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Many readers may have only a vague idea about vitamin D. This is made complicated, in part, because it is also expressed with suffixes such as vitamin D(2) or vitamin D(3). Otherwise the prefix of "active" is also occasionally used. Vitamin D is often referred to as an important nutrient for calcium intake, especially for growing children and the elderly. On the other hand, it serves as a therapeutic drug for osteoporosis and psoriasis. Recent studies have suggested an association with a number of diseases such as cancer, diabetes and Alzheimer's disease. The author has been involved in the research and development of vitamin D applications for over 25 years, and has often witnessed even world-class experts confuse the two roles - vitamin and hormone - of "substance" D. Assuming some readers are not familiar with vitamin D, D hormone or osteoporosis, an outline of vitamin D and D hormone is delineated with a particular focus on the treatment of osteoporosis. Furthermore, development of alfacalcidol as the first prodrug of D hormone (calcitriol) and eldecalcitol as a characteristic new D hormone derivative and basic relationship between calcemic activity and effect on bone in vitamin D (cholecalciferol), D hormone (calcitriol/alfacalcidol), and a new D hormone derivative (eldecalcitol) are introduced.

A novel synthesis of a radioactive compound of 1 alpha-hydroxyvitamin D3 (1 alpha OHD3) (1) and its pharmacokinetics are described. Radioactive 1 alpha OHD3 tritiated at 22 and 23 positions ([22,23-(3)H4]1 alpha OHD3) (5) was prepared via key reactions of the reduction of acetylenic side chain in the ketone (12) with tritium gas in the presence of palladium-charcoal and the subsequent Wittig reaction with the A-ring synthon (16). [22,23-(3)H4]1 alpha OHD3 (5) showed high specific radioactivity (111.5 Ci/mmol) and was used successfully in pharmacokinetics studies with rats. In the pharmacokinetics studies, the plasma concentration level of the active form of vitamin D3, 1 alpha,25-dihydroxy-vitamin D3 [1 alpha,25(OH)2D3], after oral or intravenous administration of [22,23-(3)H4]1 alpha OHD3 (5), showed longer half-life, lower maximum concentration, and lower area under the curve than those after treatment of 1 alpha,25(OH)2D3 tritiated at 26 and 27 positions (4). These results might suggest a beneficial therapeutic utility of 1 alpha OHD3 (1) over the treatment of 1 alpha,25(OH)2D3 (2).

Biological activities of a series of 2beta-substituted analogues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] were evaluated in vitro in terms of their binding affinity with regard to calf thymus cytosolic vitamin D receptor (VDR) and rat plasma vitamin D-binding protein (DBP). Additionally, reporter gene luciferase activities using either a rat 25-hydroxyvitamin D3-24-hydroxylase gene promoter, including two vitamin D-responsive elements (VDREs), in transfected rat osteoblast-like ROS17/2.8 cells, or a human VDR-GAL4 modified two-hybrid system in transfected human epitheloid carcinoma, cervix HeLa cells were examined. Binding affinity for VDR, transactivation potency on the target gene and VDR-mediated gene regulation of the hydroxyalkyl and hydroxyalkoxy 2beta-substituted analogues were almost comparable to those of 1alpha,25(OH)2D3, while the alkyl and alkenyl analogues were much less active than 1alpha,25(OH)2D3. This study investigated the biological evaluation of a series of 2beta-substituted analogues at the molecular level, with regard to the structural differences of alkyl, alkenyl, hydroxyalkyl, hydroxyalkoxy, alkoxy, hydroxy and chloro substituents at the 2beta-position of 1alpha,25(OH)2D3.

1alpha,25-Dihydroxyvitamin D3 [1alpha,25(OH)2D3] has been shown to exert both its nuclear vitamin D receptor (nVDR)-mediated genomic actions and membrane vitamin D receptor (mVDR)-mediated nongenomic actions. In this study, the effects of 1alpha,25(OH)2D3 and its analogues on transmembrane Ca2+ influx were examined in the growth phase of rat osteosarcoma ROS17/2.8 cells. Like BAYK8644 (2 x 10(-5)M), a well-known L-type Ca2+ channel agonist, 1alpha,25(OH)2D3 (10(-8)M) increased transmembrane influx of Ca2+ through voltage-dependent Ca2+ channels and increased intracellular Ca2+ concentration within 2 min of addition to the medium. The 1alpha,25(OH)2D3-induced Ca2+ influx was completely blocked by pre-treatment with nifedipine (2 x 10(-5)M), an L-type Ca2+ channel antagonist. Two vitamin D analogues, 22-oxa-1alpha,25(OH)2D3 (OCT, 10(-8) M) and 20-epi-22-oxa-24a, 26a,27a-trihomo-1alpha,25(OH)2D3 (KH1060, 10(-8)M), which were 3.8 and 3600-fold more active than 1alpha,25(OH)2D3 in stimulating differentiation on human promyelocytic leukemic HL-60 cells, respectively, also increased intracellular Ca2+ concentration, while their Ca2+ channeling activities were similar to or significantly weaker than that of 1alpha,25(OH)2D3. Furthermore, the enhanced transmembrane Ca2+ influx induced by 1alpha,25(OH)2D3 (10(-8)M) or OCT (10(-8)M) was completely blocked by pre-treatment with the respective 1beta epimer [1beta,25(OH)2D3 and 1beta-OCT] at equal concentration. These findings suggest that 1alpha,25(OH)2D3 and its analogues modulate transmembrane Ca2+ influx in osteoblast-like cells by opening L-type Ca2+ channels which can recognize 1alpha-hydroxy analogues as agonists and 1beta-hydroxy analogues as antagonists.

SUMMARY: 22-Oxa-1,25-dihydroxyvitamin D3 (OCT) is an analogue of vitamin D with less calcemic action than 1,25-dihydroxyvitamin D3 (1,25D3), and thus may be advantageous in the treatment of secondary hyperparathyroidism in dialysis patients. to further elucidate the mechanisms of less-calcemic action of OCT in chronic renal failure, we examined the effects of OCT and 1,25D3 on mRNA levels for vitamin D-dependent 9-KDa calcium binding protein (CaBP-D9K) in the intestinal mucosa and 28-KDa (CaBP-D28K) in the kidney. In Sprague-Dawley rats made uremic by 5/6 nephrectomy for three months, OCT at doses of 0.25, 1.25 and 6.25 μg/kg, or 1,25D3 at 0.025,0.125 and 0.625 μg/kg were administered intravenously three times per week for two weeks. At 24 h after the final injection, enhanced serum PTH and PTH mRNA levels were successfully suppressed both by OCT and 1,25D3 in a dose dependent manner. However, OCT induced less hypercalcemia than 1,25D3. 1,25D3 markedly upregulated the expression of CaBP-D9K and CaBP-D28K genes, while they were not affected by OCT at all. In conclusion, such attenuated effects of OCT on calcium-binding proteins may play a role in the noncalcemic action, because number of CaBP-D9K has been suggested to correlate with calcium absorption in the intestine.

SUMMARY: 22-oxa-calcitriol (OCT), a vitamin D analogue, suppresses parathyroid hormone (PTH) secretion and has less calcaemic activity than 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vivo. In this study, we evaluated the effect of OCT on PTH secretion in vitro using human hyperplastic parathyroid tissue obtained during surgery for advanced renal hyperparathyroidism and normal bovine parathyroid glands to compare the effects of 1,25(OH)2D3. 22-oxa-calcitriol suppressed PTH secretion by nodular hyperplastic parathyroid tissue and normal bovine tissue in a dose dependent manner, the same as 1,25(OH)2D3. We showed the additive effect of extracellular calcium level on suppression of PTH secretion by OCT and 1,25(OH)2D3. These results suggest that OCT suppresses PTH secretion, the same as 1,25(OH)2D3 not only in normal parathyroid cells but also on hyperplastic parathyroid cells.

1Alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] mediates its biological activities through specific binding to the nuclear vitamin D receptor (VDR). The VDR, bound to 1alpha,25(OH)2D3, forms a heterodimer with a nuclear accessory factor, retinoid X receptor (RXR), and the complex subsequently binds to specific nucleotide sequences or a vitamin D-responsive element (VDRE) to induce gene transcriptions. Thus, an ideal analogue of 1alpha,25(OH)2D3 for therapeutic applications has been considered to be one which has a high binding affinity for VDR, thus forming a stable VDR/RXR complex, and binding strongly to VDRE. By contrast, we report here evidence contrary to this hypothesis. Several singly dehydroxylated A-ring analogues of 19-nor-1alpha,25-dihydroxyvitamin D3 and 19-nor-22-oxa-1alpha,25-dihydroxyvitamin D3, all of which have an extremely low binding affinity for VDR, and some of which lack the 1alpha-hydroxyl group that is considered to be essential for VDR-mediated gene expression, have greater or equivalent potencies to 1alpha,25(OH)2D3 for inhibiting the proliferation and inducing the differentiation of HL-60 cells, as well as inducing the transactivation of a luciferase reporter gene combining a rat 25-hydroxyvitamin D3 24-hydroxylase gene promoter containing two VDREs. The present findings open interesting possibilities as to the role of the VDR in the genomic action of 1alpha,25(OH)2D3 and the development of new 19-nor-analogues of 1alpha,25(OH)2D3.

The binding properties, with blood proteins, and tissue distribution of 22-oxa-1 alpha,25-dihydroxyvitamin (22-oxacalcitriol; OCT), a noncalcemic analogue of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3], in rats were investigated. The binding affinity of OCT to plasma vitamin D binding protein (DBP) is extremely low and OCT mainly circulates in the blood as an intact form nonspecifically bound to lipoproteins especially to chylomicrons and low density lipoprotein (LDL). OCT intravenously injected into normal rats rats rapidly disappeared from the blood, and rapidly appeared in the bile as glucuronides of intact OCT and 1 alpha, 3 beta,20(S)-trihydroxy-9,10-secopregna-5,7,10(19)-triene (23,24,25,26, 27-pentanorOCT; pentanorOCT) as an OCT metabolite. When OCT or 1,25(OH)2D3 was injected into normal rats, significant amounts of OCT and 1,25(OH)2D3 were quickly detected in the thyroid and parathyroid glands, thymus, adrenals, liver, plasma, small intestine, kidneys, and calvaria. The detected amounts of OCT in the parathyroid glands, thymus, adrenals, liver, small intestine, and kidneys were significantly higher than the respective values for 1,25(OH)2D3 2 and/or 10 min after injection, while those of OCT in the plasma and calvaria were significantly lower than those of 1,25(OH)2D3. The in vivo rapid turn-over, nonspecific transportation, and incorporation of detectable amounts into the tissues are typical characteristics of OCT which may account for its specific activities.

22-Oxa-1α, 25-dihydroxyvitamin D3 (OCT) and 2β-(3-Hydroxypropoxy)-1α, 25-dihydroxyvitamin D3 (ED-71) are novel synthetic vitamin D3 analogues. In order to examine their calcemic actions on intestine and bone, we have investigated the effects of OCT and ED-71 on intestinal Ca transport, bone mobilization and plasma parathyroid hormone (PTH) level in vitamin D-deficient rats. The vitamin D-deficient rats were intravenously given either 6.25µg/kg or 0.2µg/kg of 1,25-D3, OCT or ED-71 and theirplasma Ca levels and intestinal Ca transport were measured periodically. At a high dose, 1,25(OH)2D3 and ED-71 showed a strong biphasic stimulation of intestinal Ca transport and bone mobilization, and reduced the plasma PTH levels to the normal level completely. On the other hand, OCT failed to suppress the PTH secretion although it exerted first phase action on the both intestinal Ca transport and bone mobilization in vitamin D-deficient rats. The reason why OCT failed to suppress the PTH secretion even at a high dose, has not yet been clarified, but it may be at least in part due to its weak calcemic action and short half-life in plasma.

The synthesis of 1-deoxyeldecalcitol (IV) is realized employing Trost′s methodology involving a palladium-catalyzed coupling between enyne (II) and bromoalkene (III) as key step.

Four vitamin D3 analogues, 1α, 24(S)-and 1α, 24(R)-dihydroxy-22-oxavitamin D3 (5 and 6) and their 20(R)-epimers (7 and 8) were synthesized from the 20(S)-alcohol (10). In tests of activity to induce differentiation of human myeloid leukemia cells (HL-60) to macrophages, 5 showed comparable activity to 1α, 25-dihydroxy-22-oxavitamin D3 (OCT) (2), and the other three analogues (6, 7 and 8) were less active than OCT (2). The binding properties of these analogues to the chick embryonic intestinal 1α, 25-dihydroxyvitamin D3 (1) receptor were evaluated. Furthermore, 20(R)-OCT (9) was synthesized and its biological properties were compared with those of OCT (2) and the 20(R)-epimers (7 and 8).

The combination of the regioselective propiolate-ene reaction, catalytic enantioselective epoxidation and catalytic enantioselective carbonyl-ene cyclization completes the synthesis of the A ring of the hybrid 19-nor-22-oxa D3 analogue (1), which shows the significant activity in transactivation.

Six analogues of 1 alpha,25-dihydroxy-22-oxavitamin D3 (OCT) (2), 26,27-dimethyl OCT (5), 26,27-diethyl OCT (6), 24-norOCT (7), 24-homoOCT (8), 24-dihomoOCT (9), and 24-trihomoOCT (10) were synthesized from the 20(S)-alcohol (11) as the common starting material. In the activity inducing differentiation of human myeloid leukemia cells (HL-60) into macrophages, 26,27-dimethyl OCT (5) and 24-homoOCT (8) showed the highest activities. The binding properties of these analogues to the chick embryonic intestinal 1 alpha,25-dihydroxyvitamin D3 (1) receptor are also described.

1 alpha,25-Dihydroxy-21-norvitamin D3 (3) was synthesized from 1 alpha-hydroxydehydroepiandrosterone (4). Certain biological properties of 3 were examined in comparison with those of 1 alpha,25-dihydroxyvitamin D3 (1) and 1 alpha,25-dihydroxy-21-nor-20-oxavitamin D3 (2) to evaluate the effect of the 21-methyl substituent on biological activities. The differentiation-inducing activity of 3 towards human myeloid leukemia cells was approximately one-fifth of that of 1, while in the binding affinity with chick intestinal cytosolic receptor, 3 was about one-tenth of that of 1. The rather weak effect of 3 on serum calcium levels in normal mice at a dosage of 500 micrograms/kg (intravenous administration) indicates that the essential importance of the 21-methyl moiety may lie in its effect on the regulation of calcium metabolism.

Using Trost convergent methodology, the synthesis of la,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D 3 (eldecalcitol) for the improved and industrial scale production and related putative metabolites of eldecalcitol is summarized. In addition, A-ring diastereomers at the 1- and 3-positions of eldecalcitol are described. The synthesis centers on a key palladium-catalyzed alkylative cyclization and coupling of ene-ynes that constitute the A-ring fragment to bromomethylenes comprising the C/D-ring fragment which affords the requisite triene framework of vitamin D 3 analogs.

Three vitamin D3 analogues, 1 alpha,25-dihydroxy-23-oxavitamin D3 (3), 1 alpha,25-dihydroxy-23-thiavitamin D3 (4) and 1 alpha,25-dihydroxy-23-azavitamin D3 (5) were synthesized. In the differentiation-inducing activity of human myeloid leukemia cells into macrophages in vitro, the 23-aza analogue (5) showed the least activity, while no remarkable differences were observed between the 23-oxa analogue (3) and the 23-thia analogue (4), which were less active than 1 alpha,25-dihydroxyvitamin D3 (1).

A new metabolic pathway of (24R)-24,25-dihydroxyvitamin D3 [24,25(OH)2D3] was clarified in the in vivo experiments. After the administration of 24,25(OH)2D3 to rats, a new monoglucuronide of a vitamin D metabolite was obtained from the bile together with 24,25(OH)2D3 3- and 24-glucuronides. The genin of the metabolite was identified as 3-epi-24,25(OH)2D3 in comparison with the synthetic sample based on the data from 1H-NMR, GC/MS, and LC/atmospheric pressure chemical ionization-MS. The conjugation position was determined to be the 24-hydroxy group by the LC/electrospray ionization-MS and -MS/MS/MS combined with derivatization. To our knowledge, this is the first reported instance of the epimerization of the 3-hydroxy group of vitamin D compound with no hydroxy group at the 1alpha-position.

A little over two decades ago, the vitamin D3 activation pathway was elucidated and alfacalcidol arrived on the scene as a prodrug for active vitamin D3, 1α, 25-dihydroxyvitamin D3, to remedy vitamin D3 deficiency. With the concurrent reported discovery of the differentiation-inducing effect of active vitamin D3, its diverse physiological effects have become appreciated and the research aiming to accentuate selected physiological effects by analogue synthesis has made a fresh development. Our studies aimed particularly at separating the differentiation-inducing effect/cell growth-inhibitory effect and the calcemic effect of active vitamin D3 led to the development of two characteristic analogues, OCT and ED-71. OCT, characterized by its profound differentiation-inducing effect and modest calcemic effect, is currently in practical use as an injectable therapeutic agent for secondary hyperparathyroidism as well as in clinical settings an ointment for the treatment of psoriasis vulgaris, an intractable skin disease. The other analogue, ED-71, possesses a profile inverse to that of OCT and is now under clinical development as an oral preparation for treatment of osteoporosis. The present overview focuses on the search for and synthesis of OCT and ED-71. It also refers to methodology of searching next-generation analogues, exemplifying a new analogue, DD-281, based on the knowledge accumulated through process development of OCT and ED-71.

1alpha,25-Dihydroxyvitamin D(3) (1alpha,25-D(3)) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, its calcemic effect in vivo limits its therapeutic applications. Here, we report the efficacy of 22-oxa-1alpha,25-dihydroxyvitamin D(3) (22-oxa-1alpha,25-D(3)), a low calcemic analog of vitamin D, against the development of metastatic lung carcinoma after an intravenous injection of green fluorescent protein-transfected Lewis lung carcinoma (LLC-GFP) cells in C57BL/6 mice. The mice injected with tumor cells were implanted simultaneously with osmotic minipumps containing either 1alpha,25-D(3), 22-oxa-1alpha,25-D(3) or vehicle. The 1alpha,25-D(3) treatment group had been hypercalcemic, but the 22-oxa-1alpha,25-D(3) and vehicle treatment groups remained normocalcemic for the duration of the experiment. The total number of lung metastases, lung weight and the expression of GFP mRNA in the lung were markedly decreased in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. In the in vitro experiment, 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3) reduced the expression of matrix metalloproteinase (MMP)-2, MMP-9, vascular endothelial growth factor and parathyroid hormone-related protein in LLC-GFP cells. Furthermore, in the angiogenesis assay, the number of tumor cells or basic fibroblast growth factor-induced angiogenesis was reduced in 1alpha,25-D(3) and 22-oxa-1alpha,25-D(3)-treated mice. Moreover, using a new experimental model of vitamin D receptor (VDR) null mutant (VDR(-/-)) mice with corrected hypocalcemia and hypervitaminosis D, we examine the anti-cancer effect of 22-oxa-1alpha,25-D(3) without other functions induced by 22-oxa-1alpha,25-D(3) in the host. In the VDR(-/-) mice, 22-oxa-1alpha,25-D(3) directly inhibited the metastatic activity of LLC-GFP cells in a dose-dependent manner without exerting a direct influence on the calcemic activity or other actions regulated by 22-oxa-1alpha,25-D(3) in the host. These results indicate that the inhibition of metastasis and angiogenesis-inducing activity in cancer cells seemed to be a major mechanism responsible for the anti-cancer effects of 22-oxa-1alpha,25-D(3). Our findings show that 22-oxa-1alpha,25-D(3) is beneficial for the prevention of metastasis in lung carcinoma.

Four vitamin D3 analogues (7a, 7b, 7c and 7d) bearing a hydroxyalkoxy group at the 2 beta-position were synthesized from the alpha-epoxide (5). The C-3 analogue (7b) showed the highest potency for elevating plasma calcium levels in rats. Furthermore, the 25-hydroxylated C-3 analogue (ED-71) (3), prepared from the 25-hydroxylated alpha-epoxide (9), significantly increased plasma calcium to levels much higher than those in rats administered 1 alpha,25-(OH)2-D3 (1).

22-Oxacalcitriol 3-hemiglutarate, a haptenic derivative of 22-oxacalcitriol, was synthesized to obtain a specific antibody for use in radioimmunoassay. Three antisera were elicited in rabbits against the hapten conjugated with bovine serum albumin, and their specificity was examined by cross-reaction study. One of the antisera was found to be satisfactorily specific, and expected to provide a radioimmunoassay useful for the pharmacokinetic study of 22-oxacalcitriol.

Analogs related to 1 alpha,25-dihydroxy-2 beta-(3-hydroxypropoxy)vitamin D3 (ED-71) (4), oxa-type and carba-type analogs of vitamin D3 bearing substituents at the 2 beta-position of 1 alpha,25-dihydroxyvitamin D3 (1), were synthesized from the alpha-epoxides (6 and 13). Three analogs, ED-71 (4) and two carba-type analogs (16 and 26), showed potent preventive effects on bone mineral loss in pre-osteoporosis model rats. ED-71 (4) was concluded to be an optimized analog and a promising candidate for the treatment of osteoporosis.

The synthesis of two vitamin D3 analogues, 1α, 25-dihydroxy-20-oxa-21-norvitamin D3 (2) and 1α-hydroxy-20-oxa-21-norvitamin D3(3) from dehydroepiandrosterone (4) is described.

The synthesis of a new prostaglandin synthon (14) from 5-trichloromethyl-4-oxatricyclo [4. 3. 0. 03, 7] non-8-ene (2) and its conversion to prostaglandin F2α (21) are described. Compound (2) was transformed into the acetylene derivative (5) which was then led to the C-9 acetylene lactone (14) via a 6-step, 8-stage reaction. A new synthesis of (±)-prostaglandin F2a (21) was completed through a 5-step reaction starting from the C-9 lactone (14).