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.

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.

On the synthesis of vitamin d in the darkness / Sobre la síntesis de vitamina D en la oscuridad

Published evidence reports the existence of two routes for the transformation of 7-dehydrocholecalciferol into previtamin D3: a photochemical route with the participation of UVB photons and another route that occurs in the darkness. Several reports appears to support the presence of these two routes in some mammals, birds, nonvascular plants (e.g.: mosses), vascular plants (e.g.: angiosperms) and lichens. The reviewed evidence suggests that in the darkness, the synthesis of vitamin D3 follows the same scheme of the photochemical pathway, but at a reduced rate respect to the synthesis under UVB radiation. The process of vitamin D synthesis in the dark, then, may be taken as an insurance for survival, at least for mammals and birds. The low rate of the synthesis of vitamin D3 in the absence of light produce low concentrations of vitamin D3 metabolites in plasma. Long term survival under these circumstances might be possible through upregulation of vitamin D receptors (VDRs). In mole rats (South African rodents that live in the dark in underground tunnels), the reduced rate of vitamin D3 synthesis produce low levels of plasma vitamin D3 and their metabolites 25(OH) D3 and 1α,25OH2 D3 . The fact that Kd and ßmax of the complex 1α,25(OH)2 D3 -VDR from the intestinal mucosa, kidneys and the Harderian glands of the mole rat Heterocephalus glaber are significantly different in each one of these tissues, is interpreted as an indicator that the VDRs are, in each tissue, adapted to the maintenance of normal physiological functions. (AU)

Varios trabajos publicados han informado que existen dos mecanismos para la transformación de 7-dehidrocolecalciferol en previtamina D3 : uno iniciado con el auxilio de fotones UVB y un segundo que ocurre en la oscuridad, sin el auxilio de radiación ultravioleta. Una serie de publicaciones contienen información que apoya la presencia de estos dos mecanismos en mamíferos, pájaros, plantas no vasculares (musgos), vasculares (angiospermas) y líquenes. La evidencia revisada sugiere que, en la oscuridad, la síntesis de vitamina D3 sigue el mismo esquema que la ruta fotoquímica y generalmente ocurre a una tasa reducida respecto de la síntesis bajo irradiación ultravioleta. La operación de la síntesis de vitamina D3 en la oscuridad, por lo menos para mamíferos y pájaros, puede tomarse como un reaseguro de sobrevida. La reducida tasa de síntesis de vitamina D en ausencia de luz produce bajas concentraciones en plasma de los metabolitos de la vitamina. La sobrevida saludable en estas condiciones sería posible mediante upregulation de los receptores. En las ratas topo Heterocephalus glaber (roedores sudafricanos que viven en permanente oscuridad, en túneles subterráneos), la reducida tasa de síntesis de vitamina D3 es la causa de los bajos niveles plasmáticos de la vitamina y sus metabolitos: 25(OH)D3 y 1α,25OH2 D3 . El hecho de que el Kd y ßmax del complejo 1α,25(OH)2 D3 -Vitamina-D-Receptor del intestino, riñón y glándulas de Harder de Heterocephalus glaber sean significativamente diferentes entre sí indicaría que los receptores se han modificado para mantener funciones fisiológicas normales en cada tejido. (AU)

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.

25 S-Adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D3: Synthesis, Tissue Selective Biological Activities, and X-ray Crystal Structural Analysis of Its Vitamin D Receptor Complex.

Both 25 R- and 25 S-25-adamantyl-23-yne-26,27-dinor-1α,25-dihydroxyvitamin D3 (4a and 4b) were stereoselectively synthesized by a Pd(0)-catalyzed ring closure and Suzuki-Miyaura coupling between enol-triflate 7 and alkenyl-boronic ester 8. The 25 S isomer (4b) showed high vitamin D receptor (VDR) affinity (50% of that of the natural hormone 1α,25-dihydroxyvitamin D3, 1) and transactivation potency (kidney HEK293, 90%). In endogenous gene expression, it showed high cell-type selectivity for kidney cells (HEK293, CYP24A1 160% of 1), bone cells (MG63, osteocalcin 64%), and monocytes (U937, CAMP 96%) over intestine (SW480, CYP24A1 8%) and skin (HaCaT, CYP24A1 7%) cells. The X-ray crystal structural analysis of 4b in complex with rat VDR-ligand binding domain (LBD) showed the highest Cα positional shift from the 1/VDR-LBD complex at helix 11. Helix 11 of the 4b and 1 VDR-LBD complexes also showed significant differences in surface properties. These results suggest that 4b should be examined further as another candidate for a mild preventive osteoporosis agent.

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.

Stereoselective synthesis of 25S,26-dihydroxyvitamin D3.

A convergent approach to 25S,26-dihydroxyvitamin D3 (1) has been developed in our laboratories. The A-ring and the CD-fragment are constructed from ergocalciferol and Inhoffen-Lythgoe diol, respectively. The triene system is assembled by a Wittig-Horner coupling. With this convergent synthesis, a novel hydroxylated vitamin D metabolite in our laboratory is available for biological testing.

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.

MART-10, a newly synthesized vitamin D analog, represses metastatic potential of head and neck squamous carcinoma cells.

Even with multidisciplinary treatment, the prognosis and quality of life of patients diagnosed with head and neck squamous cell carcinoma (HNSCC) are still not satisfactory. Previously, 19-Nor-2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10), the new brand 1α,25(OH)2D3 analog, has been demonstrated to be an effective drug to inhibit HNSCC growth in vitro. Since most cancer patients die of metastasis, in this study, the antimetastatic effect of MART-10 on HNSCC was investigated. Our results reveal that both 1α,25(OH)2D3 and MART-10 effectively repressed the migration and invasion of HNSCC cells, with MART-10 being much more potent than 1α,25(OH)2D3. The antimetastatic effect of 1α,25(OH)2D3 and MART-10 was mediated by attenuation of epithelial-mesenchymal transition (EMT), which was supported by the finding that the expression of EMT-inducing transcriptional factors, Sail and Twist, was inhibited by 1α,25(OH)2D3 and MART-10. The upregulation of E-cadherin and downregulation of N-cadherin in FaDu cells induced by both drugs further confirmed the repression of EMT. In addition, 1α,25(OH)2D3 and MART-10 treatment inhibited intracellular MMP-9 expression and extracellular MMP activity in FaDu cells. Collectively, our results suggest that the less-calcemia 1α,25(OH)2D3 analog, MART-10, is a promising drug for HNSCC treatment. Further clinical studies are warranted.

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.

Current Progresses and Trends in the Development of Progesterone Receptor Modulators.

The progesterone receptor (PR) is a ligand-activated steroid receptor in the nuclear receptor (NR) superfamily of transcription factor. Besides gynecological and obstetrical indications, the involvement/mechanism of PR in many other diseases, such as oncology, neurology, immunology, etc. has been revealed and studied in recent decades. Therapeutic agents that selectively activate or inhibit PR have been developed. PR agonists have generally been used in oral contraception and postmenopausal hormone replacement therapy (HRT), typically in combination with estrogens. PR antagonists and selective PR modulators (SPRMs) can be useful therapies for hormone dependent breast and prostate cancers, nonmalignant chronic conditions such as fibroids, and endometriosis. This review provides an overview and detailed discussions about the recent development of chemical structures of the PR ligands, their structural characteristics (particularly those contributing to their activity and selectivity), in vitro/in vivo studies and clinical trial outcomes, and the synthetic methodologies.

Stereoselective synthesis of 1ß,25-Dihydroxyvitamin D3 and its 26,27-hexadeuterated derivative.

A mild convergent synthesis of 1ß,25-dihydroxyvitamin D3 (3a), a metabolite of vitamin D3, and its C26,27-hexadeuterated derivative (3b) are described. The A-ring and the CD-fragments are constructed from (R)-carvone and Inhoffen-Lythgoe diol, respectively. The triene system is assembled by a Pd(0)-catalyzed process, which involves an enol-triflate (A-ring fragment) and an alkenyl boronate (CD-side chain fragment). Deuterium labeling is introduced at the last step of the synthesis.

Novel 9-Alkyl- and 9-Alkylidene-Substituted 1α,25-Dihydroxyvitamin D3 Analogues: Synthesis and Biological Examinations.

Continuing the structure-activity relationship studies in the vitamin D area, we designed and synthesized novel C-9 substituted calcitriol analogues, possessing different nonpolar groups at this position. 9α-Methyl-1α,25-(OH)2D3, both epimers of 9-methylene-10,19-dihydro-1α,25-(OH)2D3 as well as the parent vitamin with the "reversed" triene system, 9-methylene-19-nor-1α,25-(OH)2D3, were obtained from the previtamin D precursors, constructed by either Suzuki-Miyaura, Sonogashira, or Stille couplings of the corresponding A- and C,D-ring fragments. An alternative synthetic path, leading to the latter vitamin and its homologue with 9-ethylidene group, involved formation of dienynes as precursors of the respective 19-norprevitamin D compounds. 9ß-Methyl-19-nor-1α,25-(OH)2D3 was prepared by homogeneous hydrogenation with Wilkinson catalyst, and this analogue was found to be the most active in vitro. Moreover, 9α-methyl-1α,25-(OH)2D3 and 9-methylene-19-nor-1α,25-(OH)2D3 showed some in vitro activity, however, the in vivo assays indicated only weak calcemic potency of these compounds in the intestinal calcium transport.

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.

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.

Double point modified analogs of vitamin d as potent activators of vitamin D receptor.

Rational design, chemical synthesis, structural analysis, molecular modeling and biological evaluation are reviewed for all the double point modified vitamin D analogs that have been developed as potential therapeutics over the last several years. The idea of double modifications was based on the 3D structure of the ligand binding domain of the model of the vitamin D receptor. It was recently proved that structural modifications in the two remote parts of the vitamin D molecule might have additive biological effects resulting in an increased functional activity and lowered calcemic side effect. Recent in vivo experiments clearly demonstrated the potential use of these analogs in new therapeutic areas such as autoimmune and hyper-proliferative diseases, including cancer and the systemic treatment of psoriasis. Although some of these analogs are already approaching clinical trials, the molecular mechanism of action and their improved efficiency still remain to be fully understood. In this review the key steps of the convergent synthetic strategies that combine the modified A-ring and the CD-ring fragment carrying the altered side-chain are presented. The advantages of using the natural alicyclic and acyclic precursors are demonstrated as well as all the modern synthetic methodologies used for combining structural fragments. The results of molecular mechanics modeling are critically examined as well as the advantages and limitations of the use of the models of vitamin D proteins for the docking experiments and the design of new analogs. The potential use of advanced structural approaches, including high resolution X-ray crystallography, is discussed as to the prospect of providing a better understanding of the observed activity of modified analogs. Biological profiles in vitro and in vivo for groups of analogs are presented in a new tabular form to illustrate structure activity relationships.

Functionalization of non-activated C-H bonds in the synthesis of vitamin D metabolites and analogs.

The development of non-microbial methods for the selective functionalization of non-activated C-H bonds has constituted a challenge, with important economical and environmental implications, for chemists for over a century. The present review provides a comprehensive and current compendium that illustrates the power of C-H functionalization and, namely, of remote functionalization strategies, to expeditiously access vitamin D analogs with intricate structures.

Synthesis of 6-s-cis and 6-s-trans A-ring modified vitamin D analogues.

Vitamin D, through its hormonally active form 1α,25-dihydroxyvitamin D3 [1α,25- (OH)2-D3], exhibits a much broader spectrum of bio logical activities than expected in the endocrine system. However, 1α,25-(OH)2-D3 causes hypercalcemia a t pharmacologically r elevant doses wh ich forms a major obstacle in the clinical development of this compound. As a result, considerable effort has been made toward the synthesis of potential chemotherapeutic structurally related congeners with dissociation of beneficial effects from their toxic effects. Most of the analogues prepared have modifications on the upper side chain, more accessible from a synthetic point of view. Modifications of the A-ring are less extensive, although A-ring analogues exhibit a unique biological profile. This seco steroid can undergo a rotation around the 6,7 carbon-carbon single bond generating a wide array of molecular shapes, extending from the 6-s-cis to the more stable extended 6-s-trans conformation, which plays an important role in modulating the different biological activities of vitamin D. We review here, the synthetic strategies for the preparation of Vitamin D analogues with modific ations on the A-ring, including 6-s-cis locked derivatives that became of interest to further probe the less well investigate membrane actions of 1α,25-(OH)2-D3 for structure-activity relationship studies.