2,2-Functionalized analogues of 1alpha,25-dihydroxyvitamin D3, the potent inducers of cell differentiation

All four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D(3) (4) were designed and convergently synthesized. Nine-step conversion of methyl hydroxypivalate 6 provided the desired A-ring enyne synthon (13a,b) in good overall yield. Cross-coupling reaction of the A-ring synthon 13a,b with the CD-ring portion in the presence of palladium catalyst, followed by deprotection, gave the vitamin analogues (4a-d). We also synthesized four stereoisomers of 2,2-ethano-1,25-dihydroxyvitamin D(3) (5), as novel spiro-ring analogues having cyclopropane fused at the C2 position. Biological potencies of the synthesized compounds were assessed in terms of the vitamin D receptor (VDR) binding affinity, as well as the HL-60 cell differentiation-inducing activity. The 2,2-ethano analogue 5a showed a comparable activity to the natural hormone 1, while the 2,2-dimethyl analogue 4a exhibited one-third of the activity of 1 in cell differentiation, with the reduced VDR binding affinity.     

Novel synthetic approach to 1alpha,25-dihydroxyvitamin D3 and analogues

A mild and stereoconvergent synthesis of 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1a) is described. The key step is a cascade process consisting of two consecutive transformations: An initial palladium-catalyzed 6-exo-cyclocarbopalladation of vinyl triflate 4 followed by a Negishi cross-coupling reaction with alkenyl zinc 3. This approach is of interest for the rapid synthesis of a variety of new vitamin D(3) analogues of therapeutic potential, especially those modified at the triene and ring-A. The mildness of the method also allows the preparation of thermal sensitive vitamin D(3) analogues.     

Vitamin D(3): synthesis of seco-C-9,11-bisnor-17-methyl-1 alpha,25-dihydroxyvitamin D(3) analogues

The synthesis and biological properties of seco-C-9,11-bisnor-17-methyl-1 alpha,25-dihydroxyvitamin D(3) novel D-ring analogues are described.     

Vitamin D(3): synthesis of seco C-9,11,21-trisnor-17-methyl-1 alpha, 25-dihydroxyvitamin D(3) analogues

The synthesis and biological activities of seco C-9,11,21-trisnor-17-methyl-1 alpha,25-dihydroxyvitamin D(3) analogues (D-ring analogues) are described.     

A-ring hydroxymethyl 19-nor analogs of the natural hormone 1alpha,25-dihydroxyvitamin D(3): synthesis and preliminary biological evaluation

A series 5-8 of 1- and 3-CH(2)OH 19-nor analogs of the hormone calcitriol (1) has been prepared. Surprisingly, 19-nor 1alpha-CH(2)OH analog 5a is more antiproliferative at 100 nM concentration than the corresponding regioisomeric analog 6a with the natural 1alpha-OH group, and 1alpha-CH(2)OH hybrid analog 7a is similar in antiproliferative potency to calcitriol (1) even at low nanomolar concentrations.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

Monoclonal antibody for calcitriol (1 alpha,25-dihydroxyvitamin D3)

Hybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3 beta-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3 beta-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to 1 alpha,25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3 beta-glu-25-OH-D3 were demonstrated to be 3.6 X 10(9) M-1 and 2.9 X 10(9) M-1, respectively, at 4 degrees C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.     

Versatile synthesis and biological evaluation of 1,3-diamino-substituted 1alpha,25-dihydroxyvitamin D3 analogues

A concise route to 1alpha,3beta-diamino-25-hydroxy-3-deoxyvitamin D(3) (5) and 1beta,3alpha-diamino-25-hydroxy-3-deoxyvitamin D(3) (6) has been developed starting from (R)- or (S)-carvone for the construction of the modified A-ring fragments. The conversion of the hydroxyl group to amine function with complete inversion of the configuration was efficiently accomplished by Mitsunobu reaction using phthalimide as nucleophile or activation of the hydroxyl group as mesylate followed by reaction with NaN(3). Diamino 5 and 6 as well as monoamino 3, 4, 30, and 31 vitamin D(3) derivatives have shown poor binding to VDR compared with 1alpha,25-dihydroxyvitamin D(3). The most active compound in the inhibition of MCF-7 cell proliferation and HL 60 cell differentiation was 1alpha-amino analogue 3. Also, very low in vivo calcemic effects of derivatives 3 and 4 were found.     

Syntheses and biological evaluation of novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues

Novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D3 analogues were efficiently synthesized and their biological activities were evaluated. 2alpha-Methyl-1alpha,25-dihydroxyvitamin D3 (2), whose unique biological activities were previously reported, was modified to 2alpha-alkyl (ethyl and propyl) and 2alpha-hydroxyalkyl (hydroxymethyl, hydroxyethyl, and hydroxypropyl) analogues 3-7 by elongation of the alkyl chain and/or introduction of a terminal hydroxyl group. 2alpha-Hydroxypropyl-1alpha,25-dihydroxyvitamin D3 (7) exhibited an exceptionally potent calcium-regulating effect and a unique activity profile.     

Synthesis of 1alpha,25-dihydroxyvitamin D3-26,23-lactams (DLAMs), a novel series of 1 alpha,25-dihydroxyvitamin D3 antagonist

Novel vitamin D(3) analogs having a lactam structure in their side chains, 1 alpha,25-dihydroxyvitamin D(3)-26,23-lactams (DLAMs), were designed based on the principle of regulation of the folding of helix-12 in the vitamin D nuclear receptor (VDR). The new analogs were synthesized via 1,3-dipolar cycloaddition reaction and subsequent reduction of the isoxazolidine as key steps. Among the analogs, (23S,25S)-DLAM-01 (4a) and (23S,25S)-DLAM-1P (5a) bind strongly to VDR. Moreover, these analogs were found to inhibit the differentiation of HL-60 cells induced by 1 alpha,25-dihydroxyvitamin D(3).     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) in human promyelocytic leukemia (HL-60) cells: in vitro biological activities of the natural metabolites of 1alpha,25-dihydroxyvitamin D(3) produced in HL-60 cells

The secosteroid hormone, 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], induces differentiation of the human promyelocytic leukemia (HL-60) cells into monocytes/macrophages. At present, the metabolic pathways of 1alpha,25(OH)(2)D(3) and the biologic activity of its various natural intermediary metabolites in HL-60 cells are not fully understood. 1alpha,25(OH)(2)D(3) is metabolized in its target tissues via modifications of both the side chain and the A-ring. The C-24 oxidation pathway, the main side chain modification pathway initiated by hydroxylation at C-24 leads to the formation of the end product, calcitroic acid. The C-23 and C-26 oxidation pathways, the minor side chain modification pathways initiated by hydroxylations at C-23 and C-26 respectively together lead to the formation of the end product, 1alpha,25(OH)(2)D(3)-lactone. The C-3 epimerization pathway, the newly discovered A-ring modification pathway is initiated by epimerization of the hydroxyl group at C-3 to form 1alpha,25-dihydroxy-3-epi-vitamin-D(3). We performed the present study first to examine in detail the metabolism of 1alpha,25(OH)(2)D(3) in HL-60 cells and then to assess the ability of the various natural intermediary metabolites of 1alpha,25(OH)(2)D(3) in inducing differentiation and in inhibiting clonal growth of HL-60 cells. We incubated HL-60 cells with [1beta-(3)H] 1alpha,25(OH)(2)D(3) and demonstrated that these cells metabolize 1alpha,25(OH)(2)D(3) mainly via the C-24 oxidation pathway and to a lesser extent via the C-23 oxidation pathway, but not via the C-3-epimerization pathway. Three of the natural intermediary metabolites of 1alpha,25(OH)(2)D(3) derived via the C-24 oxidation pathway namely, 1alpha,24(R),25-trihydroxyvitamin D(3), 1alpha,25-dihydroxy-24-oxovitamin D(3) and 1alpha,23(S),25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S),25(OH)(3)-24-oxo-D(3)] were almost as potent as 1alpha,25(OH)(2)D(3) in terms of their ability to differentiate HL-60 cells into monocytes/macrophages. We then selected 1alpha,23(S),25(OH)(3)-24-oxo-D(3) which has the least calcemic activity among all the three aforementioned natural intermediary metabolites of 1alpha,25(OH)(2)D(3) to examine further its effects on these cells. Our results indicated that 1alpha,23(S),25(OH)(3)-24-oxo-D(3) was also equipotent to its parent in inhibiting clonal growth of HL-60 cells and in inducing expression of CD11b protein. In summary, we report that 1alpha,25(OH)(2)D(3) is metabolized in HL-60 cells into several intermediary metabolites derived via both the C-24 and C-23 oxidation pathways but not via the C-3 epimerization pathway. Some of the intermediary metabolites derived via the C-24 oxidation pathway are found to be almost equipotent to 1alpha,25(OH)(2)D(3) in modulating growth and differentiation of HL-60 cells. In a previous study, the same metabolites when compared to 1alpha,25(OH)(2)D(3) were found to be less calcemic. Thus, the findings of our study suggest that some of the natural metabolites of 1alpha,25(OH)(2)D(3) may be responsible for the final expression of the noncalcemic actions that are presently being attributed to their parent, 1alpha,25(OH)(2)D(3).     

Metabolism of 1alpha,25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha,25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes

We previously reported that 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is metabolized into 1alpha,25-dihydroxy-3-epi-vitamin D(3) [1alpha,25(OH)(2)-3-epi-D(3)] in primary cultures of neonatal human keratinocytes. We now report that 1alpha,25(OH)(2)-3-epi-D(3) itself is further metabolized in human keratinocytes into several polar metabolites. One of the polar metabolite was unequivocally identified as 1alpha,23,25-trihydroxy-3-epi-vitamin D(3) by mass spectrometry and its sensitivity to sodium periodate. Three of the polar metabolites were identified as 1alpha,24,25-trihydroxy-3-epi-vitamin D(3), 1alpha,25-dihydroxy-24-oxo-3-epi-vitamin D(3) and 1alpha,23,25-trihydroxy-24-oxo-3-epi-vitamin D(3) by comigration with authentic standards on both straight and reverse phase HPLC systems. In addition to the polar metabolites, 1alpha,25(OH)(2)-3-epi-D(3) was also metabolized into two less polar metabolites. A possible structure of either 1alphaOH-3-epi-D(3)-20,25-cyclic ether or 1alphaOH-3-epi-D(3)-24,25-epoxide was assigned to one of the less polar metabolites through mass spectrometry. Thus, we indicate for the first time that 1alpha,25(OH)(2)-3-epi-D(3) is metabolized in neonatal human keratinocytes not only via the same C-24 and C-23 oxidation pathways like its parent, 1alpha,25(OH)(2)D(3); but also is metabolized into a less polar metabolite via a pathway that is unique to 1alpha,25(OH)(2)-3-epi-D(3).     

1 alpha-hydroxy-25-fluorovitamin D3: a potent analogue of 1 alpha,25-dihydroxyvitamin D3

Chemically synthesized 1 alpha-hydroxy-25-fluorovitamin D3 was compared to 1,25-dihydroxyvitamin D3 for potency in the chick intestinal cytosol-binding protein assay, induction of intestinal calcium transport, mobilization of calcium from bone, and epiphyseal plate calcification in the rat. The 25-fluorinated analogue causes 50% displacement of 1,25-dihydroxy[23,24-3H]D3 at 1.8 X 10(-8) M in the competitive protein-binding assay, whereas only 5.6 X 10(-11) M of unlabeled 1,25-dihydroxyvitamin D3 is needed for equal competition. This 315-fold difference between and 1 alpha-hydroxy-25-fluorovitamin D3 indicates that the fluoro analogue is about equipotent with 1 alpha-hydroxyvitamin D3 in the protein-binding assay. However, 1 alpha-hydroxy-25-fluorovitamin D3 is 1/50 as active as 1,25-dihydroxyvitamin D3 in vivo in the stimulation of intestinal calcium transport and bone calcium mobilization in vitamin D deficient rats on a low-calcium diet. Likewise, 1 alpha-hydroxy-25-fluorovitamin D3 is about 40 times less active than 1,25-dihydroxyvitamin D3 in inducing endochondrial calcification in rachitic rats. No selective actions of 1alpha-hydroxy-25-fluorovitamin D3 were noted. Since the 25 position of the analogue is blocked by a fluorine atom, it appears that 25-hydroxylation of 1 alpha-hydroxylated vitamin D compounds in vivo is not an obligatory requirement for appreciable vitamin D activity.     

Chemoenzymatic synthesis and biological evaluation of C-3 carbamate analogues of 1alpha,25-dihydroxyvitamin D3

The synthesis of new analogues of 1alpha,25-dihydroxyvitamin D3 containing a carbamate function at the A-ring fragment has been described using the cross-coupling approach. The carbamate group was selectively introduced at the C-3 position by regioselective enzymatic alkoxycarbonylation of A-ring enyne 3 and subsequent treatment with ammonia, amines, amino alcohols, and amino acids. Biological studies to evaluate the potency of all five of these carbamate analogues were performed and demonstrated very low binding affinity for the vitamin D receptor compared with 1alpha,25-dihydroxyvitamin D3. Moreover, all the carbamate analogues were less active than 1alpha,25-dihydroxyvitamin D3 in inhibiting cell proliferation or stimulating cell differentiation. Of all the five analogues, the 3-O-carbamoyl-1alpha,25-(OH)2-D3 analogue 10a was the most potent one in vitro. However, all investigated carbamate analogues demonstrated lower calcemic effects in vivo than the parent compound.     

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.     

The stereochemical configuration of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone

Four possible diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were chemically synthesized and compared with the natural metabolite by high-pressure liquid chromatography. The four synthetic diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone could be separated into three peaks by high-pressure liquid chromatography. The naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum and in vitro incubation of chick kidney homogenates comigrated with 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. The four diastereoisomers of 1 alpha,25-dihydroxyvitamin D3-26,23-lactone were tested against naturally occurring 1 alpha,25-dihydroxyvitamin D3-26,23-lactone to determine their relative competition in the 1 alpha,25-dihydroxyvitamin D3-specific cytosol receptor binding assay for 1 alpha,25-dihydroxyvitamin D3. 23(S)25(S)-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone was the best competitor followed by 23(R)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone and 23(R)25(S)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone, and 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was the poorest competitor. Natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone isolated from dog serum had almost the same binding affinity as that of 23(S)25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone. These data unequivocally demonstrate that the stereochemistry of the natural 1 alpha,25-dihydroxyvitamin D3-26,23-lactone has the 23(S) and 25(R) configuration.     

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.