Design, Synthesis, Evaluation and Structure of Allenic 1α,25-Dihydroxyvitamin D3 Analogs with Locked Mobility at C-17.

Vitamin D receptor ligands have potential for the treatment of hyperproliferative diseases and disorders related to the immune system. However, hypercalcemic effects limit their therapeutical uses and call for the development of tissue-selective new analogs. We have designed and synthesized the first examples of 1α,25-dihydroxyvitamin D3 analogs bearing an allenic unit attached to the D ring to restrict the side-chain conformational mobility. The triene system was constructed by a Pd0 -mediated cyclization/Suzuki-Miyaura cross-coupling process in the presence of an allenic side chain. The allenic moiety was built through an orthoester-Claisen rearrangement of a propargylic alcohol. The biological activity and structure of (22S)-1α,25-dihydroxy-17,20-dien-24-homo-21-nor-vitamin D3 bound to binding domain of the vitamin D receptor, provide information concerning side-chain conformational requirements for biological activity.

Design, synthesis and evaluation of side-chain hydroxylated derivatives of lithocholic acid as potent agonists of the vitamin D receptor (VDR).

A high number of biologically active and low-calcemic secosteroidal ligands of the vitamin D receptor (VDR) have been developed, some of which are already used clinically although with limited success in the treatment of hyperproliferative diseases because the required pharmaceutical dosages induce toxicity. We describe here the in silico design, synthesis, structural analysis and biological evaluation of two novel active lithocholic acid derivatives hydroxylated at the side chain as highly potent inhibitors of atopic dermatitis-relevant keratinocyte inflammation of potential therapeutic interest.

Lithocholic acid-based design of noncalcemic vitamin D receptor agonists.

The hypercalcemic effects of the hormone 1α,25-dihydroxyvitamin D3 (calcitriol) and most of known vitamin D metabolites and analogs call for the development of non secosteroidal vitamin D receptor (VDR) ligands as new selective and noncalcemic agonists for treatment of hyperproliferative diseases. We report on the in silico design and stereoselective synthesis of six lithocholic acid derivatives as well as on the calcemic activity of a potent LCA derivative and its crystallographic structure in complex with zVDR LBD. The low calcemic activity of this compound in comparison with the native hormone makes it of potential therapeutic value. Structure-function relationships provide the basis for the development of even more potent and selective lithocholic acid-based VDR ligands.

Stereoselective Palladium-Catalyzed Approach to Vitamin D3 Derivatives in Protic Medium.

We describe an efficient convergent synthesis of vitamin D3 metabolites and analogues. The synthetic strategy relies on a tandem Pd-catalyzed A-ring closure and Suzuki-Miyaura coupling to the CD-side chain component to set directly the vitamin D triene system under protic conditions. This strategy enables rapid access to vitamin D3 and 3-epi-vitamin D3 metabolites and analogues modified at the side chain for biological evaluation and structural and metabolic studies.

Total synthesis of 1α,25-dihydroxyvitamin D3 analogs modified at the side chain and D-ring.

Herein, we describe a synthetic strategy to access 1α,25-dihydroxyvitamin D3 (calcitriol) analogs with natural or unnatural configuration at C20 and unsaturation at the D-ring. The synthetic approach is exemplified by the synthesis of two potent analogs, namely 1α,25-dihydroxy-16-en-23-yne-vitamin D3 and 1α,25-dihydroxy-20-epi-24a-homo-26,27-dimethyl-vitamin D3. A key feature of the synthetic strategy is the generation of an unnatural configuration at C20 by a catalytic asymmetric reduction of an α,ß,γ,δ-unsaturated ester with the CuH species in a micellar system.

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.

A new suprasterol by photochemical reaction of 1α,25-dihydroxy-9-methylene-19-norvitamin D3.

The UV-induced photochemical reaction of 1α,25-dihydroxy-9-methylene-19-norvitamin D3 has been investigated. The pentacyclic structure of the isolated product has been unequivocally established by X-ray crystallographic analysis. The possible reaction paths of the examined photochemical transformation are discussed. Biological in vivo and in vitro tests proved that the photoproduct is devoid of calcemic activity.

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.

A vitamin D-based strategy overcomes chemoresistance in prostate cancer.

BACKGROUND AND PURPOSE: Castration-resistant prostate cancer (CRPC) is a common male malignancy that requires new therapeutic strategies due to acquired resistance to its first-line treatment, docetaxel. The benefits of vitamin D on prostate cancer (PCa) progression have been previously reported. This study aimed to investigate the effects of vitamin D on chemoresistance in CRPC. EXPERIMENTAL APPROACH: Structure function relationships of potent vitamin D analogues were determined. The combination of the most potent analogue and docetaxel was explored in chemoresistant primary PCa spheroids and in a xenograft mouse model derived from a patient with a chemoresistant CRPC. KEY RESULTS: Here, we show that Xe4MeCF3 is more potent than the natural ligand to induce vitamin D receptor (VDR) transcriptional activities and that it has a larger therapeutic window. Moreover, we demonstrate that VDR agonists restore docetaxel sensitivity in PCa spheroids. Importantly, Xe4MeCF3 reduces tumour growth in a chemoresistant CRPC patient-derived xenograft. In addition, this treatment targets signalling pathways associated with cancer progression in the remaining cells. CONCLUSION AND IMPLICATIONS: Taken together, these results unravel the potency of VDR agonists to overcome chemoresistance in CRPC and open new avenues for the clinical management of PCa.

Metabolic stability of 3-epi-1α,25-dihydroxyvitamin D3 over 1 α 25-dihydroxyvitamin D3: metabolism and molecular docking studies using rat CYP24A1.

3-epi-1α,25-dihydroxyvitamin D3 (3-epi-1α,25(OH)2D3), a natural metabolite of 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), exhibits potent vitamin D receptor (VDR)-mediated actions such as inhibition of keratinocyte growth or suppression of parathyroid hormone secretion. These VDR-mediated actions of 3-epi-1α,25(OH)2D3 needed an explanation as 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, exhibits low affinity towards VDR. Metabolic stability of 3-epi-1α,25(OH)2D3 over 1α,25(OH)2D3 has been hypothesized as a possible explanation. To provide further support for this hypothesis, we now performed comparative metabolism studies between 3-epi-1α,25(OH)2D3 and 1α,25(OH)2D3 using both the technique of isolated rat kidney perfusion and purified rat CYP24A1 in a cell-free reconstituted system. For the first time, these studies resulted in the isolation and identification of 3-epi-calcitroic acid as the final inactive metabolite of 3-epi-1α,25(OH)2D3 produced by rat CYP24A1. Furthermore, under identical experimental conditions, it was noted that the amount of 3-epi-calcitroic acid produced from 3-epi-1α,25(OH)2D3 is threefold less than that of calcitroic acid, the analogous final inactive metabolite produced from 1α,25(OH)2D3 . This key observation finally led us to conclude that the rate of overall side-chain oxidation of 3-epi-1α,25(OH)2D3 by rat CYP24A1 leading to its final inactivation is slower than that of 1α,25(OH)2D3. To elucidate the mechanism responsible for this important finding, we performed a molecular docking analysis using the crystal structure of rat CYP24A1. Docking results suggest that 3-epi-1α,25(OH)2D3, unlike 1α,25(OH)2D3, binds to CYP24A1 in an alternate configuration that destabilizes the formation of the enzyme-substrate complex sufficiently to slow the rate at which 3-epi-1α,25(OH)2D3 is inactivated by CYP24A1 through its metabolism into 3-epi-calcitroic acid.

Synthesis of 9-alkylated calcitriol and two 1α,25-dihydroxy-9-methylene-10,19-dihydrovitamin D3 analogues with a non-natural triene system by thermal sigmatropic rearrangements.

1α,25-(OH)(2)-9α-Methylvitamin D(3) (4), the first known analogue of the natural hormone 1α,25-(OH)(2)D(3) (3) with an alkyl substituent at C-9, and two 1α,25-(OH)(2)-9-methylene-10,19-dihydrovitamin D(3) analogues (7 and 8) with an unprecedented non-natural triene system were synthesized by thermal isomerization of 1α,25-(OH)(2)-9-methylprevitamin D(3) (6). Three alternative approaches (Sonogashira, Stille, or stereoselective dehydration of a tertiary propargyl alcohol) have been successfully used to construct the dienyne precursors of previtamin 6 possessing two methyl groups capable of participating in the [1,7]-sigmatropic hydrogen shift.

Further Studies on the Highly Active Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3 (Calcitriol): Refinement of the Side Chain.

Current efforts in the vitamin D field are directed toward the development of highly antiproliferative yet noncalcemic analogues of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3). We have recently reported the design, synthesis, biological evaluation, and crystal structures of a series of novel analogues that both lack the steroidal C-ring and have an m-phenylene ring replacing the steroidal cyclopentane D-ring. We have now investigated the potentiating effects of incorporating selected modifications (hexafluorination and/or an internal triple bond) within the steroidal side chain in our series. An alternative synthetic strategy (Wittig-Horner approach instead of our previously used Pd-catalyzed tandem cyclization/cross-coupling) for the construction of the vitamin D triene system was found convenient for the target compounds 2, 3a, 3b, and 3c of this report. These modifications enhance vitamin D nuclear receptor (VDR) interactions and consequently VDR-associated biological properties compared to parental PG-136 compound while maintaining normal calcium levels.

Design, synthesis, evaluation, and structure of vitamin D analogues with furan side chains.

Based on the crystal structures of human vitamin D receptor (hVDR) bound to 1α,25-dihydroxy-vitamin D(3) (1,25 D) and superagonist ligands, we previously designed new superagonist ligands with a tetrahydrofuran ring at the side chain that optimize the aliphatic side-chain conformation through an entropy benefit. Following a similar strategy, four novel vitamin D analogues with aromatic furan side chains (3a, 3b, 4a, 4b) have now been developed. The triene system has been constructed by an efficient stereoselective intramolecular cyclization of an enol triflate (A-ring precursor) followed by a Suzuki-Miyaura coupling of the resulting intermediate with an alkenyl boronic ester (CD-side chain, upper fragment). The furan side chains have been constructed by gold chemistry. These analogues exhibit significant pro-differentiation effects and transactivation potency. The crystal structure of 3a in a complex with the ligand-binding domain of hVDR revealed that the side-chain furanic ring adopts two conformations.

Design, Synthesis, Biological Activity, and Structural Analysis of Novel Des-C-Ring and Aromatic-D-Ring Analogues of 1α,25-Dihydroxyvitamin D3.

The toxic calcemic effects of the natural hormone 1α,25-dihydroxyvitamin D3 (1,25D3, 1,25-dihydroxycholecalciferol) in the treatment of hyperproliferative diseases demand the development of highly active and noncalcemic vitamin D analogues. We report the development of two highly active and noncalcemic analogues of 1,25D3 that lack the C-ring and possess an m-phenylene ring that replaces the natural D-ring. The new analogues (3a, 3b) are characterized by an additional six-carbon hydroxylated side chain attached either to the aromatic nucleus or to the triene system. Both compounds were synthesized by the Pd-catalyzed tandem cyclization/cross coupling approach starting from alkyne 6 and diphenol 8. Key steps include a stereoselective Cu-assisted addition of a Grignard reagent to an aromatic alkyne and a Takai olefination of an aromatic aldehyde. The new compounds are noncalcemic and show transcriptional and antiproliferative activities similar to 1,25D3. Structural analysis revealed that they induce a large conformational rearrangement of the vitamin D receptor around helix 6.

Structure-based design of a superagonist ligand for the vitamin D nuclear receptor.

Vitamin D nuclear receptor (VDR), a ligand-dependent transcriptional regulator, is an important target for multiple clinical applications, such as osteoporosis and cancer. Since exacerbated increase of calcium serum level is currently associated with VDR ligands action, superagonists with low calcium serum levels have been developed. Based on the crystal structures of human VDR (hVDR) bound to 1alpha,25-dihydroxyvitamin D(3) and superagonists-notably, KH1060-we designed a superagonist ligand. In order to optimize the aliphatic side chain conformation with a subsequent entropy benefit, we incorporated an oxolane ring and generated two stereo diasteromers, AMCR277A and AMCR277B. Only AMCR277A exhibits superagonist activity in vitro, but is as calcemic in vivo as the natural ligand. The crystal structures of the complexes between the ligand binding domain of hVDR and these ligands provide a rational approach to the design of more potent superagonist ligands for potential clinical application.

Synthesis of 25-hydroxyvitamin D(3) and 26,26,26,27,27,27-hexadeutero-25-hydroxyvitamin D(3) on solid support.

A convenient five-step route to 25-hydroxylated vitamin D(3) compounds on (hydroxymethyl)polystyrene support is reported. A CD-side chain fragment was anchored to the solid phase through an ester group at C25 and coupled to an A ring building block to assemble the vitamin D triene system by the Wittig-Horner approach. Deprotection of the hydroxy group was carried out on the support, prior to functionalization at C25. The title compounds were released from the resin in excellent global yield by nucleophilic attack on the ester carbonyl group using commercially available organometallic reagents. This key last step offers an opportunity for the efficient generation of 26,27-labeled compounds and also for diversification at the side chain without need for a pool of side chain fragments.

Synthesis of 28,28,28-trideutero-25-hydroxydihydrotachysterol2.

As part of our program on synthesis of labeled vitamin D metabolites and analogs, we describe here an efficient and versatile synthetic approach to 28,28,28-trideutero- 25-hydroxydihydrotachysterol2 where isotopic labeling was incorporated stereoselectively in the last step of the synthesis. This deuterated compound will allow the study this analog in vitro or in vivo and to measure AT10-like compounds in serum by LC-MS/MS.

Remote induction of cellular immune response in mice by anti-meningococcal nanocochleates - nanoproteoliposomes.

New adjuvant formulations, based on proteoliposomes <40 nm and cochleates <100 nm, without Al(OH)3 adjuvant, were evaluated regarding their ability to generate Th1 immune response through a Delayed -Type Hypersensitivity Test, at the mouse model, by using a Neisseria meningitidis B protein complex as antigen. The formulations were administered by intramuscular (IM) (2 inoculations - at baseline and after 14 days) and intranasal (IN) (3 inoculations at 7 days) immunization pathways. All IM immunized groups were able to induce similar response to these formulations as well as to VA-MENGOC-BC® vaccine - containing Al(OH)3 adjuvant (used as positive control of the trial). In all groups, the induced inflammation (IP) rate was statistically higher than in the negative control group (CN) (p < 0.05). Immunogenicity, measured by HSR and CD4+ lymphocyte increase was equivalent to the control vaccine and most important, granuloma reactogenicity at the site of injection was eliminated, fact demonstrated by histological study. All groups of animals immunized by IN route showed HSR reactions and statistically significant differences with respect to the CN group. However, IP values were lower, with statistical differences (p < 0.05) for the same adjuvant formulation IM administered, except the AIF2-nCh formulation that generated statistically similar induction (p > 0.05) by both immunization pathways, suggesting it to be the best candidate for the next IN trial. Proteoliposome and cochleate formulations tested were able to mount potent Th-1 immune response, equivalent to the original vaccine formulation, with the advantage of less reactogenicity in the site of the injection, caused by the toxicity of Al(OH)3 adjuvant gel.

Synthesis of Side-Chain Locked Analogs of 1α,25-Dihydroxyvitamin D3 Bearing a C17 Methyl Group.

A convergent synthesis of side-chain locked vitamin D analogs 3 and 4, which bind strongly in silico to the vitamin D receptor (VDR), is described. The synthetic approach features an SN2'- syn displacement of carbamates by cuprates to set the challenging quaternary stereogenic center at C17 and a Pd-catalyzed construction of the triene system in the presence of a diyne moiety.