Synthesis and characterization of 20-hydroxyvitamin D3 with the A-ring modification.

Two novel 20-hydroxyvitamin D3 analogues (4a,b) with the A-ring modification have been synthesized by a convergent manner. An alternative pathway of vitamin D3 metabolism by cytochrome P450scc CYP11A1 was reported to afford 20-hydroxyvitamin D3 (3), functions of which remain to be explored. Based on the structure of the 20-hydroxy metabolite, novel analogues (4a,b) with the modifications, including the 1α-hydroxy, 25-hydroxy and 2α-methyl groups, have been designed. The side chain of the requisite CD-ring portions (9a,b) was introduced by Grignard reaction as a key step, and the stereochemistry at the C20 position was confirmed by the X-ray crystal structure analysis of the synthetic intermediate (8b). Preliminary biological characterization using the bovine thymus vitamin D receptor suggested that the introduction of the active motifs into the 20-hydroxyvitamin D3 scaffold elevated the receptor affinity.

Four Molybdenum-Dependent Steroid C-25 Hydroxylases: Heterologous Overproduction, Role in Steroid Degradation, and Application for 25-Hydroxyvitamin D3 Synthesis.

Side chain-containing steroids are ubiquitous constituents of biological membranes that are persistent to biodegradation. Aerobic, steroid-degrading bacteria employ oxygenases for isoprenoid side chain and tetracyclic steran ring cleavage. In contrast, a Mo-containing steroid C-25 dehydrogenase (S25DH) of the dimethyl sulfoxide (DMSO) reductase family catalyzes the oxygen-independent hydroxylation of tertiary C-25 in the anaerobic, cholesterol-degrading bacterium Sterolibacterium denitrificans Its genome contains eight paralogous genes encoding active site α-subunits of putative S25DH-like proteins. The difficult enrichment of labile, oxygen-sensitive S25DH from the wild-type bacteria and the inability of its active heterologous production have largely hampered the study of S25DH-like gene products. Here we established a heterologous expression platform for the three structural genes of S25DH subunits together with an essential chaperone in the denitrifying betaproteobacterium Thauera aromatica K172. Using this system, S25DH1 and three isoenzymes (S25DH2, S25DH3, and S25DH4) were overproduced in a soluble, active form allowing a straightforward purification of nontagged αßγ complexes. All S25DHs contained molybdenum, four [4Fe-4S] clusters, one [3Fe-4S] cluster, and heme B and catalyzed the specific, water-dependent C-25 hydroxylations of various 4-en-3-one forms of phytosterols and zoosterols. Crude extracts from T. aromatica expressing genes encoding S25DH1 catalyzed the hydroxylation of vitamin D3 (VD3) to the clinically relevant 25-OH-VD3 with >95% yield at a rate 6.5-fold higher than that of wild-type bacterial extracts; the specific activity of recombinant S25DH1 was twofold higher than that of wild-type enzyme. These results demonstrate the potential application of the established expression platform for 25-OH-VD3 synthesis and pave the way for the characterization of previously genetically inaccessible S25DH-like Mo enzymes of the DMSO reductase family.IMPORTANCE Steroids are ubiquitous bioactive compounds, some of which are considered an emerging class of micropollutants. Their degradation by microorganisms is the major process of steroid elimination from the environment. While oxygenase-dependent steroid degradation in aerobes has been studied for more than 40 years, initial insights into the anoxic steroid degradation have only recently been obtained. Molybdenum-dependent steroid C25 dehydrogenases (S25DHs) have been proposed to catalyze oxygen-independent side chain hydroxylations of globally abundant zoo-, phyto-, and mycosterols; however, so far, their lability has allowed only the initial characterization of a single S25DH. Here we report on a heterologous gene expression platform that allowed for easy isolation and characterization of four highly active S25DH isoenzymes. The results obtained demonstrate the key role of S25DHs during anoxic degradation of various steroids. Moreover, the platform is valuable for the efficient enzymatic hydroxylation of vitamin D3 to its clinically relevant C-25-OH form.

Design, Synthesis and Biological Activities of Novel Gemini 20S-Hydroxyvitamin D3 Analogs.

Vitamin D3 (D3) can be metabolized by cytochrome P450scc (CYP11A1) into 20S-hydroxyvitamin D3 (20D3) as a major metabolite. This bioactive metabolite has shown strong antiproliferative, antifibrotic, pro-differentiation and anti-inflammatory effects while being non-toxic (non-calcemic) at high concentrations. Since D3 analogs with two symmetric side chains (Gemini analogs) result in potent activation of the vitamin D receptor (VDR), we hypothesized that the chain length and composition of these types of analogs also containing a 20-hydroxyl group would affect their biological activities. In this study, we designed and synthesized a series of Gemini 20D3 analogs. Biological tests showed that some of these analogs are partial VDR activators and can significantly stimulate the expression of mRNA for VDR and VDR-regulated genes including CYP24A1 and transient receptor potential cation channel V6 (TRPV6). These analogs inhibited the proliferation of melanoma cells with potency comparable to that of 1α,25-dihydroxyvitamin D3. Moreover, these analogs reduced the level of interferon γ and up-regulated the expression of leukocyte associated immunoglobulin-like receptor 1 in splenocytes, indicating that they have potent anti-inflammatory activities. There are no clear correlations between the Gemini chain length and their VDR activation or biological activities, consistent with the high flexibility of the ligand-binding pocket of the VDR.

Asymmetric Synthesis of Vitamin D3 Analogues: Organocatalytic Desymmetrization Approach toward the A-Ring Precursor of Calcifediol.

A novel asymmetric synthesis has been developed for the construction of the A-ring of a chiral precursor to calcifediol. The highlights of this synthesis include (i) the introduction of the stereochemistry at the C5-position of the A-ring through the organocatalytic enantioselective desymmetrization of a prochiral cyclic anhydride using a bifunctional urea catalyst and (ii) the introduction of the exo-cyclic (Z)-dienol side chain by a tandem Claisen rearrangement/sulfoxide thermolysis of an allylic alcohol.

Total synthesis of biologically active 20S-hydroxyvitamin D3.

A total synthetic strategy of 20S-hydroxyvitamin D3 [20S-(OH)D3] involving modified synthesis of key intermediates 7 and 12, Grignard reaction to stereoselectively generate 20S-OH and Wittig-Horner coupling to establish D3 framework, was completed in 16 steps with an overall yield of 0.4%. The synthetic 20S-(OH)D3 activated vitamin D receptor (VDR) and initiated the expression of downstream genes. In addition, 20S-(OH)D3 showed similar inhibitory potency as calcitriol [1,25(OH)2D3] on proliferation of melanoma cells.

Synthesis of vitamin D3 analogues with A-ring modifications to directly measure vitamin D levels in biological samples.

C-3-substituted 25-hydroxyvitamin D3 analogues were synthesized as tools to directly measure levels of vitamin D in biological samples. The strategy involves vinyloxycarbonylation of the 3ß-hydroxy group and formation of a carbamate bond with a hydroxyl or amino group at the end of the alkyl chain. Biotinylated conjugates of synthesized derivatives were generated to be linked with vitamin D binding protein (DBP). The spacer group present in the alkyl chain is important in the binding of antibodies to the analogue-DBP complex. When compared to 25-hydroxyvitamin D3-DBP, the binding of some antibodies to the analogue-DBP complex of the 25-hydroxyvitamin D3 derivative 10 that posses an 8-aminoctyl alkyl chain is significantly reduced, but this analogue displaced [26,27-(3)H]-25-hydroxyvitamin D3 from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.

Design, synthesis, and biological action of 20R-hydroxyvitamin D3.

The non-naturally occurring 20R epimer of 20-hydroxyvitamin D3 is synthesized based on chemical design and hypothesis. The 20R isomer is separated by semipreparative HPLC, and its structure is characterized. A comparison of 20R isomer to its 20S counterpart in biological evaluation demonstrates that they have different behaviors in antiproliferative and metabolic studies.

Synthesis and biological activities of vitamin D-like inhibitors of CYP24 hydroxylase.

Selective inhibitors of CYP24A1 represent an important synthetic target in a search for novel vitamin D compounds of therapeutic value. In the present work, we show the synthesis and biological properties of two novel side chain modified 2-methylene-19-nor-1,25(OH)(2)D(3) analogs, the 22-imidazole-1-yl derivative 2 (VIMI) and the 25-N-cyclopropylamine compound 3 (CPA1), which were efficiently prepared in convergent syntheses utilizing the Lythgoe type Horner-Wittig olefination reaction. When tested in a cell-free assay, both compounds were found to be potent competitive inhibitors of CYP24A1, with the cyclopropylamine analog 3 exhibiting an 80-1 selective inhibition of CYP24A1 over CYP27B1. Addition of 3 to a mouse osteoblast culture sustained the level of 1,25(OH)(2)D(3), further demonstrating its effectiveness in CYP24A1 inhibition. Importantly, the in vitro effects on human promyeloid leukemia (HL-60) cell differentiation by 3 were nearly identical to those of 1,25(OH)(2)D(3) and in vivo the compound showed low calcemic activity. Finally, the results of preliminary theoretical studies provide useful insights to rationalize the ability of analog 3 to selectively inhibit the cytochrome P450 isoform CYP24A1.

[Synthesis and identification of artificial complete antigen 25-hydroxyvitamin D(3);].

AIM: To synthesize the 25-hydroxyvitamin D(3); artificial complete antigen and to prepare the specific antibody against 25-hydroxyvitamin D(3);. METHODS: The active group carboxyl was introduced into 25-hydroxyvitamin D(3); and formed 25-hydroxyvitamin D(3);-hemisuccinate which possessed the structure of the hapten by chemical modification. The EDC method was applied to conjugate 25-hydroxyvitamin D(3);-hemisuccinate to bovine serum albumin as an artificial immunogen. The coating antigen 25-hydroxyvitamin D(3);-hemisuccinate-OVA was obtained in the same way. Ultraviolet, SDS-PAGE and MALDI-TOF were used to identify 25-hydroxyvitamin D(3);-hemisuccinate-BSA. RESULTS: BALB/c mice were immunized with 25-hydroxyvitamin D(3);-hemisuccinate-BSA to generate the polyclonal antibody of the 25-hydroxyvitamin D(3); worth high titer and the immunogen, 25-hydroxyvitamin D(3);-hemisuccinate-BSA, was successfully prepared with coupling ratio (12±0.16):1(N=3) coupling. CONCLUSION: The high titer and good sensitivity of anti-25-hydroxyvitamin D(3); antibody are produced in sera immunized BALB/c mice, which made it possible to develop a clinical diagnostics for illness.

Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity.

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1alpha,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.

Chemotherapeutic effect of calcidiol derivative B3CD in a neuroblastoma xenograft model.

Bromoacetoxy-calcidiol (B3CD), a pro-apoptotic and cytotoxic agent in neuroblastoma (NB) cell lines, displayed therapeutic potential in vivo as an anticancer drug in a NB xenograft mouse model. Tumors of all animals treated intraperitoneally with B3CD went into regression within 10-30 days of treatment, while tumors in control animals grew aggressively. The response mechanisms of NB cells to B3CD in vitro were studied and included differential targeting of cell cycle key regulators p21 and cyclin D1 on the transcriptional and expression level leading to arrest in G0/G1 phase. In contrast to the effect in ovarian cancer cells, B3CD-induced cell death in SMS-KCNR NB cells was only marginally mediated by the p38 MAPK signaling pathway. Signaling induced by exogenous recombinant EGF leads to a partial restoration of the negative effects of B3CD on SMS-KCNR cell proliferation and survival. Upon combinational treatment of SMS-KCNR cells with B3CD and recombinant EGF, the EGF receptor (EGF-R) was highly activated. We suggest future studies to include analysis of the effects of B3CD in combination therapy with pharmacological inhibitors of cell cycle regulators or with EGF-R-targeting inhibitors, -toxins or -antibodies in vitro and their translation into in vivo models of tumor development.

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 two carboxylic haptens for raising antibodies to 25-hydroxyvitamin D3 and 1alpha,25-dihydroxyvitamin D3.

Hapten derivatives of 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) were synthesized using the Wittig-Horner approach. Both haptens bearing a carboxylic group at the side chain that can be linked to a protein for raising antibodies of potential utility for the determination of 25-hydroxyvitamin D(3), 1alpha,25-dihydroxyvitamin D(3) and 1alpha-hydroxylated vitamin D(3) analogues.

Diastereoselective synthesis, binding affinity for vitamin D receptor, and chiral stationary phase chromatography of hydroxy analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol.

A series of analogs of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol were obtained with an additional hydroxyl in the aliphatic side chain at carbon atom C-24. These analogs were synthesized by direct and diastereo-selective alpha-hydroxylation of enolates derived from respective vitamin D esters using Davies chiral oxaziridines. The use of (+)-(2R,8aS)-(8, 8-dichlorocamphoryl)sulfonyl oxaziridine resulted in (R) stereochemistry of the new asymmetric center for both series of analogs. Similarly, (-)-(2S,8aR) oxaziridine gave (S) analogs. The diastereomeric purity of hydroxy analogs was determined by high-performance liquid chromatography on a chiral stationary phase. High diastereopurity of hydroxylation of vitamin D esters was obtained without the use of any chiral auxiliary. The binding affinity of (24R)-1,24,25-trihydroxycholecalciferol for the calf thymus intracellular vitamin D receptor was one order of magnitude higher than that of the respective (24S)-diastereomer.

14-epi stereoisomers of 25-hydroxy- and 1 alpha,25-dihydroxyvitamin D3: synthesis, isomerization to previtamins, and biological studies.

The C-14 epimers of vitamin D, 14-epi-25-hydroxyvitamin D3 (4) and 14-epi-1 alpha, 25-dihydroxyvitamin D3 (5), were synthesized, and their isomerization via [1,7]-sigmatropic hydrogen shifts to the corresponding previtamin forms (4' and 5', respectively) was studied. The activation parameters of the [1,7]-sigmatropic hydrogen shifts were found to be similar to those of other vitamin D analogues, although epimerization at C-14 shifts the equilibrium of the triene chromophore to the previtamin form. The in vivo biological activities of 4, 4', 5, and 5' in the chick in terms of their ability to elicit intestinal calcium absorption and bone calcium mobilization were determined. These vitamin D analogues, the first in the natural steroid series modified at the C-14 position, were essentially devoid of activity. The relative competitive indices (RCIs), derived in an in vitro assay reflecting the ability of these analogues to bind to the chick intestinal nuclear receptor, were determined. Analogues 4, 4', 5, and 5' had RCI values of 0.08, 0.01, 15, and 1.6, respectively, in comparison to the natural ligand, 1 alpha, 25-dihydroxyvitamin D3 (3), whose value is 100 by definition. Thus, the in vivo and in vitro data were somewhat at variance, particularly for 5, which bound significantly to the chick intestinal receptor. In vitro binding studies with the human serum vitamin D binding protein (DBP) were also conducted. The RCI values for human DBP reflects the ability of an analogue to bind to this protein in comparison to the hormone 3, whose value is 100. The measured RCI values for 4, 4', 5, and 5' were 3450, 90, 12, and 2.2, respectively. It is noteworthy that analogue 4 binds approximately 35 times more effectively than the parent hormone 3, but approximately 20 times less effectively than 25-hydroxyvitamin D3 (2).

Synthesis of 25-hydroxy-[6,19,19'-2H3]vitamin D3 and 1 alpha,25-dihydroxy-[6,19,19'-2H3]vitamin D3.

Synthesis of polydeuterated analogs of 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxy vitamin D3 are described. These analogs, containing stable isotope atoms at metabolically stable positions, are potentially useful in studies involving catabolism of hydroxylated metabolites of vitamin D3.

Synthesis of 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: photoaffinity labeling of rat serum vitamin D binding protein.

Vulnerability of 25-hydroxy-[26,27-3H]vitamin D3 3 beta-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D3 (25-OH-D3) (Ray et al., 1986) toward standard conditions of carboxymethylation promoted us to synthesize 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D3, and 25-hydroxyvitamin D3 3 beta-3'-[N-(4-azido-2-nitro-[3,5-3H]phenyl)amino] propyl ether (3H-25-ANE), the radiolabeled counterpart of 25-ANE. Competitive binding assays of 25-OH-D3 and 25-ANE with rat serum demonstrated that 25-ANE competes for the 25-OH-D3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D3. These results provide strong evidence for the covalent labeling of the 25-OH-D3 binding site in rDBP by 3H-25-ANE.

Synthesis and biological activity of novel vitamin D analogues: 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 and 24,24-difluoro-1 alpha,25-dihydroxy-26,27-dimethylvitamin D3.

We synthesized 24,24-difluoro-25-hydroxy-26,27-dimethylvitamin D3 (16), and 24,24-difluoro-1 alpha, 25-dihydroxy-26,27-dimethylvitamin D3 (21), from 3 beta-hydroxy-22,23-dinorcholenic acid 3-acetate. Compound 16 was found to be a highly potent vitamin D analogue with bioactivity similar to that of 25-hydroxyvitamin D3 in vivo. Compound 16 was bound by vitamin D binding protein with an affinity slightly less than that of 25-hydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with approximately the same affinity as that of 25-hydroxyvitamin D3. In the organ-culture duodenum, 16 induced the synthesis of calcium binding protein with a potency approximately 1/20 that of 1,25-dihydroxyvitamin D3. Compound 21 was also noted to be a highly potent vitamin D analogue with bioactivity in vivo similar to that of 1,25-dihydroxyvitamin D3. It was bound to vitamin D binding protein with an affinity considerably less than that of 1,25-dihydroxyvitamin D3. It was bound to the intestinal cytosol receptor for 1,25-dihydroxyvitamin D3 with an affinity slightly less than that of the native hormone. In the organ-culture duodenum, 21 was noted to be about 3 times more active than 1,25-dihydroxyvitamin D3 in the induction of calcium binding protein. The introduction of fluorines at C-24 and extension of the sterol side chain at C-26 and C-27 by methylene groups results in vitamin D analogues that have biological activity in vivo similar to those of the respective nonfluorinated natural sterols.

The synthesis and biological activity of 25-hydroxy-26,27-dimethylvitamin D3 and 1,25-dihydroxy-26,27-dimethylvitamin D3: highly potent novel analogs of vitamin D3.

We synthesized 25-hydroxy-26,27-dimethylvitamin D3, 9, and 1,25-dihydroxy-26,27-dimethylvitamin D3, 14, from chol-5-enic acid-3 beta-ol and tested their biological activity in vivo and in vitro. 9 was found to be highly potent vitamin D analog with bioactivity similar to that of 25-hydroxyvitamin D3. 9 bound to rat plasma vitamin D binding protein with approximately one-third the affinity of 25-hydroxyvitamin D3. In a duodenal organ culture system and in a competitive binding assay with chick intestinal 1,25-dihydroxyvitamin D receptor, 9 was significantly more potent than 25-hydroxyvitamin D3. 1,25-Dihydroxy-26,27-dimethylvitamin D3, 14 was also highly active in vivo. At doses of 1000-5000 pmol/rat, its action was more sustained than that of 1,25-dihydroxyvitamin D3. 14 bound to vitamin D binding protein about 18 times less effectively than 1,25-dihydroxyvitamin D3. 14 bound to the chick intestinal cytosol receptor with an affinity one-half that of 1,25-dihydroxyvitamin D3. In a duodenal organ culture system, 14 was about half as active as 1,25-dihydroxyvitamin D3. Extension of the sterol side chain, at C-26 and C-27, by methylene groups, prolongs the bioactivity of a vitamin D sterol hydroxylated at C-1 and C-25; the corresponding sterol, hydroxylated only at C-25, does not show any alteration of its bioactivity in vivo. These newly synthesized analogs may potentially be of therapeutic use in various mineral disorders.