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-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats

Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1alpha,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.     

Isolation and identification of 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3: metabolites of 1alpha,24(R)-dihydroxyvitamin D3 produced in rat kidney

1alpha,24(R)-Dihydroxyvitamin D3 [1alpha,24(R)(OH)2D3], a synthetic vitamin D3 analog, has been developed as a drug for topical use in the treatment of psoriasis. At present, the target tissue metabolism of 1alpha,24(R)(OH)2D3 is not understood completely. In our present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in the isolated perfused rat kidney. The results indicated that 1alpha,24(R)(OH)2D3 is metabolized in rat kidney into several metabolites, of which 1alpha,24(R),25-trihydroxyvitamin D3, 1alpha,25-dihydroxy-24-oxovitamin D3, 1alpha,23(S),25-trihydroxy-24-oxovitamin D3, and 1alpha,23-dihydroxy-24,25,26,27-tetranorvitamin D3 are similar to the previously known metabolites of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. In addition to these aforementioned metabolites, we also identified two new metabolites, namely 1alpha-hydroxy-24-oxovitamin D3 and 1alpha,23-dihydroxy-24-oxovitamin D3. The two new metabolites do not possess the C-25 hydroxyl group. Thus, the metabolism of 1alpha,24(R)(OH)2D3 into both 25-hydroxylated and non-25-hydroxylated metabolites suggests that 1alpha,24(R)(OH)2D3 is metabolized in the rat kidney through two pathways. The first pathway is initiated by C-25 hydroxylation and proceeds further via the C-24 oxidation pathway. The second pathway directly proceeds via the C-24 oxidation pathway without prior hydroxylation at the C-25 position. Furthermore, we demonstrated that rat kidney did not convert 1alpha-hydroxyvitamin D3 [1alpha(OH)D3] into 1alpha,25(OH)2D3. This finding indicates that the rat kidney does not possess the classical vitamin D3-25-hydroxylase (CYP27) activity. However, from our present study it is apparent that prior hydroxylation of 1alpha(OH)D3 at the C-24 position in the 'R' orientation allows 25-hydroxylation to occur. At present, the enzyme responsible for the C-25 hydroxylation of 1alpha,24(R)(OH)2D3 is unknown. Our observation that the side chain of 1alpha,24(R)(OH)2D3 underwent 24-ketonization and 23-hydroxylation even in the absence of the C-25 hydroxyl group suggests that 1alpha,25(OH)2D3-24-hydroxylase (CYP24) can perform some steps of the C-24 oxidation pathway without prior C-25 hydroxylation. Thus, we speculate that CYP24 may be playing a dual role in the metabolism of 1alpha,24(R)(OH)2D3.     

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 a 20-methyl substituted series of vitamin D analogs by cultured human cells: apparent reduction of 23-hydroxylation of the side chain by the 20-methyl group

We describe here for the first time the effect of introducing a 20-methyl group on the side-chain metabolism of the vitamin D molecule. Using a series of 20-methyl-derivatives of 1alpha,25-(OH)2D3 incubated with two different cultured human cell lines, HPK1A-ras and HepG2, previously shown to metabolize vitamin D compounds, we obtained a series of metabolic products that were identified by comparison to chemically synthesized standards on HPLC and GC-MS. 24-Hydroxylated-, 24-oxo-hydroxylated-, and 24-oxo-23-hydroxylated products of 20-methyl-1alpha,25-(OH)2D3 were observed, but the efficiency of 23-hydroxylation was low as compared with that of the natural hormone and, in contrast to 1alpha,25-(OH)2D3, no truncated 23-alcohol was formed from the 20-methyl analog. These data, taken together with results from other analogs with changes in the vicinity of the C17-C20 positions, lead us to speculate that such changes must alter the accessibility of the C-23 position to the cytochrome P450 involved. Using the HepG2 cell line, we found evidence that the 24S-hydroxylated product of 20-methyl-1alpha,25-(OH)2D3 predominates, implying that the liver cytochrome involved in metabolism is a different isoform. Studies with a more metabolically resistant analog of the series, 20-methyl-Delta(23)-1alpha,25-(OH)2D3, gave the expected block in 23- and 24-hydroxylation, and evidence of an alternative pathway, namely 26-hydroxylation. 20-Methyl-Delta(23)-1alpha,25-(OH)2D3 was also more potent in biological assays, and the metabolic studies reported here help us to suggest explanations for this increased potency. We conclude that the 20-methyl series of vitamin D analogs offers new perspectives into vitamin D analog action, as well as insights into the substrate preferences of the cytochrome(s) P450 involved in vitamin D catabolism.     

Metabolism of 2 alpha-propoxy-1 alpha,25-dihydroxyvitamin D3 and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 by human CYP27A1 and CYP24A1.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.     

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.     

Analysis of C-3 epimerization in (24R)-24,25-dihydroxyvitamin D3 catalyzed by hydroxysteroid dehydrogenase

Studies on the C-3 epimerization in (24R)-24,25-dihydroxyvitamin D(3) [24R,25(OH)(2)D(3)] were performed using hydroxysteroid dehydrogenases (HSDs). 3-Epi-24R,25(OH)(2)D(3) was formed from 24R,25(OH)(2)D(3) by the catalysis of 3alpha- or beta-HSD. These HSDs also catalyzed the C-3 epimerization in 3-epi-24R,25(OH)(2)D(3) to form 24R,25(OH)(2)D(3). 24R,25(OH)(2)D(3) and its C-3 epimer were separated by inclusion high-performance liquid chromatography using gamma-cyclodextrin (gamma-CD) as the mobile phase additive or a gamma-CD bonded chiral column. The product derived from the intermediate during the C-3 epimerization was isolated from the incubation specimens and identified as (7Z)-(24R)-24,25-dihydroxy-9,10-secocholesta-4,7,10(19)-trien-3-one by several instrumental analyses including (1)H-nuclear magnetic resonance spectrometry. The occurrence of this compound strongly proves that the formation of the C-3 epimer by HSD involves a dehydrogenation process. The present study suggests that HSDs may catalyze the C-3 epimerization of vitamin D compounds and modulate their concentrations and biological activities in animals and humans.     

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.     

Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.     

Characterization of 3-epi-1alpha,25-dihydroxyvitamin D3 involved in 1alpha,25-dihydroxyvitamin D3 metabolic pathway in cultured cell lines

Using six different cultured cell models representing osteoblast, intestine, kidney and keratinocyte, we have demonstrated that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) is metabolized into 3-epi-1alpha,25(OH)2D3 in vitamin D-target cells. Although differences existed in the amount of 3-epi-1alpha,25(OH)2D3 formed with different cell types, it was apparent that 1alpha,25(OH)2D3 was subjected to metabolism both through the C24-oxidation and 3-epimerization pathways. Time course and dose response studies showed that the production of 3-epi-1alpha,25(OH)2D3 was enzymatic. It is interesting to note that this epimerization proceeded from 3beta towards 3alpha unidirectionally, and this conversion was not inhibited by ketoconazole. These data suggest that cytochrome P450 related enzymes including the 24-hydroxylase would not affect this reaction. The biological activity of 3-epi-1alpha,25(OH)2D3 was found to be lower than the native 1alpha,25(OH)2D3 in suppressing of proliferation of HL-60 cells, while the affinity of 3-epi-1alpha,25(OH)2D3 for vitamin D-binding protein was 2.5-fold higher than that of 1alpha,25(OH)2D3. The results indicate that 3-epimerization may change the pharmacokinetics and catabolism of 1alpha,25(OH)2D3 in vitamin D-target cells.     

Manipulation of thromboxane synthesis by novel 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 in human promyelocytic leukemia (HL-60) cells.

Using new steroidal side-chain-lengthened 26,27-dialkyl analogues of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3], we manipulated the synthesis of thromboxane and thromboxane-producing enzymes, cyclo-oxygenase and thromboxane synthase, in human promyelocytic leukemia (HL-60) cells in serum-free culture. The order of potency of the analogues for stimulating thromboxane B2 synthetic activity from arachidonic acid (reflecting combined cyclo-oxygenase activity and thromboxane synthase activity) and from prostaglandin H2 (thromboxane synthase activity only) as well as for cyclo-oxygenase induction was 1 alpha,25-(OH)2D3 > or = 1 alpha,25-(OH)2-26,27-CH3)2D3 > 1 alpha,25-(OH)2-26,27-(C2H5)2D3 > 1 alpha,25-(OH)2-26,27-(C3H7)2D3. These results suggest that there are functional and structural limits to the chain length of C-26 and C-27 dialkyl groups flanking the C-25-OH group in the 1 alpha,25-(OH)2D3 molecule for expressing thromboxane synthetic activity in HL-60 cells. Removal of the C-1 alpha-OH group from 1 alpha,25-(OH)2D3 led to markedly decreased thromboxane synthetic activity in HL-60 cells. These structure-activity relationships indicate that both the C-25-OH and C-1 alpha-OH groups in the 1 alpha,25-(OH)2D3 molecule are essential for expressing thromboxane synthesis in HL-60 cells. Also, the rank order for stimulating thromboxane synthesis correlated well with the binding affinity of these dialkyl analogues for the 1 alpha,25-(OH)2D3 receptor of HL-60 cells, suggesting a 1 alpha,25-(OH)2D3 receptor-mediated induction mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of 26,26,26,27,27,27-F6-1 alpha,23S,25-trihydroxyvitamin D3 by human UDP-glucuronosyltransferase 1A3.

26,26,26,27,27,27-Hexafluoro-1alpha,25-dihydroxyvitamin D(3) [F(6)-1alpha, 25(OH)(2)D(3)], which is now clinically used as a drug for secondary hyperparathyroidism, is a hexafluorinated analog of the active form of vitamin D(3). Our previous studies demonstrated that CYP24A1 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues and that F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3). In this study, we examined the metabolism of F(6)-1alpha,25(OH)(2)D(3),F(6)-1alpha,23S,25(OH)(3)D(3), and F(6)-23-oxo-1alpha,25(OH)(2)D(3) by human UDP-glucuronosyltransferases (UGTs). Of these compounds, F(6)-1alpha,23S,25(OH)(3)D(3) was remarkably glucuronidated both in human liver microsomes and in the recombinant system expressing human UGT. No significant interindividual differences were observed among 10 human liver samples. The recombinant system for 12 species of human UGTs revealed that F(6)-1alpha,23S,25(OH)(3)D(3) glucuronidation was specifically catalyzed by UGT1A3. The information obtained in this study seems very useful to predict the metabolism and efficacy of vitamin D analogs in human bodies before clinical trials. In addition, note that for the first time a possible probe substrate for UGT1A3 has been found.     

Polar metabolites of dihydrotachysterol3 in the rat. Comparison with in vitro metabolites of 1 alpha,25-dihydroxydihydrotachysterol3.

The metabolism of 25-hydroxydihydrotachysterol3 (25-OH-DHT3) to more polar metabolites was investigated in vivo in the rat and compared with the in vitro metabolism of 1 alpha,25-dihydroxy-DHT3 (1 alpha,25-(OH)2DHT3) in the osteosarcoma cell line UMR 106. Rats were given 2 mg of DHT3 in divided doses at 0 and 6 hr. Plasma was collected 24 hr after the initial dose, extracted, separated, and polar metabolites purified by HPLC. A number of polar metabolites were formed in vivo with mass spectrometric characteristics which suggested that they were derived from a previously isolated metabolite of 25-OH-DHT3, T3/H. Of these, four were isolated and identified as 24-oxo-T3/H, 24-hydroxy-T3/H, 26-hydroxy-T3/H and the 26,23-lactone of T3/H. In view of the identification of T3/H as a mixture of 1 alpha- and 1 beta-hydroxylated 25-OH-DHT3, osteosarcoma cells (UMR 106) were incubated with chemically synthesized 1 alpha,25-(OH)2DHT3 in an attempt to determine from which component of the T3/H mixture these metabolites were derived. Again, more polar metabolites were formed and five of these were isolated by lipid extraction, purified by HPLC and identified as 24-oxo-1 alpha,25-(OH)2DHT3, 1 alpha,23,25-(OH)3DHT3, 24-oxo-1 alpha,23,25-(OH)3DHT3, 1 alpha,24,25-(OH)3DHT3 and 1 alpha,25,26-(OH)3DHT3. Three of the in vitro metabolites were similar to those found in rat plasma but only two of these metabolites were available in sufficient amounts to allow comparison. The chromatographic characteristics, using HPLC and gas chromatography, of these two pairs of metabolites (24-oxo and 24-hydroxy) were examined and it was demonstrated that they were not the same. It is therefore suggested that the polar metabolites formed in vivo are in fact metabolites of the T3/Hb component (1 beta,25-(OH)2DHT3) rather than the T3/Ha component (1 alpha,25-(OH)2DHT3). Supporting evidence for this suggestion was obtained when a small quantity of 1 beta,25-(OH)2DHT3, obtained from chemically synthesized 1 beta-OH-DHT3 by incubation with Hep 3B cells, was further incubated in the osteosarcoma UMR 106 system. Preliminary studies indicated that the putative 24-oxo and 24-hydroxy metabolites formed from 1 beta,25-(OH)2DHT3 had chromatographic and mass spectral properties almost indistinguishable from those of corresponding metabolites of T3/H formed in vivo. All the metabolites formed in vivo and in vitro are components of two metabolic pathways described previously for 25-hydroxyvitamin D3 and also for 25-OH-DHT3.     

New vitamin D3 derivatives with unexpected antiproliferative activity: 1-(hydroxymethyl)-25-hydroxyvitamin D3 homologs.

Surprisingly, both of the synthetic 1-(hydroxymethyl)-25-hydroxyvitamin D3 diastereomers (-)-2 and (+)-3 retained the antiproliferative activity of natural calcitriol in murine keratinocytes. Preliminary studies indicated, however, that both of these synthetic diastereomers were less than 0.1% as effective as calcitriol for binding to the 1,25-(OH)2-D3 receptor and that they were less than 0.1% as potent as calcitriol for calbindin-D28K induction in organ-cultured embryonic chick duodenum. 1-(Hydroxymethyl)-25-hydroxyvitamin D3 homologs (-)-2 and (+)-3 were synthesized in a convergent manner by combining enantiomerically pure C,D-ring ketone 12 with highly enantiomerically enriched A-ring phosphine oxides (-)-11a and (+)-11b. These A-ring chirons were prepared starting from thermal [2 + 4] cycloaddition of 3-bromo-2-pyrone and acrolein.     

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.