Design and Synthesis of Fluoro Analogues of Vitamin D.

The discovery of a large variety of functions of vitamin D3 and its metabolites has led to the design and synthesis of a vast amount of vitamin D3 analogues in order to increase the potency and reduce toxicity. The introduction of highly electronegative fluorine atom(s) into vitamin D3 skeletons alters their physical and chemical properties. To date, many fluorinated vitamin D3 analogues have been designed and synthesized. This review summarizes the molecular structures of fluoro-containing vitamin D3 analogues and their synthetic methodologies.

Synthesis and vitamin D receptor affinity of 16-oxa vitamin D3 analogues.

Two novel 16-oxa-vitamin D3 analogues were synthesized using a tandem Ti(ii)-mediated enyne cyclization/Cu-catalyzed allylation, Ru-catalyzed ring-closing metathesis reaction, and a low-valent titanium (LVT)-mediated stereoselective radical reduction of 8α,14α-epoxide as the key steps for the synthesis of the 16-oxa-C,D ring unit. The vitamin D receptor-binding affinity of the synthesized analogues, 16-oxa-1α,25-(OH)2VD3 and 16-oxa-19-nor-1α,25-(OH)2VD3, was evaluated by fluorescence polarization vitamin D receptor competitor assay and time-resolved fluorescence energy transfer vitamin D receptor co-activator assay.

Studies on the Synthesis of Vitamin D Analogs with Aromatic D-Ring.

Herein, we describe our studies on the synthesis of 1α,25-dihydroxyvitamin D3 analogs possessing a benzene ring replacing the natural 5-membered D-ring by the Wittig-Horner and dienyne approaches. A key feature is the synthesis of a Cr(CO)3-complexed previtamin D derivative that enables the construction of vitamin D analogs with aromatic D-ring through a thermal [1,7]-H sigmatropic shift. This study establishes the basis for the design of new vitamin D analogs containing aromatic D-ring, complexed or uncomplexed to Cr(CO)3 type moieties for specific molecular recognition and drug research and development.

Seco-B-Ring Steroidal Dienynes with Aromatic D Ring: Design, Synthesis and Biological Evaluation.

Continuing our structure-activity studies on the vitamin D analogs with the altered intercyclic seco-B-ring fragment, we designed compounds possessing dienyne system conjugated with the benzene D ring. Analysis of the literature data and the docking experiments seemed to indicate that the target compounds could mimic the ligands with a good affinity to the vitamin D receptor (VDR). Multi-step synthesis of the C/D-ring building block of the tetralone structure was achieved and its enol triflate was coupled with the known A-ring fragments, possessing conjugated enyne moiety, using Sonogashira protocol. The structures of the final products were confirmed by NMR, UV and mass spectroscopy. Their binding affinities for the full-length human VDR were determined and it was established that compound substituted at C-2 with exomethylene group showed significant binding to the receptor. This analog was also able to induce monocytic differentiation of HL-60 cells.

Helix12-Stabilization Antagonist of Vitamin D Receptor.

To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and synthesized vitamin D analogues with bulky side chains based on the "active antagonist" concept in which antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds 3a and 3b showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and static X-ray crystal structure analyses indicated that compound 3a stabilizes H11-H12 but displaces H6-H7 so that 3a is a novel rather than "active" or "passive" type of antagonist. We classified 3a as a third type of antagonist and called it "H11-H12 stabilization antagonist". HDX-MS analysis indicated that antagonist 3b is an "active" antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse depending on each antagonist structure. Additionally, HDX-MS was proven to be very useful for investigations of protein structure alterations resulting from ligand binding.

Synthesis and biological activity of two C-7 methyl analogues of vitamin D.

Two novel vitamin D analogues of the hormone 1α,25-(OH)2D3 modified at C-7, namely, 7-methyl-1α,25-(OH)2D3 (12) and 7-methyl-1α,25-(OH)2-19-nor-D3 (26), were synthesized and biologically evaluated to gain further insights into the structure-function relationships of vitamin D. Key steps in the synthesis of 12 include the functionalization at C-7 by an efficient regioselective hydrostannylation of an allene precursor, and the construction of the triene framework by a palladium-catalyzed intramolecular cyclization-Suzuki-Miyaura coupling cascade. Since the calcitriol analogue 12 was prone to conversion into its previtamin D form by thermal equilibration, the corresponding 19-nor-compound 26 was also synthesized. The diene moiety of compound 26 was constructed by a modified Julia coupling. UV data as well as X-ray analysis indicate that introduction of the methyl group at C-7 results in a significant deviation from planarity of the 5,7-diene moiety. The new vitamin D analogues 12 and 26 retained good VDR binding ability.

Vitamin D analogue: potent antiproliferative effects on cancer cell lines and lack of hypercalcemic activity.

The active form of vitamin D3, 1α,25(OH)2D3, plays a major role in maintaining calcium/phosphate homeostasis. In addition, it is a potent antiproliferative and pro-differentiating agent. Unfortunately, it usually causes hypercalcemia in vivo when effective antitumour doses are used. It has therefore been found necessary to synthesise new analogues that retain or even increase the antitumour effects but preclude hypercalcemia. This report presents the synthesis of a novel Gemini vitamin D analogue (UVB1) and its biological evaluation. We demonstrate that this compound has potent antitumoural effects over a wide panel of tumour cell lines while showing lack of hypercalcemic activity and toxicity effects in in vivo assays.

Structural development of p-carborane-based potent non-secosteroidal vitamin D analogs.

Non-secosteroidal vitamin D receptor (VDR) ligands are promising candidates for many clinical applications. We recently developed novel non-secosteroidal VDR agonists based on p-carborane (an icosahedral carbon-containing boron cluster) as a hydrophobic core structure. Here, we report the design, synthesis and biological evaluation of carborane-based vitamin D analogs bearing various substituents at the diol moiety. Among the synthesized compounds, methylene derivative 31 exhibited the most potent vitamin D activity, which was comparable to that of the natural hormone, 1α,25(OH)2D3. This compound is one of the most potent non-secosteroidal VDR agonists reported to date, and is a promising lead for development of novel drug candidates.

Synthesis and structure-activity relationship of p-carborane-based non-secosteroidal vitamin D analogs.

1α,25-Dihydroxyvitamin D3 [1α,25(OH)2D3: 1] is a specific modulator of nuclear vitamin D receptor (VDR), and novel vitamin D analogs are therapeutic candidates for multiple clinical applications. We recently developed non-secosteroidal VDR agonists bearing a p-carborane cage (a carbon-containing boron cluster) as a hydrophobic core structure. These carborane derivatives are structurally quite different from classical secosteroidal vitamin D analogs. Here, we report systematic synthesis and activity evaluation of carborane-based non-secosteroidal vitamin D analogs. The structure-activity relationships of carborane derivatives are different from those of secosteroidal vitamin D derivatives, and in particular, the length and the substituent position of the dihydroxylated side chain are rather flexible in carborane derivatives. The structure-activity relationships presented here should be helpful in development of non-secosteroidal vitamin D analogs for clinical applications.

Design and synthesis of novel 1,25-dihydroxyvitamin D3 analogues having a spiro-oxetane fused at the C2 position in the A-ring.

Four structurally novel stereoisomeric analogues of 1,25-dihydroxyvitamin D3 (3a-d) bearing a spiro-oxetane fused at the C2 position of the A-ring have been designed and synthesised in a convergent manner. The requisite A-ring enyne precursors (13a,b) for the vitamin D analogues (3a,b) and (3c,d), respectively, were synthesised from pentaerythritol according to an eleven-step procedure. Preliminary biological evaluation of the analogues using the bovine thymus vitamin D receptor (VDR) suggested that the incorporation of the spiro-oxetane moiety instead of a gem-dimethyl group at the C2 position had a beneficial effect on the VDR affinity.

Synthesis of C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation, especially biological effect on bone by modification at the C-2 position.

In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and demonstrated the SAR of the C-2 position of VD(3). Furthermore, by using the OVX model, we found that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported compound, 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D(3)1d, and this derivative shows a possibility that calcemic activity is less than 1d in vivo.

Unravelling the details of vitamin D photosynthesis by non-adiabatic molecular dynamics simulations.

We investigate the photodynamics of vitamin D derivatives by a fully analytical implementation of the linear response time-dependent density functional theory surface hopping method (LR-TDDFT-SH). Our study elucidates the dynamics of the processes involved in vitamin D formation at the molecular level and with femtosecond resolution. We explain the major experimental findings and provide new insights that cannot directly be obtained from experiments: firstly, we investigate the dynamics of the photoinduced ring-opening of provitamin D (Pro) and cyclohexadiene (CHD) and the subsequent rotational isomerization. In agreement with recent experiments and CC2 calculations, only the bright S(1) state is involved in the ring-opening reaction. Our calculations confirm the experimentally reported 5 : 1 ratio between the excited state lifetimes of Pro and CHD. The longer lifetimes of Pro are attributed to steric constraints of the steroid skeleton and to temperature effects, both emerging directly from our simulations. For CHD and Pro, we present an explanation of the biexponential decay recently reported by Sension and coworkers [Tang et al., J. Phys. Chem., 2011, 134, 104503]: our calculations suggest that the fast and slow components arise from a reactive and an unreactive reaction pathway, respectively. Secondly, we assess the wavelength dependent photochemistry of previtamin D (Pre). Using replica exchange molecular dynamics we sample the Pre conformers present at thermal equilibrium. Based on this ensemble we explain the conformation dependent absorption and the essential features of Pre photochemistry. Consistent with the experiments, we find ring-closure to occur mostly after excitation of the cZc conformers and at lower energies, whereas Z/E isomerization of the central double bond preferably occurs after excitation at higher energies. For the isomerization we provide the first theoretical evidence of the proposed hula-twist mechanism. Our results show that LR-TDDFT-SH is a highly valuable tool for studying the photochemistry of moderately large systems, even though challenges remain in the vicinity of conical intersections.

The Synthesis and Biological Evaluation of D-Ring-Modified Vitamin D Analogues.

The vitamin D3 structure consists of the A-ring, a linker originating from the B-ring, C-ring, D-ring, and side-chain moieties. Each unit has its unique role in expressing the biological activities of vitamin D3. Many efforts have been made to date to assess the possible clinical use of vitamin D. Some organic chemists focused on the D-ring structure of vitamin D and synthesized D-ring-modified vitamin D analogues, and their biological activities were studied. This review summarizes the synthetic methodologies of D-ring-modified vitamin D analogues, except for seco-D, and their preliminary biological profiles.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Synthesis of 2alpha-propoxy-1alpha,25-dihydroxyvitamin D3 and comparison of its metabolism by human CYP24A1 and rat CYP24A1.

A new vitamin D(3) analogue, 2alpha-propoxy-1alpha,25-dihydroxyvitamin D(3) (C3O1), was synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We studied the metabolism of the new analogue by human CYP24A1 and rat CYP24A1 to learn of species-based differences and found that the former has multiple metabolic pathways, but the latter has only a single pathway.

'Novel alkyl side chain sulfone 1alpha,25-dihydroxyvitamin D3 analogs: a comparison of in vitro antiproliferative activities and in vivo calcemic activities'.

The replacement of a t-butyl group with a trifluoromethyl group has profound effects on the biological profile of 1alpha,25-dihydroxyvitamin D(3) sulfone analogs. Investigation of whether the improved biological activities are due to steric and electronic factors of the trifluoromethyl group led to the design, synthesis and biological evaluation of two analogous alkyl sulfone molecules, methyl sulfone (AU-16-ene-25-SO(2)-CH(3)) and isopropyl sulfone (AU-16-ene-25-SO(2)-i-Pr). These alkyl sulfones are sterically comparable to, but electronically very different from a trifluoromethyl group. The syntheses, antiproliferative activities and calcemic activities of these new alkyl sulfones are presented herein. In comparing the in vitro antiproliferative profiles of the new alkyl sulfone 1alpha,25-dihydroxyvitamin D(3) analogs with the trifluoromethylsulfone and an analogous t-butyl sulfone, the activities increase in the following order: CH(3) < t-Bu approximately = i-Pr < CF(3). In contrast to the calcemic t-butyl sulfone, the novel alkyl sulfones and trifluoromethyl sulfone display desirable low calcemic levels.

Synthesis of 1α,25-dihydroxyvitamin D3 analogues with α,α-difluorocycloketone at the CD-ring side chains and their biological properties in ovariectomized rats.

Two novel 1α,25-dihydroxyvitamin D3 derivatives containing a α,α-difluorocyclopentanone (3) or α,α-difluorocyclohexanone (4) moiety at the CD-ring side chains were designed, synthesized, and evaluated for their biological properties on restoring bone mass in ovariectomized (OVX) rats with established osteopenia. The synthesis of compounds 3 and 4 utilized the Wittig-Horner coupling to build up the vitamin D conjugated triene system, followed by the introduction of the cycloketone fragments at the side chain, and subsequent α,α-difluorination of the ketone by the treatment of the derived silyl enol ether with Selectfluor, as the key synthetic steps. In comparison with the natural 1α,25-dihydroxyvitamin D3 (calcitriol; 200 ng/kg/day), oral administration of compounds 3 and 4 at the dose of 25 ng/kg/day for 6 weeks led to much improved bone mass and bone density related parameters, while maintaining normal serum calcium and serum phosphorus levels. The immunohistochemistry results showed that both compounds remarkably decreased in osteoclast number and moderately decreased in osteoblast number on trabecular bone surface. Therefore, our findings suggested that compounds 3 and 4 successfully rescue bone loss by suppression on bone turnover in OVX rat models.

Structure-activity relationship studies on vitamin D lactam derivatives as vitamin D receptor antagonist.

Structure-activity relationship studies on 1alpha,25-dihydroxyvitamin D(3)-26,23-lactams (DLAMs), antagonists of vitamin D, were conducted, focusing on the substituents of the phenyl group. One of the derivatives (23S,25S)-DLAM-1P-3,5(OEt)(2), showed potent antagonistic activity with an IC(50) of 90nM.

2-Methylene 19-nor-25-dehydro-1alpha-hydroxyvitamin D3 26,23-lactones: synthesis, biological activities and molecular basis of passive antagonism.

To investigate the molecular mechanism of vitamin D receptor (VDR) antagonists having no structurally bulky group interfering with helix 12 of the ligand-binding domain of the VDR, we have synthesized four diastereomers at C(20) and C(23) of 19-nor-1alpha-hydroxyvitamin D(3) 25-methylene-26,23-lactone bearing a 2MD-type A-ring. All four analogs showed significant VDR affinity. Transactivation was tested by using Cos7 cells and HEK293 cells. In both types of cells, LAC67a showed little transactivation potency and inhibited the activation induced by the natural hormone concentration-dependently, indicating that LAC67a works as an antagonist for the VDR in these cells. LAC67b, LAC82a and LAC82b similarly acted as VDR antagonists in Cos7 cells, but in HEK293 cells they behaved as potent VDR agonists. Docking of four lactones into the VDR-LBD, followed by structural analysis, demonstrated that each lactone lacks the hydrophobic interaction with helix12 necessary for maintaining the active conformation of the VDR, indicating that these lactones are passive-type antagonists. Furthermore, each docking structure explained the characteristic transactivation profiles of the four lactones. On the basis of our present findings, we suggest that the ligand acts as an agonist if there are appropriate coactivators in the cells to bind to the looser VDR-ligand complex, and as an antagonist if there are no such appropriate coactivators. The molecular basis of the passive antagonism is discussed in detail.

Synthesis and biological activity of previtamin D(3) analogues with A-ring modifications.

Synthesis of two novel 6-s-cis analogues of 1alpha,25-dihydroxyvitamin D3 are described using shikimic acid and its 4-epi isomer as versatile chiral starting materials. These derivatives contain a 2beta-(3'-hydroxypropoxy) moiety or a 2beta,3beta-epoxy group into 1alpha,25-OH(2)-19-nor-pre-D3. The synthesized analogues were found to be not suitable for binding to the vitamin D receptor and showed weak binding affinity toward the vitamin D-binding protein. The new derivatives failed to inhibit cell proliferation.