Synthesis, biological evaluation, and conformational analysis of A-ring diastereomers of 2-methyl-1,25-dihydroxyvitamin D(3) and their 20-epimers: unique activity profiles depending on the stereochemistry of the A-ring and at C-20

All eight possible A-ring diastereomers of 2-methyl-1, 25-dihydroxyvitamin D(3) (2) and 2-methyl-20-epi-1, 25-dihydroxyvitamin D(3) (3) were convergently synthesized. The A-ring enyne synthons 19 were synthesized starting with methyl (S)-(+)- or (R)-(-)-3-hydroxy-2-methylpropionate (8). This was converted to the alcohol 14 as a 1:1 epimeric mixture in several steps. After having been separated by column chromatography, each isomer led to the requisite A-ring enyne synthons 19 again as 1:1 mixtures at C-1. Coupling of the resulting A-ring enynes 20a-h with the CD-ring portions 5a,b in the presence of a Pd catalyst afforded the 2-methyl analogues 2a-h and 3a-h in good yield. In this way, all possible A-ring diastereomers were synthesized. The synthesized analogues were biologically evaluated both in vitro and in vivo. The potency was highly dependent on the stereochemistry of each isomer. In particular, the alpha alpha beta-isomer 2g exhibited 4-fold higher potency than 1 alpha,25-dihydroxyvitamin D(3) (1) both in bovine thymus VDR binding and in elevation of rat serum calcium concentration and was twice as potent as the parent compound in HL-60 cell differentiation. Furthermore, its 20-epimer, that is, 20-epi-alpha alpha beta 3g, exhibited exceptionally high activities: 12-fold higher in VDR binding affinity, 7-fold higher in calcium mobilization, and 590-fold higher in HL-60 cell differentiation, as compared to 1 alpha,25-dihydroxyvitamin D(3) (1). Accordingly, the double modification of 2-methyl substitution and 20-epimerization resulted in unique activity profiles. Conformational analysis of the A-ring by (1)H NMR and an X-ray crystallographic analysis of the alpha alpha beta-isomer 2g are also described.     

Metabolism of 2-methyl analogs of 1alpha,25-dihydroxyvitamin D3 in rat osteosarcoma cells (UMR 106)

Several novel A-ring modified analogs of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] have been synthesized in order to investigate the structure-function relationships of 1alpha,25(OH)2D3. We synthesized A-ring modified analogs which contain a methyl group on C-2 of the A-ring. There are eight 2-methyl diastereomers, which differ in the stereochemistry of the methyl group on C-2 and the hydroxyl groups on C-1 and C-3. Further our biological activity studies of the 2-methyl diastereomers indicated that the potency of each analog is highly dependent on the stereochemistry of the A-ring substituents [Konno et al., Biorg. Med. Chem. Letts. 8(2), 151-156 (1998); Nakagawa et al., Biochem. Pharmacol. 60(12), 1937-1947 (2000)]. For example, the VDR binding affinities exhibited by the 1alpha-isomers are significantly higher than those exhibited by the 1beta-isomers. Furthermore, out of all the 1alpha-isomers, the 2alpha-methyl isomers, when compared to the corresponding 2beta-methyl isomers, showed much higher potency in inducing cell differentiation of HL-60 cells, but failed to stimulate apoptosis. In contrast the 2beta-methyl isomers strongly stimulated apoptosis. At present it is unknown how the addition of the 2-methyl modification to the hormone, 1alpha,25(OH)2D3 alters its metabolism in target tissues. Previously, we reported that 1alpha,25(OH)2D3 is metabolized in rat osteosarcoma (UMR 106) cells via both the C-24 oxidation and the C-3 epimerization pathways. Therefore, we studied the metabolism of the four 1alpha,2-methyl diastereomers in UMR 106 cells. Our results indicated that in UMR 106 cells, all four diastereomers were metabolized into several polar metabolites via the C-24 oxidation pathway. Thus, the presence of the 2-methyl group on the A-ring did not inhibit the metabolism of the analogs via the C-24 oxidation pathway. However, it is significant to note that the 2-methyl group prevented the metabolism of the analogs via the C-3 epimerization pathway. In summary, we report that the 2-methyl group interferes with the action of the enzyme(s) involved in C-3 epimerization, but not with the enzyme 1alpha,25(OH)2D3-24-hydroxylase, which is responsible for C-24 oxidation pathway.     

Analysis of molecular mechanism of cancer cell differentiation and apoptosis induced by vitamin D3 analogs on the basis of molecular recognition of vitamin D receptor ligand binding domain

1 alpha,25-Dihydroxyvitamin D3 [1 alpha,25 (OH)2D3] has antiproliferative, differentiation and apoptosis-inducing effects on many malignant cells. These properties have raised the possibility of its use as a therapeutic agent in cancer. Our recent studies using stereoisomers of the A-ring of monohydroxylated 19-nor or 2-methyl substituted 1 alpha,25 (OH)2D3 have clearly demonstrated that the A-ring analogs that contain 1 alpha-hydroxy or 3 beta-hydroxy group are potent inducers of HL-60 cell differentiation. In contrast, the A-ring analogs that contain 1 beta-hydroxy or 3 alpha-hydroxy group are potent stimulators of HL-60 cell apoptosis. It was interesting to note that the analogs could induce differentiation or apoptosis of HL-60 cells on the basis of the stereochemistry of both hydroxy groups at positions 1 and 3 of the A-ring. To further elucidate the possible roles of both the hydroxy groups in regulating cell differentiation and apoptosis, we have synthesized all possible diastereomers of the A-ring of 1 alpha,25 (OH)2D3 and examined their molecular mechanism of differentiation and apoptosis-inducing actions of HL-60 cells in vitro. This study shows that differentiation and apoptosis of HL-60 cells are strictly controlled by the stereochemistry of both hydroxy groups at positions 1 and 3 of the A-ring of 1 alpha,25 (OH)2D3, and the proteins responsible for the regulation of cell cycle and mitochondrial membrane potential are the major targets of 1 alpha,25 (OH)2D3 analogs. These findings provide useful information not only for structure-function studies of 1 alpha,25 (OH)2D3 analogs but also for the development of therapeutic agents for the treatment of cancer.     

Structure-specific control of differentiation and apoptosis of human promyelocytic leukemia (HL-60) cells by A-ring diastereomers of 2-methyl-1alpha,25-dihydroxyvitamin D(3) and its 20-epimer

1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) has been shown to modulate not only proliferation and differentiation but also apoptosis of malignant cells, indicating that it would be useful for the treatment of hyperproliferative diseases such as cancer and psoriasis. Little information is available concerning structural motifs of the 1alpha,25(OH)(2)D(3) molecule responsible for modulation of differentiation and apoptosis. We synthesized all possible A-ring diastereomers of the 2-methyl-1alpha,25(OH)(2)D(3) and its 20-epimer and evaluated their biological activities in human promyelocytic leukemia (HL-60) cells. Surprisingly, the potent analogues could be clearly divided into two groups: (i) those bearing the 1alpha- and 3beta-hydroxyl groups on the A-ring were potent inducers of differentiation and growth inhibitors of HL-60 cells and (ii) those bearing the 1beta-hydroxyl group together with either 3alpha- or 3beta-hydroxyl groups on the A-ring were potent stimulators of apoptosis in these cells. We have clearly identified for the first time the structural motifs on the basis of the stereochemistry of both hydroxyl groups at positions 1 and 3 of the A-ring of the 1alpha,25(OH)(2)D(3) molecule responsible for the induction of differentiation and apoptosis of HL-60 cells. These findings provide useful information not only for structure-function studies of 1alpha,25(OH)(2)D(3) analogues but also for the development of therapeutic agents for the treatment of leukemia and other cancers.     

Vitamin D receptor ligands for osteoporosis

1alpha,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the biologically active metabolite of vitamin D, mediates its actions via the vitamin D receptor (VDR), a member of the superfamily of steroid/thyroid hormone/retinoid receptors. 1,25-(OH)2D3 is required for calcium and phosphorus homeostasis, and for normal skeletal development as well as maintenance of skeletal architecture. Two VDR ligands, calcitriol (1,25-(OH)2D3) and its synthetic analog alfacalcidol (1alpha-hydroxyvitamin D3), have been approved for the treatment of osteoporosis. However, the use of calcitriol and alfacalcidol is limited by their major side effect, hypercalcemia, which is mediated mainly by VDR activity in the small intestine. In order to identify VDR ligands with less hypercalcemia liability, a number of pharmaceutical companies are pursuing efforts to develop synthetic vitamin D analogs. This review discusses the mechanism of action of vitamin D, and summarizes the currently approved anti-osteoporotic VDR agonists and compounds that are under development. The future directions of vitamin D research for the discovery of novel VDR agonists for osteoporosis are also discussed.     

Further synthetic and biological studies on vitamin D hormone antagonists based on C24-alkylation and C2alpha-functionalization of 25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactones

An efficient synthesis and the biological evaluation of 80 novel analogs of 25-dehydro-1alpha-hydroxyvitamin D3-26,23S-lactone 2 (TEI-9647) and its 23R epimer (3) in which the lactone ring was systematically functionalized by introduction of a C1 to C4 primary alkyl group at the C24 position (5 sets of 4 diastereomers), together with their C2alpha-methyl, 3-hydroxypropyl, and 3-hydroxypropoxy-substituted derivatives were described. The triene structure of the vitamin D3 was constructed using palladium-catalyzed alkenylative cyclization of the A-ring precursor enyne with the CD-ring counterpart bromoolefin having the C24-alkylated lactone moiety on the side chain. The CD-ring precursors having 23,24-cis lactones were prepared by using a chromium-mediated syn-selective allylation-lactonization process, and the 23,24-trans lactone derivatives were derived from these via inversion of the C23 stereochemistry. The biological evaluation revealed that both binding affinity for chick vitamin D hormone receptor and antagonistic activity (inhibition of vitamin D hormone induced HL-60 cell differentiation) were affected by the orientation and chain-length of the primary alkyl group on the lactone ring. Furthermore, the C2alpha-functionalization of the C24-alkylated vitamin D3 lactones dramatically enhanced their biological activities. The most potent compound to emerge, (23S,24S)-2alpha-(3-hydroxypropoxy)-24-propyl exhibited almost 1000-fold stronger antagonistic activity (IC50=7.4 pM) than 2 (IC50=6.3 nM).     

Recent results on A-ring modification of 1alpha,25-dihydroxyvitamin D3: design and synthesis of VDR-agonists and antagonists with high biological activity

The structure-activity relationships of 1alpha,25-dihydroxyvitamin D3 on simultaneous modification at both C2alpha and CD-ring side chain, including 20-epimerization, double side chain (gemini), and vitamin D receptor (VDR) antagonists TEI-9647 and TEI-9648 lactone rings, and also on simultaneous modifications at both C2 and C10 positions, i.e., C2 modified active 19-norvitamin D3, have been studied in our laboratory to find new seeds of B-seco-steroidal medicine for treating bone diseases, psoriasis, secondary hyperparathyroidism, and certain kinds of cancers. We developed an efficient and systematic route to the 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogs, i.e., VDR-agonists (20-epi-2-4, double side chain 13a-c, 19-nor 15a-c) and antagonists (36a-c, 37a-c). The A-ring precursors (11a-o) for these analogs were synthesized from D-glucose as a chiral template. In the 19-nor series, we used radical coupling reaction for preparing the A-ring parts from (-)-quinic acid, and the resulting 2-substituted A-ring moiety was coupled with 25-hydroxy Grundmann's ketone utilizing Julia olefination to connect between the C5 and C6 positions. We also synthesized the highly potent VDR-antagonists by introducing the 2alpha-functional group to the TEI-9647 and TEI-9648 skeletons.     

Simultaneous determination of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in human plasma by liquid chromatography-tandem mass spectrometry employing derivatization with a Cookson-type reagent.

A rapid and highly sensitive LC-MS-MS method for simultaneous determination of 25-hydroxyvitamin D2 125(OH)D21 and 25-hydroxyvitamin D3 125(OH)D3] in human plasma has been developed using derivatization with a Cookson-type reagent, 4-12-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalyl)ethyll-1,2,4-triazoline-3,5-dione (DMEQTAD). The derivatization with DMEQTAD significantly improved the ionization efficiencies of 25(OH)D2 and 25(OH)D3 with detection limits of 20 and 12.5 fmol (8 and 5 pg) per injection, respectively. The method employed two steps of solvent extraction but did not require chromatographic purifications for sample pretreatment. The determination was carried out by mass chromatography of the protonated molecular ions formed by atmospheric pressure chemical ionization operating in the positive-ion mode after the derivatization, and 25-hydroxyvitamin D4 was used as an internal standard. The intra-assay coefficients of variation were below 4.02 and 3.24% for 25(OH)D2 and 25(OH)D3, respectively, and the analytical recoveries of both compounds were quantitative. Assay linearity was obtained in the range of 0.05-1 ng per tube and the determination limit was 3 ng/ml for a 20 microl plasma aliquot, for each compound. The developed method was applied to plasma samples obtained from volunteers, two of whom had received vitamin D2 supplementation, and gave satisfactory results.     

Metabolism of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 and 26,27-hexafluoro-1 alpha,23(S)25-trihydroxyvitamin D3 in ROS17/2.8 cells transfected with a plasmid expressing CYP24

1. To clarify the possibility that the metabolism of 26,27-hexafluoro-1 alpha,25-dihydroxyvitamin D3 [F6-1,25(OH)2D3] to 26,27-hexafluoro-1 alpha,23(S),25-trihydroxyvitamin D3 [F6-1,23,25(OH)3D3 and that of F6-1,23,25(OH)3D3 to 26,27-hexafluoro-23-oxo-1 alpha,25-dihydroxyvitamin D3 [F6-23-oxo-1,25(OH)2D3] are catalysed by 25-hydroxyvitamin D3 24-hydroxylase (CYP24), ROS17/2.8 cells transfected with a plasmid expressing CYP24 [pSVL-CYP24(+)] and a corresponding blank plasmid [pSLV-CYP24R(-)] were used. 2. Incubation of [1 beta-3H]-F6-1,25(OH)2D3 for 2 and 5 days with ROS17/2.8 cells transfected with pSVL-CYP24(+) generated a metabolite that co-migrated with authentic F6-1,23,25(OH)3D3 in both normal phase and reversed-phase HPLC systems. 3. Incubation of [1 beta-3H]-F6-1,23,25(OH)3D3 for 5 days with pSVL-CYP24(+)- transfected ROS 17/2.8 cells generated a metabolite that co-migrated with authentic F6-23-oxo-1,25(OH)2D3. In contrast, the metabolites F6-1,23,25(OH)3D3 or F6-23-oxo-1,25(OH)2D3 were not generated in the cells transfected with pSVL-CYP24R(-). 4. The results indicate that CYP24 catalyses the conversion of F6-1,25(OH)2D3 to F6-1,23,25(OH)3D3 and that of F6-1,23,25(OH)3D3 to F6-23-oxo-1,25(OH)2D3.     

Estradiol-induced stimulation of 25-hydroxyvitamin D3-1-hydroxylase in vitamin D-deficient Japanese quail

The dose-response relationship of estradiol benzoate (E2B)-induced increase of in vitro 1,25-dihydroxyvitamin D3 - 1,25-(OH)2D3 - production in Japanese quail raised on a vitamin D-deficient diet was investigated. 4-week-old male and female Japanese quail, initially raised on a normal calcium and vitamin D diet, were placed on a vitamin D-deficient diet for an additional 4 weeks. At 8 weeks of age, E2B was injected intramuscularly at three different doses (0.03, 0.3 and 3.0 mg/kg) daily for 5 days. Two control groups received an equal volume of vehicle only (0.5 ml/kg, 95% ethanol). Kidney homogenates were incubated with tritiated 25-hydroxyvitamin D3 (25-OH-D3). All three doses of E2B stimulated 1,25-(OH)2D3 production beyond the basal level in both sexes but to a much greater extent in the female. Maximal stimulation was observed with the lowest dose (0.03 mg/kg) of E2B. There was no detectable 24,25-dihydroxyvitamin D3 production in any group. A rise in plasma calcium and phosphorus was seen at all three dose levels of E2B in both sexes. The femur dry weight increased significantly only at the two higher doses of E2B in both sexes. It is concluded that E2B injection in vitamin D-deficient Japanese quail can stimulate 25-OH-D3-l-hydroxylase activity beyond the already stimulated basal.     

Synthesis, biological activity, and conformational analysis of four seco-D-15,19-bisnor-1alpha,25-dihydroxyvitamin D analogues, diastereomeric at C17 and C20.

The synthesis of four CD-ring-modified 19-nor-1alpha, 25-dihydroxyvitamin D(3) derivatives lacking C15, referred to as 6C analogues, and diastereomeric at C17 and C20 is described. The synthesis involves an Ireland-Claisen rearrangement of a 3-methyl-substituted ester of (1R)-3-methyl-2-cyclohexen-1-ol as the key step, followed by elaboration of the side chain, transformation into a C8 cyclohexanone derivative, and final Wittig-Horner coupling with the 19-nor A-ring phosphine oxide. Despite possessing a more flexible side chain than the parent hormone, biological evaluation showed an unexpected superagonistic antiproliferative and prodifferentiating activity (10-50 times higher as compared to that of 1alpha,25(OH)(2)D(3)) for the diastereomer with the "natural" configuration at C17 and C20. The other diastereomers exhibit a 25-90% decrease in activity. All four analogues show a decreased binding affinity (45% or less), and their calcemic activity is 4-400 times less than that of 1alpha,25(OH)(2)D(3). The conformational behavior of their side chain was studied using molecular mechanics calculations, and the result is presented as volume maps. A relative activity volume was determined by subtraction of the volume map of the least active analogue from the volume map of the most active one. This shows three regions corresponding to preferred orientations in space of the side chain of the active analogue. One of these regions was found to overlap with the region that is preferentially occupied by the most active of the four diastereomeric 22-methyl-substituted 1alpha,25(OH)(2)D(3) analogues.     

Noteworthy idiosyncrasies of 1α,25-dihydroxyvitamin D3 kinetics for extrapolation from mouse to man: Commentary

Calcitriol or 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is the active ligand of the vitamin D receptor (VDR) that plays a vital role in health and disease. Vitamin D is converted to the relatively inactive metabolite, 25-hydroxyvitamin D₃ [25(OH)D₃], by CYP27A1 and CYP2R1 in the liver, then to 1,25(OH)₂D₃ by a specific, mitochondrial enzyme, CYP27B1 (1α-hydroxylase) that is present primarily in the kidney. The degradation of both metabolites is mostly carried out by the more ubiquitous mitochondrial enzyme, CYP24A1. Despite the fact that calcitriol inhibits its formation and degradation, allometric scaling revealed strong interspecies correlation of the net calcitriol clearance (CL estimated from dose/AUC_∞), production rate (PR), and basal, plasma calcitriol concentration with body weight (BW). PBPK-PD (physiologically based pharmacokinetic-pharmacodynamic) modeling confirmed the dynamic interactions between calcitriol and Cyp27b1/Cyp24a1 on the decrease in the PR and increase in CL in mice. Close scrutiny of the literature revealed that basal levels of calcitriol had not been taken into consideration for estimating the correct AUC_∞ and CL after exogenous calcitriol dosing in both animals and humans, leading to an overestimation of AUC_∞ and underestimation of the plasma CL. In humans, CL was decreased in chronic kidney disease but increased in cancer. Collectively, careful pharmacokinetic data analysis and improved definition are achieved with PBPK-PD modeling, which embellishes the complexity of dose, enzyme regulation, and disease conditions. Allometric scaling and PBPK-PD modeling were applied successfully to extend the PBPK model to predict calcitriol kinetics in cancer patients.     

Reduction of circulating 25-hydroxyvitamin D by antipyrine.

1 Twenty-four Asian vegetarians had significantly lower 25-hydroxyvitamin D (25-OHD) levels and longer antipyrine half-lives than twenty white non-vegetarians (P less than 0.001). 2 Treatment with oral antipyrine over 4 or 5 weeks in seven vegetarian Asians and five racially different non-vegetarians increased drug oxidation significantly in both groups as measured by a fall in antipyrine half-lives and a rise in serum gamma-glutamyltranspeptidase levels and urinary 6 beta-hydroxycortisol/17-hydroxycorticosteroid ratios. 3 Antipyrine treatment produced a fall in circulating 25-hydroxyvitamin D of around 60% in all subjects in whom pretreatment levels could be measured, independent of race and diet. 4. In the Caucasian non-vegetarian group 1,25 dihidroxyvitamin D levels, the most active metabolite of vitamin D, were also measured and remained unaltered despite a substantial fall in 25-hydroxy substrate. 5 The acute fall in 25-hydroxyvitamin D concentration with a maintained level of 1,25 dihidroxyvitamin D may represent the early changes of drug-induced osteomalacia.     

Mechanism of the differentiating action of 25-hydroxyvitamin D3 endoperoxides in human myeloid leukemia cells (HL-60)

The action of 25-hydroxy-6,19-dihydro-6,19-epidioxyvitamin D3 [25-(OH)D3 endoperoxides, 2a and 3a] in inducing differentiation of human myeloid leukemia cells (HL-60) was studied by using their radioactive derivatives (2a' and 3a'). When HL-60 cells were incubated with the labeled endoperoxides (2a' and 3a') in serum-free RPMI 1640 medium, no radioactivity was incorporated into either the cytosol or the chromatin fraction of the cells. When the radioactive endoperoxide (2a') was incubated in the culture medium for 3 days, with or without HL-60 cells, about 45% of the compound was similarly converted to 19,25-dihydroxy-6,19-dihydro-6,19-epoxyvitamin D3 (4a) and about 10% to 25-hydroxy-6,19-epoxyvitamin D3 (6a). These two new vitamin D derivatives were synthesized chemically and tested for their biological activities. Both compounds (4a and 6a) were about 2 times as active as 25-(OH)D3 endoperoxides (2a and 3a) and about 7 times as active as 25-hydroxyvitamin D3 (1a) in inducing differentiation of HL-60 cells. The differentiating activity of these compounds was well correlated with their activity in binding to the cytosol receptor for 1 alpha, 25-dihydroxyvitamin D3 in HL-60 cells. The in vitro bone-resorbing activity of 25-hydroxy-6,19-epoxyvitamin D3 (6a) and 25-(OH)D3 endoperoxide (2a) was higher than that of 25-hydroxyvitamin D3 (1a), indicating that the differentiating activity also paralleled the bone-resorbing activity in these vitamin D derivatives. These results suggest that 25-(OH)D3 endoperoxides (2a and 3a) induce differentiation of HL-60 cells and bone resorption after being converted to these two compounds.     

Kinetic studies of 25-hydroxy-19-nor-vitamin D3 and 1 alpha,25-dihydroxy-19-nor-vitamin D3 hydroxylation by CYP27B1 and CYP24A1.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.     

First human evidence of d-amphetamine induced displacement of a D2/3 agonist radioligand: A [11C]-(+)-PHNO positron emission tomography study.

Imaging the competition between D(2/3) radioligands and endogenous dopamine is so far the only way to measure dopamine release in the living human brain. The dopamine D(2) receptor exists in a high (D(2)(high)) and a low-affinity state for dopamine. Under physiological conditions, dopamine is expected to bind to D(2)(high) only. [(11)C]-(+)-4-propyl-9-hydroxynaphthoxazine ((+)-PHNO) is the first D(2/3) agonist radioligand for positron emission tomography (PET) imaging in humans. Since [(11)C]-(+)-PHNO is expected to bind preferentially to D(2)(high), it should be particularly vulnerable to competition with endogenous dopamine. Nine healthy subjects participated in two PET scans, one after administration of d-amphetamine and one after placebo. [(11)C]-(+)-PHNO PET test re-test variability was determined in 11 healthy subjects. Binding potentials (BPs) were calculated for caudate, putamen, ventral striatum, and globus pallidus. d-Amphetamine led to a significant decrease of [(11)C]-(+)-PHNO BPs in caudate (-13.2%), putamen (-20.8%), and ventral striatum (-24.9%), but not in globus pallidus (-6.5%). d-Amphetamine-induced displacement correlated with serum d-amphetamine levels in all regions but caudate. This is the first report on competition between endogenous dopamine and a D(2/3) agonist radioligand in humans. [(11)C]-(+)-PHNO PET might be a superior measure for release of endogenous dopamine than PET employing conventional D(2/3) antagonist radioligands.     

Stereospecific synthesis, assignment of absolute configuration, and biological activity of the enantiomers of 3-[[[3-[2-(7-chloroquinolin-2-yl)-(E)-ethenyl]phenyl] [[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid, a potent and specific leukotriene D4 receptor antagonist

The enantiomers of the leukotriene D4 antagonist 3-[[[3-[2-(7-chloroquinolin-2-yl)-(E)-ethenyl]phenyl] [[3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]propionic acid (L-660,711)(MK-571) have been prepared, their absolute stereochemistry has been assigned as S for (+)-1 and R for (-)-1 by X-ray analysis of a synthetic intermediate (5), and the biological activity of the enantiomers has been explored. Unexpectedly, the enantiomers are both comparably biologically active with (+)-1 slightly more intrinsically active at the LTD4 receptor in vitro.     

2-Ethyl and 2-ethylidene analogues of 1alpha,25-dihydroxy-19-norvitamin D(3): synthesis,conformational analysis,biological activities,and docking to the modeled rVDR ligand binding domain

Novel 19-nor analogues of 1alpha,25-dihydroxyvitamin D(3) were prepared and substituted at C-2 with an ethylidene group. The synthetic pathway was via Wittig-Horner coupling of the corresponding A-ring phosphine oxides with the protected 25-hydroxy Grundmann's ketones. Selective catalytic hydrogenation of 2-ethylidene analogues provided the 2alpha- and 2beta-ethyl compounds. The 2-ethylidene-19-nor compounds with a methyl group from the ethylidene moiety in a trans relationship to the C(6)-C(7) bond (E-isomers) were more potent than the corresponding Z-isomers and the natural hormone in binding to the vitamin D receptor. Both geometrical isomers (E and Z) of (20S)-2-ethylidene-19-norvitamin D(3) and both 2alpha-ethyl-19-norvitamins (in the 20R- and 20S-series) have much higher HL-60 differentiation activity than does 1alpha,25-(OH)(2)D(3). Both E-isomers (20R and 20S) of 2-ethylidene vitamins are characterized by very high calcemic activity in rats. The three-dimensional structure model of the rat vitamin D receptor and the computational docking of four synthesized (20R)-19-norvitamin D(3) analogues into its binding pocket are also reported.