Biological activities and binding properties of 23,23-difluoro-25-hydroxyvitamin D3 and its 1 alpha-hydroxy derivative

23,23-Difluoro-25-hydroxyvitamin D3 is 5-10 times less active than 25-hydroxyvitamin D3 in stimulating intestinal calcium transport, bone calcium mobilization, increasing serum phosphorus, mineralization of rachitic bone, and binding to the plasma transport protein in rats. It is converted to 23,23-difluoro-1 alpha, 25-dihydroxyvitamin D3 by chick renal 25-hydroxyvitamin D-1-hydroxylase. This compound is one-seventh as active as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal receptor for 1,25-dihydroxyvitamin D3. Thus, fluoro substitution on carbon-23 of vitamin D has an unexpected and unexplained suppressive action on plasma binding and biological activity. However, since this substitution does not block the biological response of 25-hydroxyvitamin D3, these results provide additional evidence that 23-hydroxylation of vitamin D is not involved in biological function.     

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3, a novel synthetic vitamin D3 derivative, on calcium metabolism

Regulatory activities of 2 beta-(3-hydroxypropoxy)-1 alpha, 25-dihydroxyvitamin D3 [ED-71], a novel synthetic vitamin D3 derivative, on calcium metabolism were investigated. The compound behaved similar to 1 alpha, 25-dihydroxyvitamin D3 [1,25(OH)2D3] in the ex vivo intestinal calcium transport using rat everted gut sac and the in vivo bone mobilization using vitamin D-deficient rats. By means of Raisz's assay method, 45Ca releasing activity of ED-71 was not greater than that of 1,25(OH)2D3. The time course curve of ED-71 in plasma made a mild round shape compared with that of 1,25(OH)2D3 and the former's plasma concentration remained increased longer than the latter's. The therapeutic effect of ED-71 for the animal models with osteoporosis seemed to be better than that of 1,25(OH)2D3. The results suggest that ED-71 may be a promising drug for therapy of osteoporosis.     

Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.     

26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3: a highly potent, long-lasting analog of 1,25-dihydroxyvitamin D3

A new fluoro analog of 1,25-dihydroxyvitamin D3, i.e., 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3, has been compared with the native hormone, 1,25-dihydroxyvitamin D3, in its biological potency, duration of action, and binding to the vitamin D transport protein and intestinal receptor protein. The fluoro analog is about 5 times more active than the native hormone in healing rickets and elevating serum inorganic phosphorus levels of rachitic rats. It is about 10 times more active than 1,25-dihydroxyvitamin D3 in increasing intestinal calcium transport and bone calcium mobilization of vitamin D-deficient rats fed a low-calcium diet. Furthermore, the higher biopotency is manifested in animals after oral dosing. Of great importance is that the action of the fluoro analog is longer lasting than that of 1,25-dihydroxyvitamin D3. This is especially apparent in the elevation of serum phosphorus and bone mineralization responses. The fluoro analog is only slightly less competent than 1,25-dihydroxyvitamin D3 in binding to the vitamin D transport protein in rat blood, and is one-third as competent as 1,25-dihydroxyvitamin D3 in binding to the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D3. These results suggest that the basis for increased potency of this analog is likely the result of less rapid metabolism.     

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.     

Biological activity of 1 alpha, 25-dihydroxyvitamin D derivatives--24-epi-1 alpha, 25-dihydroxyvitamin D-2 and 1 alpha,25-dihydroxyvitamin D-7

Biological activity of 24-epi-1 alpha,25-dihydroxyvitamin D-2 (24-epi-1,25(OH)2D2) and 1 alpha,25-dihydroxyvitamin D-7 (1,25(OH)2D7), the 22,23-dihydro derivative of the former compound, was investigated. Both of the vitamin D derivatives stimulated intestinal calcium transport and calcium mobilization from bones in rats; however, the effect was about 50% of that of 1 alpha,25-dihydroxyvitamin D-3 (1,25(OH)2D3). On the other hand, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 inducement of HL-60 human leukemia cell differentiation was comparable to that of 1,25(OH)2D3. Accordingly, the differentiation-inducing activity of 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 was much greater than their ability to stimulate calcium metabolism. In contrast to 1,25(OH)2D3, 24-epi-1,25(OH)2D2 and 1,25(OH)2D7 exerted little hypercalcemic activity in mice. These results suggest that both vitamin D derivatives will be useful as anti-tumor agents.     

1 alpha, 25-difluorovitamin D3: an inert vitamin D analog

1α,25-Difluorovitamin D₃ has been synthesized by reacting 1,25-dihydroxyvitamin D₃-3-acetate with diethylaminosulfurtrifluoride followed by hydrolysis. Retention of configuration of the fluoro group in this reaction was demonstrated by physical studies using 1α-fluoro and 1β-fluorovitamin D₃ models. The 1,25-difluorovitamin D₃ compound possessed no vitamin D-like activity demonstrating the importance of 1α- and 25-hydroxylations of vitamin D for activity. However, 1,25-difluorovitamin D₃ had no anti-25-hydroxylation activity and no antivitamin D activity. Since 25-fluorovitamin D₃ has anti-25-hydroxylase activity, it appears the introduction of a fluoro group on the 1 position diminishes interaction of the vitamin D molecule with the 25-hydroxylase system.     

New highly calcemic 1 alpha,25-dihydroxy-19-norvitamin D(3) compounds with modified side chain: 26,27-dihomo- and 26,27-dimethylene analogs in 20S-series

New highly potent 2-substituted (20S)-1 alpha,25-dihydroxy-19-norvitamin D(3) analogs with elongated side chain were prepared by Wittig-Horner coupling of A-ring phosphine oxide with the corresponding protected (20S)-25-hydroxy Grundmann's ketones. Biologic evaluation in vitro and in vivo of the synthesized compounds was accomplished. All the synthesized vitamins possessing a 25-hydroxylated saturated side chain were slightly less active (3-5X) than 1 alpha,25-dihydroxyvitamin D(3) in binding to the porcine intestinal vitamin D receptor and significantly more potent (12-150X) in causing differentiation of HL-60 cells. In vivo, 2-methylene-26,27-dihomo and 2 alpha-methyl-26,27-dimethylene analogs were at least 10 times more active, and 2 alpha-methyl-26,27-dihomo compound at least 5 times more active than the vitamin D hormone both in stimulating intestinal calcium transport and bone calcium mobilization (serum calcium increase). It was also established that a 260 pmol dose of the corresponding 2 beta-methyl analogs had a similar effect on intestinal calcium transport and a much more pronounced effect on bone calcium mobilization as the same dose of 1 alpha,25-dihydroxyvitamin D(3).     

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

25-Azavitamin D3, an inhibitor of vitamin D metabolism and action.

25-Azavitamin D3 inhibited both the bone calcium mobilization and intestinal calcium transport responses of rats to vitamin D3 but not to 25-hydroxyvitamin D3. Although 25-azavitamin D3 had no effect on the response of bone to 1alpha,25-dihydroxyvitamin D3, it did diminish the response of the intestine to that metabolite. 25-Azavitamin D3 increased liver vitamin D content and reduced the concentration of 25-hydroxyvitamin D3 required to inhibit the metabolism of vitamin D3 (75 and 200 microgram) were similar to the doses of 25-azavitamin D3 required to inhibit the action of vitamin D3 in vivo (50 and 150 microgram). 25-Azavitamin D3 is thus a vitamin D antagonist, acting for the most part via inhibition of the liver 25-hydroxylation of vitamin D3.     

Biological activity assessment of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone in the rat

1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [1 alpha,25(OH)2D3-26,23-lactone] was compared to 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3] in terms of their stimulation, in vivo, of intestinal calcium transport and mobilization of calcium from bone in the rat (the two classic vitamin D-mediated responses), and their relative binding to the chick intestinal receptor for 1 alpha,25(OH)2D3, 1 alpha,25-(OH)2D3-26,23-lactone was found to be only one-thirtieth as active as 1 alpha,25-(OH)2D3 in the stimulation of intestinal calcium transport and was found to mediate a significant reduction in the steady-state serum calcium levels. Associated with the reduction in serum calcium was a significant increase in urinary calcium excretion for 24 h after the administration of the steroid. Prior administration of 1 alpha,25(OH)2D3-26,23-lactone partially blocked the actions of a subsequently administered dose of 1 alpha,25(OH)2D3 in increasing serum calcium levels, but did not affect the action of 1 alpha,25(OH)2D3 in stimulating intestinal calcium transport. The binding affinity of 1 alpha,25(OH)2D3-26,23-lactone to the chick intestinal cytosol receptor protein was observed to be 670 times lower than that of 1,25-(OH)2D3 which indicates that perturbation of the 25-hydroxylated side chain by formation of the 26,23-lactone causes a significant reduction in ligand affinity for the receptor.     

Synthesis of [3 beta-3H]-3-epivitamin D3 and its metabolism in the rat

3-Epivitamin D3, the 3 alpha epimer of vitamin D3, was synthesized, and its biological activity in the rat was evaluated. It was found to be approximately 4 times less active on a weight basis than vitamin D3 with respect to intestinal calcium transport, bone calcium mobilization, and calcification score as determined by the line-test assay. Tritiated 3-epivitamin D3 was prepared, and its metabolism in the rat was compared with that of vitamin D3 to investigate the reasons for this diminished activity. 3-Epivitamin D3 was converted to two polar metabolites, for which the chromatographic properties and the origin of biosynthesis (in the liver and kidney, respectively) correspond to 25-hydroxy-3-epivitamin D3 and 1 alpha,25-dihydroxy-3-epivitamin D3. The fact that the concentration of 1 alpha,25-dihydroxy-3-epivitamin D3 in the intestine is half that of 1 alpha,25-dihydroxyvitamin D3 may be one explanation for the reduced biological activity of this epimer.     

1,25-Dihydroxyvitamin D3 increases the expression of the CaT1 epithelial calcium channel in the Caco-2 human intestinal cell line

Background  

The active hormonal form of vitamin D (1,25-dihydroxyvitamin D) is the primary regulator of intestinal calcium absorption efficiency. In vitamin D deficiency, intestinal calcium absorption is low leading to an increased risk of developing negative calcium balance and bone loss. 1,25-dihydroxyvitamin D has been shown to stimulate calcium absorption in experimental animals and in human subjects. However, the molecular details of calcium transport across the enterocyte are not fully defined. Recently, two novel epithelial calcium channels (CaT1/ECaC2 and ECaC1/CaT2) have been cloned and suggested to be important in regulating intestinal calcium absorption. However, to date neither gene has been shown to be regulated by vitamin D status. We have previously shown that 1,25-dihydroxyvitamin stimulates transcellular calcium transport in Caco-2 cells, a human intestinal cell line.     

Biological activity of 24,24-difluoro-25-hydroxyvitamin D3. Effect of blocking of 24-hydroxylation on the functions of vitamin D.

To examine the question of whether 24-hydroxylation plays and importance role in the physiological functions of vitamin D, the biological activity of 24,24-difluoro-25-hydroxyvitamin D was compared with that of 25-hydroxyvitamin D in vitamin D-deficient rats. These two compounds were found almost identically active in the stimulation of intestinal calcium transport, the mobilization of calcium from bone, the healing of rachitic epiphyseal plate cartilage, the elevation of serum inorganic phosphorus, the mineralization of rachitic bone, and in the prevention of rachitogenesis in rats. Little or no difference was detected in the time course of response of intestinal calcium transport or bone calcium mobilization to the two forms of vitamin D. Therefore, in the rat no support could be obtained for the idea that 24,25-dihydroxyvitamin D3 plays an important role in the known physiological responses to the vitamin.     

Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3

In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue.     

Biological activity assessment of the 26,23-lactones of 1,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 and their binding properties to chick intestinal receptor and plasma vitamin D binding protein

The binding of the natural and unnatural diastereoisomers 25-hydroxyvitamin D3-26,23-lactone and 1,25 dihydroxyvitamin D3-26,23-lactone to the vitamin D-binding protein (DBP) and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] chick intestinal receptor have been investigated. Also, the biological activities, under in vivo conditions, of these compounds, in terms of intestinal calcium absorption (ICA) and bone calcium mobilization (BCM), in the chick are reported. The presence of the lactone ring in the C23-C26 position of the seco-steroid side chain increased two to three times the ability of both 25(OH)D3 and 1,25(OH)2D3 to displace 25(OH)[3H]D3 from the D-binding protein; however, the DBP could not distinguish between the various diastereoisomers. In contrast, the unnatural form (23R,25S) of the 25-hydroxy-lactone was found to be 10-fold more potent than the natural form, and the unnatural (23R,25S)1,25(OH)2D3-26,23-lactone three times more potent than the natural 1,25-dihydroxy-lactone in displacing 1,25(OH)2[3H]D3 from its intestinal receptor. While studying the biological activity of these lactone compounds, it was found that the natural form of the 25-hydroxy-lactone increased the intestinal calcium absorption 48 h after injection (16.25 nmol), while bone calcium mobilization was decreased by the same dose of the 25-hydroxy-lactone. The 1,25-dihydroxyvitamin D3-26,23-lactone in both its natural and unnatural forms was found to be active in stimulating ICA and BCM. These results suggest that the 25-hydroxy-lactone has some biological activity in the chick and that 1,25(OH)2D3-26,23-lactone can mediate ICA and BCM biological responses, probably through an interaction with 1,25-(OH)2D3 specific receptors in these target tissues.     

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.     

24,24-Difluoro-1,25-dihydroxyvitamin D3: In vitro production,isolation, and biological activity

24,24-Difluoro-1,25-dihydroxyvitamin D₃ has been synthesized by in vitro incubation of vitamin D-deficient chick kidney homogenates with 24,24-difluoro-25-dihydroxyvitamin D₃. The compound produced was isolated and purified by successive high-performance liquid chromatographic steps and then identified by means of ultraviolet absorption spectrophotometry and mass spectrometry. The difluoro analog of 1,25-dihydroxyvitamin D₃ is found to be highly active in stimulating intestinal calcium transport and bone calcium mobilization in vitamin D₃-deficient rats.     

23-Keto-25-hydroxyvitamin D3: a vitamin D3 metabolite with high affinity for the 1,25-dihydroxyvitamin D specific cytosol receptor

A new metabolite of 23,25-dihydroxyvitamin D3 has been generated with kidney homogenates prepared from vitamin D treated chicks. The metabolite was purified with three high-performance liquid chromatographic steps and was identified as 23-keto-25-hydroxyvitamin D3 by ultraviolet absorption spectroscopy, mass spectrometry, and chemical reactivity. The R stereoisomer of 23,25-dihydroxyvitamin D3 was 10-fold more effective as an in vitro precursor to 23-keto-25-hydroxyvitamin D3 than was the naturally occurring S stereoisomer. Approximately 500 ng of 23-keto-25-hydroxyvitamin D3 was necessary to produce the same degree of intestinal-calcium transport as 25 ng of vitamin D3--a difference of about 20-fold. 23-Keto-25-hydroxyvitamin D3 was not active at stimulating bone calcium resorption at the doses and times tested. This new vitamin D3 metabolite, however, had greater affinity than 25-hydroxyvitamin D3 to both the rat plasma vitamin D binding protein and the 1,25-dihydroxyvitamin D specific cytosol receptor. Heretofore, only 1 alpha-hydroxylated metabolites of 25-hydroxyvitamin D3 or analogues possessing a pseudo 1 alpha-hydroxy group were known to bind to the 1,25-dihydroxyvitamin D receptor with higher affinity than 25-hydroxyvitamin D3. Ketone formation at the 23 position, therefore, is the first side-chain modification of 25-hydroxyvitamin D3 that results in enhanced binding to the 1,25-dihydroxyvitamin D receptor binding protein.     

6-Fluoro-vitamin D3: a new antagonist of the biological actions of vitamin D3 and its metabolites which interacts with the intestinal receptor for 1 alpha,25(OH)2-vitamin D3

The biological activity and the binding affinity for the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] intestinal receptor of a new fluorine-containing vitamin D compound, namely 6-fluoro-vitamin D3 (6-F-D3), is reported. A significant interaction of 6-F-D3 with the 1,25(OH)2D3 receptor was found, with a relative competitive index (RCI) of 0.26 +/- 0.04, which is intermediate between 25-hydroxyvitamin D3 (0.14 +/- 0.01) and 1 alpha-hydroxyvitamin D3 (0.46 +/- 0.08), where the RCI of 1,25(OH)2D3 is defined to be 100. In contrast, vitamin D3 was unable to interact with the 1,25(OH)2D3 receptor. Also, the biological activity of 6-F-D3 was assessed in vivo in the vitamin D-deficient chick. 6-F-D3 at doses up to 130 nmol displayed no biological action on either intestinal calcium absorption (ICA) or bone calcium mobilization (BCM) over the time interval of 14-48 h after dosing. However, when 130 nmol 6-F-D3 was given 2 h before and 6 h after vitamin D3 (1.62 nmol), a significant inhibition of vitamin D-mediated ICA was noted. Also, a dose of 130 nmol 6-F-D3 given 2 h before and 6 h after 1,25(OH)2D3 (0.26 nmol) significantly inhibited ICA, as measured at 12 h. 6-F-D3 is the first vitamin D analog found which has an ability to both bind to the 1,25(OH)2D3 receptor and to antagonize the production of biological responses by 1,25(OH)2D3.