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.     

Chemical synthesis and biological activity of 3-deoxy-1alpha-hydroxyvitamin D3 an analog of 1alpha,25-(OH)2-D3, the active form of vitamin D3

The chemical synthesis of 3-deoxy-1α-hydroxyvitamin D₃ from cholesterol is described. This steroid is a highly important analog of the hormonally active form of vitamin D, 1α, 25-dihydroxyvitamin D₃; it is the only analog presently available for structure-function studies which lacks the 3β-hydroxyl but retains the key 1α-hydroxyl of 1α, 25-dihydroxyvitamin D₃. The new steroid is highly biologically active; it stimulated intestinal calcium absorption significantly more rapidly than vitamin D₃ and as rapidly as 1α, 25-dihydroxyvitamin D₃.     

Biological activity of 1alpha-hydroxyvitamin D3 in the rat.

The biological activity of 1α-hydroxyvitamin D₃ has been determined in vitamin D-deficient rats. In the accumulation of mineral in bone and cartilage, maintenance of serum calcium, and in efficiency of calcium absorption the 1α-hydroxyvitamin D₃ was approximately two to five times more active than vitamin D₃ or 80–200 units of activity per microgram.     

Synthesis of 1-deoxy-3-C-methyl-d-fructose and 1-deoxy-3-C-methyl-d-sorbose

Hydroxylation of trans-1,3,4-trideoxy-5,6-O-isopropylidene-3-C-methyl-d-glycero-hex-3-enulose with osmium tetraoxide gave a mixture of 1-deoxy-5,6-O-isopropylidene-3-C-methyl-d-arabino- and -d-xylo-hexulose that was partially resolved by acetonation to give 1-deoxy-2,3:4,5-di-O-isopropylidene-3-C-methyl-β-d-fructopyranose (4), 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-keto-d-fructose (5), and 1-deoxy-2,3:4,6-di-O-isopropylidene-3-C-methyl-α-d-sorbofuranose (6). Treatment of a mixture of 4 and 5 with sodium borohydride gave, after column chromatography, 4 and 1-deoxy-3,4:5,6-di-O-isopropylidene-3-C-methyl-d-manno- and -d-gluco-hexitol. Deuterated derivatives corresponding to 4–6 were obtained when isopropylidenation was carried out with acetone-d₆. Deacetonation of 4 and 5 yielded 1-deoxy-3-C-methyl-d-fructose, and 6 similarly afforded 1-deoxy-3-C-methyl-d-sorbose.     

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.     

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 vitamin D3-sulfate

Vitamin D3-3 beta-sulfate has been synthesized using pyridine sulfur trioxide as the sulfate donor. It has been shown to be pure by high performance liquid chromatography and spectral methods. Unlike previous reports, the product has been identified unambiguously as the 3 beta-sulfate ester of vitamin D3 by its ultraviolet, nuclear magnetic resonance, infrared, and mass spectra. The biological activity of vitamin D3-sulfate was then determined in vitamin D-deficient rats. Vitamin D3-sulfate has less than 5% of the activity of vitamin D3 to mobilize calcium from bone and approximately 1% of the ability of vitamin D3 to stimulate calcium transport, elevate serum phosphorus, or support bone calcification. These results disprove previous claims that vitamin D3-sulfate has potent biological activity, and they further do not support the contention that vitamin D-sulfate represents a potent water-soluble form of vitamin D in milk.     

Synthesis and biological properties of 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones--weak agonists

In a continuing effort to explore the 2-methylene-1alpha-hydroxy-19-norvitamin D(3) class of pharmacologically important vitamin D compounds, two novel 2-methylene-19-nor-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones, GC-3 and HLV, were synthesized and biologically tested. Based on reports of similarly structured molecules, it was hypothesized that these compounds might act as antagonists, at least in vitro. The pathway designed to synthesize these compounds was based on two key steps: first, the Lythgoe-type Wittig-Horner coupling of Windaus-Grundmann-type ketone 18, with phosphine oxide 15, followed, later in the synthesis, by the Zn-mediated Reformatsky-type allylation of aldehyde 20 with methylbromomethylacrylate 8. Our biological data show that neither compound has antagonistic activity but acts as weak agonists in vitro and in vivo.     

Metabolism of 1alpha-hydroxyvitamin D3 in the chick.

Chicks convert both orally and intravenously administered 1alpha-hydroxy[6-3H]vitamin D3 rapidly to 1alpha,25-dihydroxy[6-3H]vitamin D3. The maximal accumulation of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine precedes the intestinal absorption response to 1alpha-hydroxyvitamin D3 by at least 2 hours. Oral administration results in the highest concentrations of 1alpha,25-dihydroxy[6-3H]vitamin D3 in intestine, giving a level about 1.5 times that achieved with an intravenous dose. On the other hand, an oral dose of 1alpha-hydroxy[6-3H]vitaminD3 gives much lower amounts of both 1alpha-hydroxy[6-3H]vitamin D3 and 1alpha,25-dihydroxy[6-3H]vitamin D3 in bone and blood than an intravenous dose, which suggests that the 1alpha-hydroxy[6-3H]vitamin D3 may not be utilized as well by the oral route as by an intravenous route. Liver homogenates from both rat and chick convert 1alpha-hydroxy[6-3H]vitamin D3 to 1alpha,25-dihydroxy[6-3H]vitamin D3. However, intestinal homogenates from chick, but not rat, can also cary out this conversion, which may account for the higher concentration of 1alpha,25-dihydroxy[6-3H]vitamin D3 found in the intestine of chicks given an oral dose of 1alpha-hydroxy[6-3H]vitamin D3.     

Biological activity of 1 alpha-hydroxyvitamin D2 in the rat.

The biological activity of 1α-hydroxyvitamin D₂ has been determined in vitamin D-deficient rats. In the calcification of the rachitic epiphyseal plate, 1α-hydroxyvitamin D₂ is more active than 25-hydroxyvitamin D₃, while it is equally active in stimulating intestinal calcium absorption. On the other hand, it is much less active (one-third to one-fifth) than 25-hydroxyvitamin D₃ in the mobilization of calcium from bone. In both the intestinal and bone responses, 1α-hydroxyvitamin D₂ (312 pmol) is active in nephrectomized rats while 25-hydroxyvitamin D₃ is not.     

Synthesis,biological activity and tubulin binding poses of 1-deoxy-9-(R)-dihydrotaxane analogs

1-Deoxy-9α-dihydrotaxane analogs 9 and 10 were semi-synthesized from 1-deoxybaccatin VI, isolated from Taxus mairei, and tested for cytotoxic activity. Taxane 9 is 10-fold less cytotoxic than paclitaxel, while 10 is equally active. In the tubulin polymerization assay (ED₅₀ values), 10 is 4-fold less effective than paclitaxel, but 3-fold superior to 9. These observations can be explained by analysis of the corresponding taxane/β-tubulin complexes.     

1 alpha-Hydroxyvitamin D2 is less toxic than 1 alpha-hydroxyvitamin D3 in the rat

An LD50 of 0.2 mg/kg body wt has been determined for 1 alpha-hydroxyvitamin D3 in the rat. In comparison, the LD50 for 1 alpha-hydroxyvitamin D2 is between 3.5 and 6.5 mg/kg. In terms of chronic toxicity, 1 alpha-hydroxyvitamin D3 at a dose of 5 micrograms/kg/day causes death of one-half the animals in a 4-week period. On the other hand, 20 micrograms/kg/day of 1 alpha-hydroxyvitamin D2 is required to induce similar toxicity. The body weight record and renal calcium accumulation during chronic treatment support the above conclusion. It therefore appears that 1 alpha-hydroxyvitamin D2 is between 5 and 15 times less toxic than 1 alpha-hydroxyvitamin D3. This surprising result prompted a reexamination of the relative biological activity of 1 alpha-hydroxyvitamin D2 and 1 alpha-hydroxyvitamin D3. Both compounds are equally potent in the stimulation of intestinal calcium transport, bone calcium mobilization, in the elevation of serum phosphorus, and in the healing of rickets in the rat. The reason for lower toxicity of 1 alpha-hydroxyvitamin D2 is unknown. The results suggest that 1 alpha-hydroxyvitamin D2 might represent a therapeutically superior compound.     

Synthesis and biological evaluation of L-alpha-phosphatidyl-D-3-deoxy-3-heteromethyl-myo-inositols as phosphoinositide 3-kinase inhibitors.

We have synthesized a series of 3-deoxy-3-heteromethyl derivatives of L-alpha-phosphatidyl-D-myo-inositol as part of our effort to develop specific, reversible inhibitors of phosphoinositide (PI) 3-kinase. Among various derivatives examined, phosphatidyl-D-3-deoxy-3-aminomethyl-myo-inositol displays the highest potency in inhibiting PI 3-kinase both in vitro and in cells. It effectively suppressed antigen-stimulated degranulation in mast cells (IC(50), 17 microM), suggesting a potential application of this PI 3-kinase inhibitor as a mast cell-stabilizing agent.     

Synthesis of 3,4-anhydro-1-deoxy-3-C-methyl-d-hexulose derivatives

Epoxidation of (E)-1,3,4-trideoxy-5,6-O-isopropylidene-3-C-methyl-d-glycero-hex-3-enulose by alkaline hydrogen peroxide gave a mixture of 3,4-anhydro-1-deoxy-5,6O-isopropylidene-3-C-methyl-d-arabino- (2) and -d-xylo-hexulose (3) that was resolved by chromatography. From the reaction of 2 with 3-chloroperbenzoic acid, the Baeyer-Villiger rearrangement product (2R)-2-O-acetyl-2,3-anhydro-1-deoxy-4,5-O-isopropylidene-d-eythro-pentulose hydrate was isolated. The structures and configurations of the above products were established on the basis of chemical transformations and anlytical and spectroscopic data.     

Metabolism of 1alpha-hydroxyvitamin D3 to 1alpha, 25-dihydroxyvitamin D3 in perfused rat liver.

The metabolism of 1α-hydroxyvitamin D₃ (1α-OH-D₃) was studied in rat liver perfused with [³H]-1α-OH-D₃. [³H]-1α-OH-D₃ was converted very rapidly to a more polar metabolite, which was identified as 1α,25-dihydroxy-vitamin D₃ [1α,25-(OH)₂-D₃] by co-chromatography with synthetic 1α,25-(OH)₂-D₃ as well as by gas chromatography-mass spectrometry. [³H]-1α,25-(OH)₂-D₃ appeared in the perfusate as early as 20 min after addition of [³H]-1α-OH-D₃, and its level in the perfusate increased linearly for at least 120 min. These data strongly indicate that 1α-OH-D₃ is metabolized to 1α,25-(OH)₂-D₃, which exerts biological effects on bone and intestine.     

Synthesis of active metabolite(s) from 1 alpha-hydroxyvitamin D3 by human monocytic leukemia cells.

Synthesis of the biologically active metabolite(s) from 1 alpha-hydroxyvitamin D3 (1 alpha(OH)D3) was examined in various types of human leukemia cell lines. Untreated monocytoid leukemia cells (U937 and HEL/S) metabolized 1 alpha (OH)D3 to the active metabolite(s), possibly 1 alpha, 24- and/or 1 alpha, 25-dihydroxyvitamin D3, and these cells were efficiently induced to differentiate by treatment with 1 alpha (OH)D3. However, the other types of leukemia cells did not efficiently metabolize it and were not induced to differentiate by 1 alpha (OH)D3. The possible therapeutic advantage of 1 alpha (OH)D3 in the treatment of monocytic leukemia is discussed.