronate inhibit osteoclast-mediated bone resorption and are therefore used in the prevention and treatment of osteoporosis. Also vitamin D3 and calcium supplementation is commonly used in the prevention or treatment of osteoporosis. Combined therapy of risedronate with 1,25(OH)2D3, the active metabolite of vitamin D3, may be advantageous over the use of either monotherapy, but bears a risk of causing hypercalcemia thereby decreasing the therapeutic window for osteoporosis treatment. In this study, we evaluated the effect on bone mass of the combination of risedronate with the 17-methyl 19-nor five-membered D-ring vitamin D3 analog WY 1048 in a mouse ovariectomy model for postmenopausal osteoporosis. Ovariectomy-induced bone loss was restored by administration of risedronate or a combination of risedronate with 1,25(OH)2D3. However, the combination of WY 1048 with risedronate induced an even higher increase on total body and spine bone mineral density and on trabecular and cortical bone mass. Our data indicate that combination therapy of risedronate with WY 1048 was superior in restoring and improving bone mass over a combination of risedronate with 1,25(OH)2D3 with minimal calcemic side effects.

Possibility of [1,5] sigmatropic shifts in bicyclo[4.2.0]octa-2,4-dienes.

The thermal equilibration of the methyl esters of endiandric acids D and E was subject to a computational study. An electrocyclic pathway via an electrocyclic ring opening followed by a ring flip and a subsequent electrocyclization proposed by Nicolaou [ Nicolaou , K. C. ; Chen , J. S. Chem. Soc. Rev. 2009 , 38 , 2993 ], was computationally explored. The free-energy barrier for this electrocyclic route was shown to be very close to the bicyclo[4.2.0]octa-2,4-diene reported by Huisgen [ Huisgen , R. ; Boche , G. ; Dahmen , A. ; Hechtl , W. Tetrahedron Lett. 1968 , 5215 ]. Furthermore, the possibility of a [1,5] sigmatropic alkyl group shift of bicyclo[4.2.0]octa-2,4-diene systems at high temperatures was explored in a combined computational and experimental study. Calculated reaction barriers for an open-shell singlet biradical-mediated stepwise [1,5] sigmatropic alkyl group shift were shown to be comparable with the reaction barriers for the bicyclo[4.1.0]hepta-2,4-diene (norcaradiene) walk rearrangement. However, the stepwise sigmatropic pathway is suggested to only be feasible for appropriately substituted compounds. Experiments conducted on a deuterated analogous diol derivative confirmed the calculated (large) differences in barriers between electrocyclic and sigmatropic pathways.

1,25-Dihydroxyvitamin D3 and its analog TX527 promote a stable regulatory T cell phenotype in T cells from type 1 diabetes patients.

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+ CD25high CD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+ CD25high CD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+ CD25high CD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.

Cell cycle arrest and apoptosis induced by 1α,25(OH)2D3 and TX 527 in Kaposi sarcoma is VDR dependent.

We have previously shown that 1α,25(OH)2-Vitamin D3 [1α,25(OH)2D3] and its less calcemic analog TX 527 inhibit the proliferation of endothelial cells transformed by the viral G protein-coupled receptor associated to Kaposi sarcoma (vGPCR) and this could be partially explained by the inhibition of the NF-κB pathway. In this work, we further explored the mechanism of action of both vitamin D compounds in Kaposi sarcoma. We investigated whether the cell cycle arrest and subsequent apoptosis of endothelial cells (SVEC) and SVEC transformed by vGPCR (SVEC-vGPCR) elicited by 1α,25(OH)2D3 and TX 527 were mediated by the vitamin D receptor (VDR). Cell cycle analysis of SVEC and SVEC-vGPCR treated with 1α,25(OH)2D3 (10nM, 48h) revealed that 1α,25(OH)2D3 increased the percentage of cells in the G0/G1 phase and diminished the percentage of cells in the S phase of the cell cycle. Moreover, the number of cells in the S phase was higher in SVEC-vGPCR than in SVEC due to vGPCR expression. TX 527 exerted similar effects on growth arrest in SVEC-vGPCR cells. The cell cycle changes were suppressed when the expression of the VDR was blocked by a stable transfection of shRNA against VDR. Annexin V-PI staining demonstrated apoptosis in both SVEC and SVEC-vGPCR after 1α,25(OH)2D3 and TX 527 treatment (10nM, 24h). Cleavage of caspase-3 detected by Western blot analysis was increased to a greater extent in SVEC than in SVEC-vGPCR cells, and this effect was also blocked in VDR knockdown cells. Altogether, these results suggest that 1α,25(OH)2D3 and TX 527 inhibit the proliferation of SVEC and SVEC-vGPCR and induce apoptosis by a mechanism that involves the VDR.

The vitamin D analog TX527 ameliorates disease symptoms in a chemically induced model of inflammatory bowel disease.

The vitamin D system plays a critical role in inflammatory bowel disease as evidenced by the finding that both vitamin D deficient mice and vitamin D receptor knockout mice are extremely sensitive to dextran sodium sulfate (DSS)-induced colitis. Moreover, the active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is an important immunomodulator that ameliorates the pathogenesis of inflammatory bowel disease. However, therapeutic application of 1,25(OH)2D3 is hampered by its calcemic activity. Previous work illustrated that the analog 1α,25(OH)2-19-nor-14,20-bisepi-23-yne-vitamin D3 (TX527) has potent antiproliferative effects with limited calcemic activity. In the present study we demonstrated that TX527 ameliorated disease symptoms in a DSS-induced model of inflammatory bowel disease. TX527 significantly attenuated disease scores, by suppressing bleeding and diarrhea. Colon length was significantly elevated at the end of the experiment. Histological examination indicated that TX527 diminished mucosal damage and crypt loss and suppressed the infiltration of immune cells in DSS-induced colitis mice. Furthermore, transcript levels of inflammatory cytokines such as IL-1, IL-6, IFN-γ and TNF-α were significantly down-regulated in colonic mucosa of mice with colitis. Moreover, transcript levels of the gastrointestinal glutathione peroxidase 2, which acts as a radical scavenger, were significantly down-regulated after TX527 treatment in DSS-colitis mice. These results indicate that TX527 may have a therapeutic value in the setting of inflammatory bowel disease. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

CD-ring modified vitamin D3 analogs and their superagonistic action.

Non-steroidal analogs of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] represent a most particular class of analogs because they are either not directly derived from the core 1,25(OH)2D3-structure or they have modifications in the core structure that are so drastic that the steroidal structure is lost. Non-steroidal CD-ring analogs of 1,25(OH)2D3 have been developed to study the role of the central rigid CD-ring system in the biological activity of 1,25(OH)2D3. Here we review the different classes of CD-ring analogs and highlight some representative analogs such as the fluorinated D-ring analogs CD578, WU515 and WY1113 which show markedly increased differentiating activity on human SW480-ADH colon cancer cells, characterized by a stronger induction of the invasion suppressor E-cadherin and a stronger repression of the beta-catenin/TCF target oncogene c-Myc. Correspondingly, CD578, WU515 and WY1113 are more potent inhibitors of beta-catenin/TCF signaling than 1,25(OH)2D3 and induce stronger VDR-coactivator interactions. Underlying the increased biological potency of analog CD578 are additional contacts between the side chain fluorine atoms of the analog with specific residues of helix 12 (H12) of the Vitamin D Receptor (VDR) and subsequent stronger VDR-coactivator interactions.

Antiproliferative and calcemic actions of trans-decalin CD-ring analogs of 1,25-dihydroxyvitamin D3.

BACKGROUND: 1,25-Dihydroxyvitamin D3 [1,25(OH)2D3] has potent antiproliferative actions but calcemic effects obstruct its application in the treatment of hyperproliferative disorders. Therefore, analogs of 1,25-(OH)2D3 are designed with a clear dissociation between both effects. Here the biological activity of the trans-decalin CD-ring analog CY10012 is discussed. MATERIALS AND METHODS: Proliferation/differentiation/transactivation assays as well as mouse models were used to determine the activity of CY10012 in vitro and in vivo. RESULTS: CY10012, has ten-fold higher antiproliferative actions than 1,25(OH)2D3 but is also twice as calcemic. To determine the role of the Vitamin D Receptor (VDR) in mediating the calcemic actions of CY10012, the analog was daily administered to VDRwt and VDRko mice. This treatment caused drastic weight loss and death in VDRwt mice but not in VDRko mice. CONCLUSION: Analog CY10012 has greater antiproliferative action but also two-fold higher calcemic effects which depended entirely on VDR-mediated signalling pathways.

Microarray analysis of MCF-7 breast cancer cells treated with 1,25-dihydroxyvitamin D3 or a 17-methyl-D-ring analog.

BACKGROUND: 1alpha,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] is the biological active form of vitamin D. Its antiproliferative capacities make it a potential drug to treat diseases such as cancer. The clinical use of 1,25-(OH)2D3 as an antiproliferative agent is hampered by its calcemic effects. Hence, structural analogs such as the seco-9,11-bisnor-17-methyl analog, WY1112, have been developed with superagonistic capacities. This study aims to distinct the molecular activities of 1,25-(OH)2D3 and WY1112 and identify possible differences in gene expression. MATERIALS AND METHODS: Total RNA was extracted from MCF-7 breast cancer cells treated with 1,25-(OH)2D3 or WY1112 and was used for microarray analysis. RESULTS: The experiments revealed that WY1112 induces the same genes as 1,25-(OH)2D3, but the induction level of the individual genes is higher. Microarray analysis did not reveal genes that were exclusively regulated by WY1112. CONCLUSION: The superagonistic vitamin D analog WY1112 induces the same set of genes as 1,25-(OH)2D3, but the level of induction of the individual genes is higher.

Synthesis of 1alpha,25-dihydroxyvitamin D analogues featuring a S(2)-symmetric CD-ring core.

Three analogues of 1a,25-dihydroxyvitamin D(3) (calcitriol), featuring a trans-fused decalin C,D-core with local S(2)-symmetry, and possessing identical side-chain and seco-B,A-ring structures, have been synthesized starting from readily available (4aR,8aS)-octahydronaphthalene-1,5-dione (7). The very short sequences involve the simultaneous introduction of the side-chain and seco-B,A-ring fragments via Suzuki and Sonogashira coupling reactions. The analogues are devoid of relevant biological activity.

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.

Nonenzymic polycyclization of analogues of oxidosqualene with a preformed C-ring.

Some nonenzymic epoxide-initiated polyolefin cyclization are reported. The presented molecules are partially constrained analogues of (3S)-oxidosqualene, the natural substrate to many important cyclase enzymes. These model compounds feature a preformed C-ring with built-in stereochemical information. The experimental results allow for an instructive comparison with the enzymic processes, particularly those of the cyclases in steroid biosynthesis (i.e. lanosterol synthase).

Synthesis of 3,4,7,8-tetrahydronaphthalene-1,5(2H,6H)-dione.

A short and high yielding route for the preparation of the title compound, starting from commercially available 1,5-dihydroxynaphthalene, is described. The key step in the sequence is the air oxidation of a bis(trimethylsilyloxy)diene precursor.

Asymmetric synthesis of the epimeric (3S)-3-((E)-hex-1-enyl)-2-methylcyclohexanones.

The asymmetric rhodium-catalysed 1,4-addition of alkenylzirconium reagents to 2-cyclohexenone can be useful in the synthesis of 3-alkenyl-2-methylcyclohexanones, provided that formaldehyde is used in trapping the intermediate zirconium enolates. In this manner a four-step sequence leading to the two epimeric 3-hexenyl-2-methylcyclohexanones in enantiomeric form was developed.

The development of CD-ring modified analogs of 1alpha,25-dihydroxyvitamin D.

During a 20-year collaboration the laboratories of UGent and KU Leuven have developed different series of Vitamin D analogs characterized by structural modifications in the central CD-ring system. Modifications have first involved the introduction of substituents at C11 and the epimerization at C14, and subsequently more drastic changes consisting in both ring deletion and enlargement relative to the natural CD-ring system. Lately, the focus has shifted towards the synthesis of analogs featuring a symmetrical CD-ring core. As an illustration two different series are presented.

Removal of C-ring from the CD-ring skeleton of 1alpha,25-dihydroxyvitamin D3 does not alter its target tissue metabolism significantly.

It is now well established that 1alpha,25(OH)2D3 is metabolized in its target tissues through the modifications of both side chain and A-ring. The C-24 oxidation pathway is the side chain modification pathway through which 1alpha,25(OH)2D3 is metabolized into calcitroic acid. The C-3 epimerization pathway is the A-ring modification pathway through which 1alpha,25(OH)2D3 is metabolized into 1alpha,25(OH)2-3-epi-D3. During the past two decades, a great number of vitamin D analogs were synthesized by altering the structure of both side chain and A-ring of 1alpha,25(OH)2D3 with the aim to generate novel vitamin D compounds that inhibit proliferation and induce differentiation of various types of normal and cancer cells without causing significant hypercalcemia. Previously, we used some of these analogs as molecular probes to examine how changes in 1alpha,25(OH)2D3 structure would affect its target tissue metabolism. Recently, several nonsteroidal analogs of 1alpha,25(OH)2D3 with unique biological activity profiles were synthesized. Two of the analogs, SL 117 and WU 515 lack the C-ring of the CD-ring skeleton of 1alpha,25(OH)2D3. SL 117 contains the same side chain as that of 1alpha,25(OH)2D3, while WU 515 contains an altered side chain with a 23-yne modification combined with hexafluorination at C-26 and C-27. Presently, it is unknown how the removal of C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 would affect its target tissue metabolism. In the present study, we compared the metabolic fate of SL 117 and WU 515 with that of 1alpha,25(OH)2D3 in both the isolated perfused rat kidney, which expresses only the C-24 oxidation pathway and rat osteosarcoma cells (UMR 106), which express both the C-24 oxidation and C-3 epimerization pathways. The results of our present study indicate that SL 117 is metabolized like 1alpha,25(OH)2D3, into polar metabolites via the C-24 oxidation pathway in both rat kidney and UMR 106 cells. As expected, WU 515 with altered side chain structure is not metabolized via the C-24 oxidation pathway. Unlike in rat kidney, both SL 117 and WU 515 are also metabolized into less polar metabolites in UMR 106 cells. These metabolites displayed GC and MS characteristics consistent with A-ring epimerization and were putatively assigned as C-3 epimers of SL 117 and WU 515. In summary, we report that removal of the C-ring from the CD-ring skeleton of 1alpha,25(OH)2D3 does not alter its target tissue metabolism significantly.

Application of the B-alkyl suzuki-miyaura cross-coupling reaction to the stereoselective synthesis of analogues of (3S)-oxidosqualene.

[reaction: see text] A general method is described for the direct and stereoselective synthesis of epoxypolyenes via Suzuki-Miyaura cross-coupling reaction of 1-iodoalkenes with B-alkylboron compounds. It allows for the straightforward and convergent assembly of compounds that are structurally similar to (3S)-oxidosqualene, an important intermediate in steroid biosynthesis.

Synthesis of spiro[4.5]decane CF-ring analogues of 1 alpha,25-dihydroxyvitamin D3.

A novel series of analogues of calcitriol (1) is developed featuring a spirocyclic central core resulting from C18/C21-connection and C15/C16-deletion (2a, 2b). The synthesis of the key intermediate involves an Eschenmoser rearrangement of an enantiomerically pure bromo-substituted cyclohexenol.

Vitamin D analogs and coactivators.

The secosteroid hormone 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] has potent antiproliferative and prodifferentiating actions on a wide variety of normal as well as malignant cell types. Strong calcemic effects obstruct the actual application of 1,25-(OH)2D3 for the treatment of hyperproliferative disorders such as cancer. To overcome this problem, structural analogs of 1,25-(OH)2D3 have been designed with a clear dissociation between antiproliferative and calcemic effects. This review focuses on the molecular mode of action of different 1,25-(OH)2D3 analogs and, in particular, on the recruitment of cofactor molecules to the vitamin D receptor by these analogs.

Application of the solid-phase Julia-Lythgoe olefination in vitamin D side-chain construction.

An example of the Julia-Lythgoe attachment of the vitamin D side chain to a solid-phase linked Inhoffen-Lythgoe diol derived CD-ring fragment is reported.

Asymmetric synthesis of (7aS)-7a-methyl-4,5,7,7a-tetrahydro-1H-indene-2,6-dione and useful derivatives thereof.

The enantioselective synthesis of the title compound, using Meyers' bicyclic lactam methodology, is described. This compound and a few of its derivatives are useful intermediates in natural product synthesis.