Synthesis of vitamin D5: its biological activity relative to vitamins D3 and D2.

The chemical synthesis, spectral characterization, and biological activity of vitamin D₅ in vitamin D-deficient rats is reported. Vitamin D₅ is about 180-fold less active than vitamin D₃ in calcification of rachitic cartilage and about 100- to 200-fold less active in induction of bone-calcium mobilization. In stimulation of intestinal-calcium transport, vitamin D₅ is about 80-fold less active than vitamin D₃. Vitamins D₂ and D₃ appear to be equiactive in all three responses when low doses are administered.     

Synthesis and biological activity of 3 beta-fluorovitamin D3: comparison of the biological activity of 3 beta-fluorovitamin D3 and 3-deoxyvitamin D3

The alteration in the biologic activity of the vitamin D3 molecule resulting from the replacement of a hydrogen atom with a fluorine atom is a subject of fundamental interest. To investigate this problem we synthesized 3 beta-fluorovitamin D3 6 and its hydrogen analog, 3-deoxyvitamin D3 7, and tested the biologic activity of each by in vitro and in vivo methods. Contrary to previous reports which showed that 3 beta-fluorovitamin D3 was as active as vitamin D3 in vivo, we found that the fluoro-analog was less active than vitamin D3. With regard to stimulation of intestinal calcium transport and bone calcium mobilization in the D-deficient hypocalcemic rat, 3 beta-fluorovitamin D3 showed significantly greater biologic activity than its hydrogen analog, 3-deoxyvitamin D3. In the organ-cultured, embryonic chick duodenum, 3 beta-fluorovitamin D3 was approx 1/1000th as active as the native hormone, 1,25-dihydroxyvitamin D3, while 3-deoxyvitamin D3 was inactive even at microM concentrations, in the induction of the vitamin D-dependent, calcium-binding protein. With regard to in vitro activity in displacing radiolabeled 25-hydroxyvitamin D3 from vitamin D binding protein and radiolabelled 1,25-dihydroxyvitamin D3 from a chick intestinal cytosol receptor, 3 beta-fluorovitamin D3 and 3 beta-deoxyvitamin D3 both showed very poor binding efficiencies when compared with vitamin D3. Our results show that the substitution of a fluorine atom for a hydrogen atom at the C-3 position of the vitamin D3 molecule results in a fluorovitamin 6 with significantly more biological activity than its hydrogen analog, 3-deoxyvitamin D3 7.     

Synthesis and biological activity of 1alpha-hydroxyvitamin D3

Hydroboration of cholesta-1,5-diene-3β-ol followed by alkaline-peroxide oxidation resulted in the formation of 1α- and 2α-hydroxy derivatives of cholesterol in nearly equal amounts. 1α-Hydroxycholesterol was then transformed to 1α-hydroxyvitamin D₃, via 1α-hydroxycholest-5,7-diene-3β-ol. 1α-Hydroxyvitamin D₃ was as active as 25-hydroxyvitamin D₃ in the stimulation of intestinal calcium transport and bone mineral mobilization in intact rats, and moreover was able to produce both response in anephric rats similar to 1α,25-dihydroxyvitamin D₃, the active metabolite of vitamin D₃, as reported originally by DeLuca's group.     

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 3β-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 [26,27-³H]-25-hydroxyvitamin D₃ from DBP. In contrast, the 8-hydroxyoctyl alkyl chain analogue 9 showed less displacement.     

The synthesis and biological activity of 22-fluorovitamin D3: a new vitamin D analog

We synthesized 22-fluorovitamin D3 from (22S) cholest-5-ene-3 beta, 22-diol-3 beta-acetate 2. Compound 2 was treated with diethylaminosulfur trifluoride to give 22-fluorocholest-5-en-3 beta-acetate 3 and (E) 22-dehydrocholest-5-en-3 beta-acetate. Compound 3 was treated with N-bromosuccinimide to give a mixture of the respective 5,7- and 4,6-dienes. The 5,7-diene of 3 was separated from the 4,6-diene using the dienophile 4-phenyl-1,2,4-triazoline-3, 5-dione. 22-Fluoro-5 alpha,8 alpha-(3,5-dioxo-4-phenyl-1, 2,4-triazolino)-cholest-6-en-3 beta-acetate 4 was purified by flash chromatography and treated with lithium aluminum hydride to generate 22-fluorocholesta-5,7-dien-3 beta-ol 5. Photolysis of the diene 5, followed by thermal equilibration, resulted in the synthesis of 22-fluorovitamin D3 7. The vitamin 7 increased active intestinal calcium transport only at a dose of 50,000 pmol/rat, whereas vitamin D3 increased intestinal calcium transport at a dose of between 50 and 500 pmol/rat. 22-Fluorovitamin D3 7 did not mobilize bone and soft tissue calcium at a dose as high as 50,000 pmol/rat, whereas vitamin D3 was successful in doing so at a dose of 500 pmol/rat. When tested in the duodenal organ culture system, 22-fluorovitamin D3 7 had approximately 1/25th the potency of vitamin D3. It did not antagonize the activity of 1,25-dihydroxyvitamin D3. 22-Fluorovitamin D3 7 bound to the rat plasma vitamin D binding protein less avidly than vitamin D3. 22-Fluorovitamin D3 was bound very poorly to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. We conclude that the introduction of fluorine at the C-22 position results in a vitamin D sterol with decreased biologic activity when compared to vitamin D3. The presence of a fluorine group at C-22 position inhibits the binding of the vitamin to rat vitamin D binding protein when compared to the binding of its hydrogen analog, vitamin D3.     

Synthesis and biological activity of 1 alpha,23,25,26-tetrahydroxyvitamin D3

The metabolic pathway from 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone includes the formation of 1 alpha,23,25-26-tetrahydroxyvitamin D3 [1 alpha,23,25,26-(OH)4D3]. The aim of the current study was to explore the as yet unknown biological properties of this vitamin D3 sterol. The four diastereoisomers of 1 alpha,23,25,26-(OH)4D3 were chemically synthesized. They were compared to 1 alpha,25-(OH)2D3 in terms of their affinity for the chick intestinal 1 alpha,25-(OH)2D3 receptor and their biologic activity in vivo (stimulation of intestinal calcium absorption and mobilization of calcium from bone in vitamin D-deficient rats). The 1,25-(OH)2D3 receptor binding affinities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4 D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 were 11, 100, 216, and 443 times weaker than the binding affinity of 1 alpha,25-(OH)2D3, respectively. Compared to 1 alpha,25-(OH)2D3, the relative capacities of the 1 alpha,23,25,26-(OH)4D3 compounds to stimulate intestinal calcium absorption were 1/4 for 1 alpha,23(R)25(R)26-(OH)4D3; 1/19 for 1 alpha,23(S)25(S)26-(OH)4D3; 1/90 for 1 alpha,23(S)25(R)26-(OH)4D3; and 1/136 for 1 alpha,23(R)25(S)26-(OH)4D3. Maximal stimulation of intestinal calcium transport occurred 8 h after administration of vitamin D3 metabolites. Mobilization of calcium from bone was quantitated by serum calcium concentration measurements. The activities of 1 alpha,23(R)25(R)26-(OH)4D3, 1 alpha,23(S)25(S)26-(OH)4D3, 1 alpha,23(S)25(R)26-(OH)4D3, and 1 alpha,23(R)25(S)26-(OH)4D3 to increase serum calcium were estimated to be 4, 13, 43, and 69 times weaker than that of 1 alpha,25-(OH)2D3, respectively. These results illustrate the stereospecificity of the chicken intestine 1 alpha,25-(OH)2D3 receptor for binding of 1 alpha,23,25,26-(OH)4D3 and suggest that the 1 alpha,23,25,26-(OH)4D3 exerts its biological activity in the rat through an interaction with 1,25-(OH)2D3 receptors. In summary, the 1 alpha,23,25,26-(OH)4D3 had a markedly lower biological activity than 1 alpha,25-(OH)2D3.     

The synthesis and biological activity of 25,26-dihydroxy-cholecalciferol, a polar metabolite of vitamin D3

The synthesis of 25,26-dihydroxycholecalciferol, a biologically active metabolite of cholecalciferol (vitamin D₃) is described. 3β-Hydroxy-27-nor-5-cholesten-25-one was converted in three steps to 5,7-cholestadiene-3β,25 (RS), 26-triol. The latter compound was irradiated with ultraviolet light to give 25 (RS), 26-dihydroxyprecholecalciferol; this compound underwent thermal isomerisation to yield 25 (RS), 26-dihydroxycholecalciferol. The structure of the final product was confirmed by ultra-violet spectroscopy, mass spectroscopy and by periodate degradation to the known 25-oxo-27-nor-cholecalciferol. 25 (RS), 26-Dihydroxycholecalciferol was able to stimulate the intestinal absorption of calcium but had little or no effect on the healing of rickets.     

Synthesis and biological activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one: a side chain analogue of vitamin D3

The synthesis, biological and antagonistic activity of 3 beta-hydroxy-9,10-secopregna-5,7,10[19]-triene-20-one (20-oxopregnacalciferol, 7) a shortened side chain analogue of vitamin D3, are described. At the highest dose tested the analogue was found to have small though significant bone and soft tissue mobilization activity; no significant increase in intestinal calcium transport was noted. The compound was found to possess no antagonistic activity against vitamin D3.     

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.     

Synthesis and biological evaluation of a new vitamin D(2) analogue

A new vitamin D(2) analogue was synthesized using the Julia-Kocienski olefination. It has antiproliferative effects on cell lines from squamous cell carcinomas of colon and head and neck, but is also as hypercalcaemic as calcitriol in vivo.     

Synthesis and biologic activity of 3 beta-thiovitamin D3

We synthesized 3 beta-thiovitamin D3 from 7-dehydrocholesterol and tested its biological activity and protein binding properties. The thiovitamin was found to be a weak vitamin D agonist at high doses in vivo. It was poorly bound by both vitamin D-binding protein as well as by the intestinal cytosol receptor for 1,25-dihydroxyvitamin D. It did not increase the synthesis of calcium binding protein in the chick embryonic duodenum and did not block the activity of 1,25-dihydroxyvitamin D3 in this system. We conclude that 3 beta-thiovitamin D3 is a weak vitamin D agonist in vivo with no agonist activity or antagonist activity to 1,25-dihydroxyvitamin D3 in the chick embryonic duodenum.     

An evaluation of the biologic activity and vitamin D receptor binding affinity of the photoisomers of vitamin D3 and previtamin D3

Skin is in the site of previtamin D3 and vitamin D3 synthesis and their isomerization in response to ultraviolet irradiation. At present, little is known about the function of the photoisomers of previtamin D3 and the vitamin D3 in skin cells. In this study we investigated the antiproliferative activity of the major photoisomers and their metabolites in the cultured human keratinocytes by determining their influence on 3H-thymidine incorporation into DNA. Our results demonstrated at both 10(-8) and 10(-6) M in a dose-dependent manner. Lumisterol, tachysterol3, 5,6-trans-vitamin D3, and 25-hydroxy-5,6-trans-vitamin D3 only induced significant inhibition at 10(-6) M. 25-Hydroxytachysterol3 was approximately 10- to 100-fold more active than tachysterol3. 7-Dehydrocholesterol was not active even at 10(-6) M. The dissociation constants of vitamin D receptor (VDR) for 25-hydroxytachysterol3, 25-hydroxy-5,6-trans-vitamin D3, and 5,6-trans-vitamin D3 were 22, 58, and 560 nM, respectively. The dissociation constants for 7-dehydrocholesterol, tachysterol, and lumisterol were greater than 20 microM. In conclusion, vitamin D3, its photoisomers and the photoisomers of previtamin D3 have antiproliferative activity in cultured human keratinocytes. However, the antiproliferative activity did not correlate with their binding affinity for VDR. The results suggest that some of the photoproducts may be metabolized to their 25-hydroxylated and 1 alpha,25-dihydroxylated counterparts before acting on VDR. Alternatively, a different receptor may recognize these photoproducts or another mechanism may be involved in modulating the antiproliferative activity of the photoisomers examined.     

Measurement and characterization of C-3 epimerization activity toward vitamin D3

Recently, epimerization of the hydroxyl group at C-3 has been identified as a unique metabolic pathway of vitamin D compounds. We measured C-3 epimerization activity in subcellular fractions prepared from cultured cells and investigated the basic properties of the enzyme responsible for the epimerization. C-3 epimerization activity was detected using a NADPH-generating system containing glucose-6-phosphate, NADP, glucose-6-phosphate dehydrogenase, and Mg(2+). The highest level of activity was observed in a microsomal fraction prepared from rat osteoblastic UMR-106 cells but activity was also observed in microsomal fractions prepared from MG-63, Caco-2, Hep G2, and HUH-7 cells. In terms of maximum velocity (V(max)) and the Michaelis constant (K(m)), 25-hydroxyvitamin D(3) [25(OH)D(3)] exhibited the highest specificity for the epimerization at C-3 among 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], 25(OH)D(3), 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)], and 22-oxacalcitriol (OCT). The epimerization activity was not inhibited by various cytochrome P450 inhibitors and antiserum against NADPH cytochrome P450 reductase. Neither CYP24, CYP27A1, CYP27B1 nor 3(alpha-->beta)hydroxysteroid epimerase (HSE) catalyzed the epimerization in vitro. Based on these results, the enzyme(s) responsible for the epimerization of vitamin D(3) at C-3 are thought to be located in microsomes and different from cytochrome P450 and HSE.     

Chromatographic separation of vitamin D3 sulfate and vitamin D3.

This paper describes a simple chromatographic technique on Sephadex LH20 for the separation of vitamin D3 sulfate from free vitamin D3 and its metabolites. This technique has been used in the study of vitamin D3 sulfate metabolism in rats. Seven hours after injection of vitamin D3 sulfate (35S or 35S and 3H) only the peak of vitamin D sulfoconjugate was found in chromatographic elution of serum extracts.