Synthesis of 9-alkylated calcitriol and two 1α,25-dihydroxy-9-methylene-10,19-dihydrovitamin D3 analogues with a non-natural triene system by thermal sigmatropic rearrangements.

1α,25-(OH)(2)-9α-Methylvitamin D(3) (4), the first known analogue of the natural hormone 1α,25-(OH)(2)D(3) (3) with an alkyl substituent at C-9, and two 1α,25-(OH)(2)-9-methylene-10,19-dihydrovitamin D(3) analogues (7 and 8) with an unprecedented non-natural triene system were synthesized by thermal isomerization of 1α,25-(OH)(2)-9-methylprevitamin D(3) (6). Three alternative approaches (Sonogashira, Stille, or stereoselective dehydration of a tertiary propargyl alcohol) have been successfully used to construct the dienyne precursors of previtamin 6 possessing two methyl groups capable of participating in the [1,7]-sigmatropic hydrogen shift.

Synthesis and biological activities of 1α,4α,25- and 1α,4ß,25-trihydroxyvitamin D(3) and their metabolism by human CYP24A1 and UDP-glucuronosyltransferase.

A previous report has demonstrated the existence of a C4-hydroxylated vitamin D(2) metabolite in serum of rats treated with pharmacological doses of vitamin D(2). However, the biological significance and metabolic fate of this metabolite have not been described. To explore its potential biological activities, we therefore synthesized 1α,4α,25-trihydroxyvitamin D(3) and its diastereoisomer, 1α,4ß,25-trihydroxyvitamin D(3), using Trost Pd-mediated coupling reaction, and studied their vitamin D receptor (VDR) binding affinity, osteocalcin promoter transactivation activity, and their further metabolism by human CYP24A1 as well as by human liver microsomal fraction based on CYP- and UDP-glucuronosyltransferases (UGTs)-reactions.

Synthesis of (6-3H)-1alpha-hydroxyvitamin D3 and its metabolism in vivo to (3H)-1alpha,25-dihydroxyvitamin D3.

[6-3H]-1alpha-Hydroxyvitamin D3 was chemically synthesized and its full biological activity and radiochemical purity were demonstrated. With the use of this preparation it has been possible to demonstrate in vivo that in rats the [6-3H]-1Alpha-hydroxyvitamin D3 is converted to [6-3H]-1alpha,25-dihydroxyvitamin D3, the natural hormone. In fact, in the intestine and bone of rats given 32 picomoles of [6-3H]-1alpha-hydroxyvitamin D3 each day for 6 days, more than 80 percent of the lipid-soluble radioactivity exists as [6-3H]-1alpha,25-dihydroxyvitamin D3, a finding that suggests that much of the biological effectiveness of 1alpha-hydroxyvitamin D3 is due to its conversion to 1alpha,25-dihydroxyvitamin D3.

Concise synthesis of 23-hydroxylated vitamin D3 metabolites.

Three 23-hydroxylated vitamin D3 derivatives, which are metabolites of 25-hydroxyvitamin D3 produced by CYP24A1 and a related diastereomer, were efficiently synthesized. Each C23 hydroxy unit was constructed by the Claisen condensation reaction with ethyl acetate or the Grignard reaction with 2-methylallymagnesium chloride. Stereochemistry at the C23 position was determined by a modified Mosher's method. The triene structures were constructed by the Wittig-Horner reaction utilizing the A-ring phosphine oxide moiety.

The stereochemical configuration of the natural 23,25,26-trihydroxyvitamin D3.

Four possible diastereoisomers of 23,25,26-trihydroxyvitamin D3 were synthesized and compared with the natural metabolite. The 4 synthetic diastereoisomers could be separated into 4 peaks by high-performance liquid chromatography. The natural 23,25,26-trihydroxyvitamin D3 comigrated with 23(S),25(R),26-trihydroxyvitamin D3. This result unequivocally demonstrates that the stereochemistry of the natural 23,25,26-trihydroxyvitamin D3 has the 23(S) and 25(R) configuration.

Studies on vitamin D (calciferol) and its analogues. 10. Side-chain analogues of 25-hydroxyvitamin D3.

A homologous series of side-chain analogues of 25-hydroxyvitamin D3 (25-hydroxycholecalciferol) in which the length of the side chain is modified while maintaining its characteristic tertiary hydroxyl moiety has been synthesized. The following five analogues have been prepared and characterized: pentanor-25-OH-D3 (2a), trinor-25-OH-D3 (2b), dinor-25-OH-D3 (2c), nor-25-OH-D3 (2d), and homo-25-OH-D3 T2e). Biological assays in vivo of intestinal calcium absorption and bone calcium mobilization in the chick of the five analogues revealed that the homo analogue 2e exhibited a significant biological response relative to the -D (vitamin D3) control. Compared to the natural vitamin D3, 2e is as active in its ability to mobilize bone calcium and is about half as effective in stimulating intestinal calcium transport. The remaining analogues (2a-d) exhibited no significant activity in either assay, although the nor analogue 2d was previously observed to exhibit antimetabolite activity.

A concise enantioselective synthesis of a key A-ring synthon for 1 alpha-hydroxyvitamin D3 compounds.

[figure: see text] This report describes a concise enantioselective synthesis of the A-ring synthon for the synthesis of 1 alpha-hydroxyvitamin D3 compounds. The synthesis involves two notable transformations: (I) stereoselective construction of the enol triflate from the vinyl ketone by Michael addition of Ph2P(O)Li followed by in situ triflation of the resulting enolate and (II) palladium-catalyzed Heck type cyclization of the enol triflate.

A novel specific assay of 25-hydroxy vitamin D.

The synthesis of 25-hydroxy-[26-2H3]vitamin D3 is described. A fixed amount of this compound (usually 250 ng) is added to a fixed amount of serum (usually 2.5 ml) and the mixture is extracted with a chloroform/methanol mixture. The extract is chromatographed on a Sephadex LH-20 column together with a trace amount of 25-hydroxy-[263H3]vitamin D3. The chromatographic fraction corresponding to 25-hydroxy vitamin D3 is converted into trimethylsilyl ether and the amount of unlabeled 25hydroxy vitamin D3 is determined from the ratio between the mass fragmentographic recording at m/e 131 (base peak of unlabeled 25-hydroxy vitamin D3) and m/e 134 (base peak 25-hydroxy-[26-2H3]vitamin D3). The relative standard deviation of the method was about 5%.

The synthesis of 25-hydroxycholecalciferol-23,23,24,24-t4 of high specific activity.

Catalytic tritium reduction of cholest-5-en-23-yne-3beta,25-diol diacetate (VIII) gave cholest-5-ene-3 beta,25-diol diacetate-23,23,24,24-t4 (IX) having a specific activity of 92 Ci/mmol. Bromination, dehydrobromination and hydrolysis of the labelled material gave cholesta-5,7-diene-3beta,25-diol-23,23,24,24-t4 (XI) which was photolyzed to the previtamin and then thermally equilibrated to 25-hydroxycholecalciferol-23,23,24,24-t4 (I).

Improved and efficient synthesis of 1alpha-hydroxy-[6-(2)H] and 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives.

Improved and efficient procedures for deuterium-labeling at the 6,19,19 positions of 1alpha-hydroxyvitamin D3 derivatives via its sulfur dioxide-adduct by using a base-catalyzed H-D exchange reaction are described. Application of the known procedure using tBuOK/DMF-D2O, which is effective for labeling vitamin D3 derivatives, to 1alpha-hydroxy compounds gave only poor results because of isomerization and decomposition. We found that this procedure is improved by the use of iPrONa/iPrOD. During this study, we also found that the 6-monodeuterated product was selectively obtained when MeONa/CD3OD was employed instead of iPrONa/iPrOD. On the other hand, simple addition of 1,3-dimethyl-2-imidazolidinone as a co-solvent to the above conditions was effective for 1alpha,25-dihydroxy compounds. These improved procedures were successfully applied to the synthesis of 1alpha-hydroxy-[6,19,19-(2)H]vitamin D3 derivatives 4 and 1alpha-hydroxy-[6-(2)H]vitamin D3 derivatives 6 from the corresponding 1alpha-hydroxyvitamin D3 derivatives 1 via its sulfur dioxide-adducts 2, 3 and 5 in good over-all yield with high deuterium incorporation.

Synthesis and biological activity of 25xi,26-dihydroxycholecalciferol.

25xi,26-Dihydroxycholecalciferol (25xi,26-dihydroxyvitamin D3), a metabolite of vitamin D3 preferentially active on intestine has been synthesized. This compound was prepared by converting 3beta-hydroxy-27-norcholest-5-en-25-one to 25xi,26-epoxy-5-cholesten--3beta-ol and base-catalyzed hydrolysis of the latter to 5-cholestene-3beta,25xi,26-triol; allylic bromination of the corresponding triacetate, and dehydrobromination gave the required 5,7-diene which yielded the vitamin derivative upon photolysis (Figure 3). The synthetic product shows the same activity pattern as the natural metabolite: at dose levels of 0.25 mug, the compound stimulates intestinal calcium transport, but has no effect on bone calcium mobilization in rats maintained on a vitamin D-deficient, low calcium diet. Higher doses (2.5 mug) elicit a more pronounced intestinal calcium transport response, but also have no significant effect on the bone mobilization system. The compound exhibits no biologial activity in nephrectomized animals.

The synthesis and activity in vitro of 25-masked 1alpha-hydroxylated vitamin D3 analogs.

1alphaHydroxylated-25-masked-vitamin D3 and analogs were synthesized as probes to help evaluate the role of 25-hydroxyl group in hormone-receptor interactions of 1alpha,25-dihydroxyvitamin D3 (13a). Synthetic work on model systems showed that the steroidal 25-hydroxyl group could be easily fluorinated in high yield with diethylaminosulfur trifluoride. Treatment of 25-fluoro compound with acetic acid resulted in both elimination and displacement of fluorine by acetate. The desired 1alpha-hydroxy-25-fluoro-vitamin D3 (14b) was obtained efficiently by fluorination and subsequent deacetylation of 1alpha,25-dihydroxyvitamin D3 1,3-diacetate (13b). Also obtained was a mixture of 1alpha-hydroxyvitamin D3-24- and 25-enes (15b). Both 14b and 15b were 300-400 times less active than 13a in the chick intestinal cytosol protein binding assay, making these analogs similar in potency to 1alpha-hydroxyvitamin D3 in vitro. The essentially equivalent activity of 14b and 15b with 1alpha-hydroxyvitamin D3 indicates that in the absence of a 25-hydroxyl group some alterations to the side chain carbons of 13a may be tolerated without further weakening analog-protein interactions. The fluoroanalog 14b was also about 250 times less potent than 13a in stimulating bone resorption in vitro. These compounds should prove to be valuable tools in aiding understanding of the salient structural features of the vitamin D3 metabolites.

Synthesis and biological activities of the two C(23) epimers of 1alpha,23,25-trihydroxy-24-oxo-19-nor-vitamin D(3): novel analogs of 1alpha,23(S),25-trihydroxy-24-oxo-vitamin D(3), a natural metabolite of 1alpha,25-dihydroxyvitamin D(3).

In a previous report, we indicated that 1alpha,23(S), 25-trihydroxy-24-oxovitamin D(3) [1alpha,23(S), 25(OH)(3)-24-oxo-D(3)], a natural metabolite of 1alpha, 25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] is almost equipotent to 1alpha,25(OH)(2)D(3) in suppressing parathyroid hormone (PTH) secretion (Lee et al., 1997. Biochemistry 36, 9429-9437). Also, 1alpha,23(S),25(OH)(3)-24-oxo-D(3) has been shown to possess only weak in vivo calcemic actions. Thus, vitamin D(3) analogs structurally related to 1alpha,23(S),25(OH)(3)-24-oxo-D(3) may have therapeutic value. Furthermore, biologic activity studies of various synthetic analogs of 1alpha,25(OH)(2)D(3) showed that the removal of carbon-19 (C-19) reduces the calcemic activity of 1alpha, 25(OH)(2)D(3.) Therefore, in an attempt to produce vitamin D(3) analogs with a better therapeutic index, we synthesized C(23) epimers of 1alpha,23,25(OH)(3)-24-oxo-19-nor-vitamin D(3) [1alpha,23, 25(OH)(3)-24-oxo-19-nor-D(3)]. The two epimers were compared to 1alpha,25(OH)(2)-19-nor-D(3) and 1alpha,25(OH)(2)D(3) in their ability to generate biologic activities in several in vitro assay systems. In the assay measuring the suppression of parathyroid hormone (PTH) secretion in bovine parathyroid cells, 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) was as potent as 1alpha, 25(OH)(2)-19-nor-D(3) but was less potent than 1alpha,25(OH)(2)D(3). In the same assay 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) exhibited greater potency than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). In the assays measuring the ability of vitamin D compounds to inhibit clonal growth and to induce differentiation of human promyelocytic leukemia (HL-60) cells, 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) was less potent than 1alpha,25(OH)(2)-19-nor-D(3) but was equipotent to 1alpha, 25(OH)(2)D(3). More importantly, in the same assays, 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was more potent than 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3) and was equipotent to 1alpha, 25(OH)(2)-19-nor-D(3). Also, the vitamin D receptor-mediated transcriptional activity of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3) was almost equal to that of 1alpha, 25(OH)(2)-19-nor-D(3), but higher than that of 1alpha,23(S), 25(OH)(3)-24-oxo-19-nor-D(3). This finding explains in part the greater in vitro biologic activities of 1alpha,23(R), 25(OH)(3)-24-oxo-19-nor-D(3). In summary, our results indicate that 1alpha,23(R),25(OH)(3)-24-oxo-19-nor-D(3) and to a lesser extent 1alpha,23(S),25(OH)(3)-24-oxo-19-nor-D(3) are potent 19-nor vitamin D(3) analogs, which suppress PTH secretion in bovine parathyroid cells and strongly inhibit clonal growth and induce differentiation of HL-60 cells in vitro.

Synthesis of 1alpha-hydroxyvitamin D5 using a modified two wavelength photolysis for vitamin D formation.

[reaction: see text] 1Alpha-hydroxyvitamin D5 (1) is a promising chemopreventive agent for breast cancer and is being developed as a drug. We report a synthesis for this vitamin D analogue which uses a photochemical method for the B-ring opening, leading to the conjugated triene system. The precursor 7-dehydrositosteryl acetate (4) obtained through a one-pot, five-step procedure, was completely free of the 4,6-diene isomer that usually forms in the 5,7-diene synthesis. The pre-vitamin isomer (11) was generated using a modified two-wavelength photolysis procedure that increases the yield for this step more than 3-fold compared to classically used photolysis. The 1alpha-hydroxylation step was performed on the 3-triethylsilyl-trans-vitamin D5 (17) obtained via the sulfur dioxide adduct of cis-vitamin D5, in an overall yield of 48%. Photoisomerization and deprotection completed the synthesis.