Concise synthesis of 23-hydroxylated vitamin D3 metabolites.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

Synthesis and Biological Evaluation of Vitamin D3 Metabolite 20S,23S-Dihydroxyvitamin D3 and Its 23R Epimer.

The vitamin D3 metabolite, 20S,23S-dihydroxyvitamin D3, was chemically synthesized for the first time and identified to be the same as the enzymatically produced metabolite. The C23 absolute configurations of both 20S,23S/R-dihydroxyvitamin D3 epimers were unambiguously assigned by NMR and Mosher ester analysis. Their kinetics of CYP27B1 metabolism were investigated during the production of their 1α-hydroxylated derivatives. Bioactivities of these products were compared in terms of vitamin D3 receptor activation, anti-inflammatory, and antiproliferative activities.

Synthesis and Biological Activity of 2-Methylene Analogues of Calcitriol and Related Compounds.

In an attempt to prepare vitamin D analogues that are superagonists, (20R)- and (20S)-isomers of 1α-hydroxy-2-methylenevitamin D3 and 1α,25-dihydroxy-2-methylenevitamin D3 have been synthesized. To prepare the desired A-ring dienyne fragment, two different approaches were used, both starting from the (-)-quinic acid. The obtained derivative was subsequently coupled with the C,D-ring enol triflates derived from the corresponding Grundmann ketones, using the Sonogashira reaction. Moreover, (20R)- and (20S)-1α,25-dihydroxy-2-methylenevitamin D3 compounds with an (5E)-configuration were prepared by iodine catalyzed isomerization. All four 2-methylene analogues of the native hormone were characterized by high in vitro activity. As expected, the 25-desoxy analogues were much less potent. Among the synthesized compounds, two of them, 1α,25-dihydroxy-2-methylenevitamin D3 and its C-20 epimer, were found to be almost as active as 2-methylene-19-nor-(20S)-1α,25-dihydroxyvitamin D3 (2MD) on bone but more active in intestine.

Phosphonate analogues of 1α, 25 dihydroxyvitamin D3 are promising candidates for antitumoural therapies.

The active metabolite of vitamin D, 1α, 25 dihydroxyvitamin D3 (calcitriol) is classically known to regulate calcium and phosphate homeostasis and bone mineralization. In addition, calcitriol has also been documented to act as a potent anticancer agent in multiple cell culture and animal models of cancer. However, major side effects, such as hypercalcemia, hinder broad-spectrum therapeutic uses of calcitriol in cancer chemotherapy. Synthesis of calcitriol analogues with the same or increased antiproliferative and pro-differentiating activities, and with reduced undesired effects on calcium and bone metabolism, is getting significant attention towards rational therapeutics to treat cancer. In this regard, phosphonate analogues have been shown to display a certain degree of dissociation between the vitamin D activity in vitro and undesired hypercalcemia in vivo. However, few phosphonates have been described in the literature and fewer of them tested for antitumoral effects. Our group has synthesized a novel vitamin D analogue (EM1) bearing an alkynylphosphonate moiety that combines the low calcemic properties of phosphonates with the decreased metabolic inactivation due to the presence of a triple bond between C-23 and C-24. Biological assays demonstrated that this analogue has potent antiproliferative effects in a wide panel of tumour cell lines, even in those resistant to calcitriol treatment. Importantly, EM1 does not show toxic effects in animals, even administered at high doses and for extended periods of time. In the current review we discuss the effects and the potential application in cancer of vitamin D and its derivatives, with an emphasis on phosphonate analogues.

Synthetic strategy and biological activity of A-ring stereoisomers of 1,25- dihydroxyvitamin D3 and C2-modified analogues.

The hormonally active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (1a), has a wide variety of biological activities and its major molecular target is considered to be the vitamin D receptor (VDR). The A-ring stereoisomers of 1a as well as its C2-modified analogues, which have different stereochemistry at the C1 and/or C3 hydroxy groups, are of interest since recent metabolic studies have shown that catabolism could occur through A-ring modification. In this review, a practical and versatile synthesis of the A-ring enyne precursors by the convergent method of Trost and coworkers, which is needed to construct all possible A-ring stereoisomers of 1,25-dihydroxyvitamin D3 (1a-d), and the C2-modified analogues (4a-d, 5a-d, 6a-d and 7a-d) is described. A strategy for the synthesis and evaluation of all possible A-ring stereoisomers of 1a and their A-ring modified analogues is important, and this will stimulate synthesis and biological studies into vitamin D.

Production of 22-hydroxy metabolites of vitamin d3 by cytochrome p450scc (CYP11A1) and analysis of their biological activities on skin cells.

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D(3), producing 20S-hydroxyvitamin D(3) [20(OH)D(3)] and 20S,23-dihydroxyvitamin D(3) [20,23(OH)(2)D(3)] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D(3), 17,20-dihydroxyvitamin D(3), and 17,20,23-trihydroxyvitamin D(3). In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D(3) and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D(3) [22(OH)D(3)] and 20S,22-dihydroxyvitamin D(3) [20,22(OH)(2)D(3)]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D(3) to 20,22(OH)(2)D(3). The 20,22(OH)(2)D(3) could also be produced from 20(OH)D(3) and was metabolized to a trihydroxyvitamin D(3) product. We compared the biological activities of these new derivatives with those of 20(OH)D(3), 20,23(OH)(2)D(3), and 1α,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. 1,25(OH)(2)D(3), 20(OH)D(3), 22(OH)D(3), 20,23(OH)(2)D(3), and 20,22(OH)(2)D(3) significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)(2)D(3), 20,22(OH)(2)D(3), 20(OH)D(3), and 1,25(OH)(2)D(3), with 22(OH)D(3) having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)(2)D(3). All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D(3) and 20,22(OH)(2)D(3) having less effect than 1,25(OH)(2)D(3) and 20(OH)D(3). Thus, we have identified 22(OH)D(3) and 20,22(OH)(2)D(3) as products of CYP11A1 action on vitamin D(3) and shown that, like 20(OH)D(3) and 20,23(OH)(2)D(3), they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D(3).

DNA binding alters coactivator interaction surfaces of the intact VDR-RXR complex.

The vitamin D receptor (VDR) functions as an obligate heterodimer in complex with the retinoid X receptor (RXR). These nuclear receptors are multidomain proteins, and it is unclear how various domains interact with one another within the nuclear receptor heterodimer. Here, we show that binding of intact heterodimer to DNA alters the receptor dynamics in regions remote from the DNA-binding domains (DBDs), including the coactivator binding surfaces of both co-receptors, and that the sequence of the DNA response element can determine these dynamics. Furthermore, agonist binding to the heterodimer results in changes in the stability of the VDR DBD, indicating that the ligand itself may play a role in DNA recognition. These data suggest a mechanism by which nuclear receptors show promoter specificity and have differential effects on various target genes, providing insight into the function of selective nuclear receptor modulators.

Identification of a unique subset of 2-methylene-19-nor analogs of vitamin D with comedolytic activity in the rhino mouse.

The active metabolite of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), and a series of 2-methylene-19-nor analogs of 1,25(OH)(2)D(3) were evaluated for their ability to reduce the size of utricles (comedolytic activity) in a rhino mouse model of acne. All analogs tested, as well as the native hormone, increased the skin epidermal thickness. In contrast, only a subset of analogs that lacked a full side chain and 25-hydroxyl group were found to possess comedolytic activity. A reduction in comedone area could be achieved without adversely affecting serum calcium levels. Although all compounds that contained a side chain ranging from 2 to 5 carbons in length had similar potency as comedolytic agents, increasing the length of the side chain resulted in a progressive increase in calcemic liability. Dose-response studies of the comedolytic analogs showed that an increase in epidermal thickness was achieved at a lower dose than that needed to induce comedolysis. Thus, we have identified a unique subset of vitamin D analogs that produce comedolysis in the absence of hypercalcemia. Further, the activity of vitamin D analogs in causing epidermal hyperproliferation has been distinguished from that resulting in a reduction in utricle size.

Stereoselective synthesis of C24-hydroxylated vitamin D3 analogs: a practical and expeditius route to calcipotriol.

The synthesis of the clinically important drug calcipotriol (2, MC903) is described as an example of a new and efficient approach to C24-hydroxylated analogs and metabolites of vitamin D3 (1). The key step of the process is the generation of the C24 stereocenter by DAIB [(-)-3-exo-(dimethylamino)isoborneol]-catalyzed addition of the alkenylzinc derivative of alkyne 3 to cyclopropylcarboxaldehyde.

Inhibitory effects of 1,25-dihydroxyvitamin D3 and its low-calcemic analogues on staurosporine-induced apoptosis.

The active form of vitamin D3 and some of its related compounds show neuroprotective effects in various models of neuronal damage, however, mechanism of their anti-apoptotic action has not been elucidated. Therefore, the present study was designed to investigate the effects of 1,25-dihydroxyvitamin D3 and its low-calcemic analogues, PRI-2191, PRI-1890 and PRI-1901 on staurosporine-induced apoptosis in human neuroblastoma SH-SY5Y cells. Twenty-four hour incubation with staurosporine (1 microM) enhanced the caspase-3 activity, decreased mitochondrial membrane potential and increased the number of apoptotic cells as visualized by Hoechst staining. 1,25-Dihydroxyvitamin D3 and PRI-2191 attenuated the staurosporine-induced caspase-3 activity at 5, 50 and 500 nM, whereas PRI-1890 and PRI-1901 were active only at higher concentrations. Furthermore, 1,25-dihydroxyvitamin D3 (50 and 500 nM) and PRI-2191 (500 but not 50 nM) reversed the staurosporine-evoked decrease in mitochondrial membrane potential. Hoechst and calcein staining confirmed the neuroprotective effects of the secosteroids under study. Further study revealed that a selective inhibitor of phosphatidylinositol 3-kinase (PI3-K), wortmannin, at concentration of 100 nM antagonized the effect of 1,25-dihydroxyvitamin D3 and PRI-2191 on staurosporine-induced caspase-3 activation. These data indicate that 1,25-dihydroxyvitamin D3 and its low-calcemic analogues at nanomolar concentrations inhibited mitochondrial pathway of apoptosis in SH-SY5Y neuronal cells, though with different potency. Moreover, the activation of PI3-K/Akt signaling pathway appears to play a role in anti-apoptotic effects of the secosteroids.

Metabolism of 2 alpha-propoxy-1 alpha,25-dihydroxyvitamin D3 and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 by human CYP27A1 and CYP24A1.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.

Dimer and superstructure of the active form of a vitamin D3; 1 alpha,24(R)dihydroxy-vitamin D3 monohydrate, C27O3H44.H2O.

The crystals of 1alpha24(R)dihydroxy-vitamin D3 monohydrate, C27O3H44.H2O are orthorhombic in the space group P2(1)2(1)2(1) with cell dimensions a=25.719, b=42.572, c=9.851A and Z=16. The asymmetric unit consists of two subunits with b/8, and each subunit contains a dimer in which two molecules are hydrogen-bonded through water molecules into non-crystallographical symmetry of C2. The two-fold axes are the straight lines, x=1/2, z=0.256 and x=1/2, z=0.623. The two dimers are the same in the rigid ring part, but differ in the conformation of the flexible chains. The dimers further make C2 symmetry between the rigid ring parts to form a superstructure, and the two-fold axis of the straight line, y=1/8, z=0.435 goes through a point that is a little apart from the hypercenter (1/2, 1/8, 1/2). The structure was solved by integrated Patterson and direct methods and refined on Fo2 under restraints. The final R1 is 0.228 on Fo for 1623 reflections with Fo>3sigma, resolutions 1.0-3.0 A, 313 restraints, 490 parameters and average Ueq=0.120. Not all the atoms of the chains appeared nor the hydrogen atoms. The missing atoms of the dimer were modeled from another pair molecule by C2 symmetry and hydrogen atoms were added. The structure of the dimer was optimized by ab initio molecular orbital of HF/6-31G.

Recent developments in vitamin D analogs.

Within the past decade it has been shown that psoriasis can be treated topically with analogs of vitamin-D3. Impaired differentiation and increased proliferation of keratinocytes are key features in psoriatic lesions together with a local activation of T lymphocytes. Evidence has accumulated showing that analogs of vitamin D3 increase differentiation and inhibit proliferation of keratinocytes. Therefore, analogs of vitamin D3 have been investigated in a number of trials showing improvement of psoriasis. It has been shown that vitamin D analogs are better than their vehicle and show the same potency as potent topical steroids. However, vitamin D analogs have been proven efficacious and without side effects also when used on long term basis. Vitamin D analogs can be used both as monotherapy and in combination topical steroids, UVB, PUVA, retinoids and cysclosporine. The vitamin D3 analog calcipotriol has been investigated in most detail and is available as an ointment, a creme and as a scalp solutation. From clinical studies involving thousands of patients, it can be concluded that calcipotriol is efficacious, safe, well tolerated and can be used on a long term basis. Other analogs are available, however, these analogs have not been studied in greater details yet.

Characterization of new conjugated metabolites in bile of rats administered 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3).

The characterization of new conjugated vitamin D metabolites in rat bile was performed using HPLC, liquid chromatography/tandem mass spectrometry combined derivatization, and GC-MS. After the administration of 24,25-dihydroxyvitamin D(3) to rats, 23, 25-dihydroxy-24-oxovitamin D(3) 23-glucuronide, 3-epi-24, 25-dihydroxyvitamin D(3) 24-glucuronide, and 24,25-dihydroxyvitamin D(3) 3-sulfate were obtained as new biliary metabolites together with 24,25-dihydroxyvitamin D(3) 3- and 24-glucuronides. The above metabolites, except 24,25-dihydroxyvitamin D(3) 3-glucuronide, were obtained from rats dosed with 25-hydroxyvitamin D(3). 23, 25-Dihydroxyvitamin D(3) 23-glucuronide was also obtained from the bile of rats administered 25-hydroxyvitamin D(3) in addition to its 3-glucuronide, 25-glucuronide, and 3-sulfate. Thus, it was found that 24,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were directly conjugated as glucuronide and sulfate, whereas at the C-23 position, they were hydroxylated and then conjugated. Furthermore, we found that the C-3 epimerization acts as one of the important pathways in vitamin D metabolism.

Differentiation-related pathways of 1 alpha,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived Caco-2 cells: production of 1 alpha,25-dihydroxy-3epi-cholecalciferol.

We used the human colon adenocarcinoma-derived cell line Caco-2, which spontaneously differentiates in vitro, as a model system to investigate the metabolism of 1 alpha,25-dihydroxycholecalciferol in colon cancer cells. Subconfluent proliferating and confluent differentiating cells were incubated with 1 microM 1 alpha,25-dihydroxycholecalciferol for a period of 24 to 48 h. HPLC analysis of the lipid extract of both cells and media was performed to isolate and identify the various metabolites of 1 alpha,25-dihydroxycholecalciferol. Undifferentiated, highly proliferating Caco-2 cells metabolized 1 alpha, 25-dihydroxycholecalciferol into several side chain modified metabolites formed through the C-24 oxidation pathway. In contrast, no metabolites of the C-24 oxidation pathway were identified in differentiated Caco-2 cells. However, differentiated cells produced significant amounts of a metabolite which was less polar than 1 alpha, 25-dihydroxycholecalciferol on a straight phase HPLC system. This metabolite was identified as 1 alpha,25-dihydroxy-3alpha-cholecalciferol by comigration with a synthetic standard on two different HPLC systems and gas chromatography--mass spectrometry. Thus, we were able to demonstrate that the state of differentiation has a profound influence on 1 alpha,25-dihydroxycholecalciferol metabolism in colon cancer cells.

In vivo metabolism of 24R,25-dihydroxyvitamin D3: structure of its major bile metabolite.

In vivo metabolism of 24R,25-dihydroxyvitamin D3 (24,25-(OH)2D3) in female dogs has been studied thoroughly, and its major bile metabolite identified. After single oral administration of 24,25-(OH)2 [6,19,19-3H]D3 the plasma concentrations of radioactive metabolites were monitored for 504 h, and the metabolites in the bile collected and analyzed. The concentration of 24,25-(OH)2D3 in plasma reached a maximum after 6 h and decayed in two distinct phases; a fast-phase with a half-life of 17 h, followed by a slow-phase with a 17-day half-life. The area under the concentration/time curve (AUC) was 78-84% (0-504 h). The only detectable metabolite in the plasma was 25-hydroxy-24-oxovitamin D3 whose AUC was less than 5%. At 504 h, about 50% of administered radioactivity has been excreted, of which about 90% was found in the feces, indicating most of the administered 24,25-(OH)2D3 to be excreted in bile. A major metabolite, which constituted 23% of the total bile radioactivity at 504 h, was found in the bile. This metabolite was efficiently deconjugated by beta-glucuronidase to afford an aglycone which was identified as 23S,25-dihydroxy-24-oxovitamin D3 (23S,25-(OH)2-24-oxo-D3), by co-chromatography on HPLC with synthetic standards. The glucuronide was isolated from the bile of dogs given large doses of 24,25-(OH)2D3, and the structure determined being 23-(beta-glucuronide) of 23S,25-(OH)2-24-oxo-D3, by analyzing its negative ion mass spectrum and the positive ion mass spectrum of its derivatives. Thus it was concluded that, in dogs, 24,25-(OH)2D3 is a long lasting vitamin D metabolite, is mainly excreted in bile when metabolized to 23S,25-(OH)2-24-oxo-D3 and is conjugated at 23-OH as glucuronide.