Synthesis of new aromatic (C17-C20)-locked side-chain analogues of calcitriol (1alpha,25-dihydroxyvitamin D(3))

The synthesis of four new analogues of calcitriol (1alpha, 25-(OH)(2)-D(3)) possessing aromatic and conjugated double bond units at the side chain are described. The triene system is introduced using the Lythgoe-Hoffmann La Roche convergent Wittig-Horner approach. The key steps in the preparation of the requisite upper fragments are the introduction of the side chain with the E-conjugated aromatic system and its photochemical conversion to the Z counterpart.     

6-s-cis locked analogues of the steroid hormone 1alpha, 25-dihydroxyvitamin D(3). Synthesis Of novel A-ring stereoisomeric 1, 25-dihydroxy-3-epi-19-nor-previtamin D(3) derivatives

Efficient syntheses of A-ring synthons 24 and 32 are described from hydroxy ester 16, which is easily available on a preparative scale from (-)-quinic acid. Key features of the syntheses were (a) the ability to selectively perform desilylations in the presence of p-nitrobenzoate esters and (b) the excellent yield and complete stereospecificity with which the configuration of alcohols 16, 18, and 26 could be inverted under Mitsunobu conditions. Thus, A-ring synthons 24 and 32 were both prepared in 35-38% yield (eight steps) from the common precursor 16. The coupling of A-ring synthons 24 and 32 with the appropriate CD-ring/side chain fragment 7 provides access to novel 6-s-cis locked analogues of steroid hormone 1alpha, 25-dihydroxyvitamin D(3): 1alpha, 25-dihydroxy-3-epi-19-nor-previtamin D(3) (37) and 1beta, 25-dihydroxy-3-epi-19-nor-previtamin D(3) (38), which are unable to undergo rearrangement to the respective vitamin D form by virtue of the absence of the C-19 methyl group. Compounds 37 and 38 can be used as tools for studying the genomic and nongenomic mechanisms of action of the previtamin form of the hormone 1alpha, 25-dihydroxyvitamin D(3).     

Functionalization at C-12 of 1alpha,25-dihydroxyvitamin D(3) strongly modulates the affinity for the vitamin D receptor (VDR)

The first synthesis of analogues of the natural hormone 1alpha,25-dihydroxyvitamin D(3) (1a) with substituents at C-12 is reported. The following are the relative affinities of the novel compounds for the vitamin D receptor (VDR) compared to that of 1a (100%): 1alpha,12alpha,25-(OH)(3)-D(3) (1b, 1%), 1alpha,25-(OH)(2)-12-methylene-D(3) (1c, 50%), and 1alpha,25-(OH)(2)-12beta-methyl-D(3) (1d, 440%). [structure: see text]     

Efficient and versatile synthesis of novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D(3) analogues and their docking to vitamin D receptors.

Novel 2alpha-substituted 1alpha,25-dihydroxyvitamin D(3) analogues with 2alpha-alkyl and 2alpha-hydroxyalkyl groups were systematically synthesized from D-xylose. Their conformation on binding to the ligand binding domain (LBD) of the vitamin D receptor was analyzed. It has been found that the 2alpha-hydroxypropyl group best fits the cavity of the LBD, and the binding activity is three times higher than that for the natural hormone.     

UVB-induced conversion of 7-dehydrocholesterol to 1 alpha,25-dihydroxyvitamin D3 (calcitriol) in the human keratinocyte line HaCaT

We have previously shown that keratinocytes in vitro can convert biologically inactive vitamin D3 to the hormone calcitriol. The present study was initiated to test whether ultraviolet B (UVB)-induced photolysis of provitamin D3 (7-dehydrocholesterol, [7-DHC]) which results in the formation of vitamin D3 also leads to the generation of calcitriol in keratinocytes. Submerged monolayers of HaCaT keratinocytes were preincubated with 7-DHC (25 microM) at 37 degrees C and irradiated with monochromatic UVB at different wavelengths (effective UV-doses: 7.5-60 mJ/cm2), or a narrow-band fluorescent lamp Philips TL-01 (UVB-doses: 125-1500 mJ/cm2). Irradiation with both sources of UVB resulted in the generation of different amounts of previtamin D3 in our in vitro model followed by time-dependent isomerization to vitamin D3 and consecutive formation of calcitriol in the picomolar range. Unirradiated cultures or cultures exposed to wavelengths > 315 nm generated no or only trace amounts of calcitriol. The conversion of vitamin D3 generated after UVB irradiation to calcitriol is inhibited by ketoconazole indicating the involvement of P450 mixed function oxidases in this chemical reaction. The generation of calcitriol was wavelength- and UVB dose dependent and reached approximately 18-fold higher levels after irradiation at 297 nm than at 310 nm (effective UVB dose: 30 mJ/cm2). Hence, keratinocytes may be a potential source of biologically active calcitriol within epidermis, when irradiated with therapeutical doses of UVB.     

Synthesis of novel analogues of 1alpha,25-dihydroxyvitamin D(3) with side chains at C-18

Novel analogues of the hormone 1alpha,25-(OH)(2)-D(3) with side chains attached to C-18 were synthesized by a versatile route in which key steps were the remote radical-induced functionalization of the 18-methyl by the C-8beta-hydroxyl group and the introduction of the side chains by Wittig reactions on a C-18-aldehyde. The triene system of the novel analogues was constructed by the convergent Lythgoe-Hoffmann la Roche approach, which involves reaction of a phosphine oxide (the ring A fragment) with a ketone (the upper fragment).     

2 alpha-(3-hydroxypropyl)- and 2 alpha-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 accessible to vitamin D receptor mutant related to hereditary vitamin D-resistant rickets

Hereditary vitamin D-resistant rickets (HVDRR) is a genetic disorder caused by mutations in the vitamin D receptor, which lead to resistance to 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. We found that the A ring-modified analogues, 2alpha-(3-hydroxypropyl)- and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)(2)D(3), (O1C3 and O2C3) can bind better than the natural hormone to the mutant VDR (R274A), which similar to the HVDRR mutant, R274L, had lost the hydrogen bond to the 1alpha-hydroxyl group of 1alpha,25(OH)(2)D(3).     

Efficient synthesis of novel 1alpha-amino and 3beta-amino analogues of 1alpha,25-dihydroxyvitamin d(3)

Convenient synthetic routes to 1alpha-amino-25-hydroxyvitamin D(3) (3) and 3beta-amino-3-deoxy-1alpha,25-dihydroxyvitamin D(3) (4), novel analogues of vitamin D(3) bearing an amino group at the C-1 or C-3 position, have been developed starting from (S)-(+)-carvone. Construction of the A-ring fragments was accomplished by selective enzymatic hydrolysis of a diester intermediate and introduction of the amino group under Mitsunobu conditions.     

Synthesis of spiro[4.5]decane CF-ring analogues of 1 alpha,25-dihydroxyvitamin D3

A novel series of analogues of calcitriol (1) is developed featuring a spirocyclic central core resulting from C18/C21-connection and C15/C16-deletion (2a, 2b). The synthesis of the key intermediate involves an Eschenmoser rearrangement of an enantiomerically pure bromo-substituted cyclohexenol.     

Efficient convergent synthesis of 1alpha,25-dihydroxyvitamin D3 and its analogues by Suzuki-Miyaura coupling

[reaction: see text] 1alpha,25-Dihydroxyvitamin D(3) was synthesized by the Suzuki-Miyaura coupling of the A-ring intermediate 1, which was efficiently prepared from readily available 1,7-enyne 2, with the corresponding boronate compound of the C,D-ring portion. The method was applied to prepare des-C,D analogues of 1alpha,25-dihydroxyvitamin D(3).     

Design and synthesis of a 1 alpha,25-dihydroxyvitamin D3 dimer as a potential chemical inducer of vitamin D receptor dimerization

[formula: see text] A dimer comprising two 1 alpha,25-dihydroxyvitamin D3 units linked by an alkyl side chain at C-11 was synthesized with a view to the simultaneous binding of two vitamin D receptor (VDR) molecules and the consequent induction of VDR dimerization. The short, convergent synthesis uses a stereoselective cuprate addition to introduce the linking side chain and a key ruthenlum olefin metathesis as the dimerization step.     

Development of analogues of 1alpha,25-dihydroxyvitamin D3 with biased side chain orientation: methylated des-C,D-homo analogues

The discovery that 1alpha,25-dihydroxyvitamin D3 is effective in the inhibition of cellular proliferation and in the induction of cellular differentiation has led to a search for analogues in which these activities and the classical calcemic activity of this hormone are separated. In this context, the synthesis and biological evaluation are reported of the three stereoisomeric CD-ring modified structural analogues in order to enforce a particular and different orientation of the 25-hydroxylated side chain. Comparison of the results of the biological evaluation and conformational analysis of the side chain suggests one defined and "active" geometry.     

Synthetic studies of vitamin D analogues. XI. Synthesis and differentiation-inducing activity of 1 alpha,25-dihydroxy-22-oxavitamin D3 analogues.

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.     

Pd-catalyzed carbocyclization-Negishi cross-coupling cascade: a novel approach to 1alpha,25-dihydroxyvitamin D3 and analogues

[reaction: see text] A mild palladium-catalyzed cascade has been used for the synthesis of the hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol, 1a) and its analogues 1b and 1c. This one-pot process involves two consecutive transformations at room temperature: An initial palladium-catalyzed 6-exo-cyclocarbopalladation of vinyl triflates followed by a Negishi cross-coupling reaction with an alkenyl zinc. This novel strategy opens new possibilities for the preparation of a variety of new vitamin D analogues of therapeutic potential, particularly with modifications at the triene and/or ring-A.     

Efficient synthesis of the A-ring phosphine oxide building block useful for 1 alpha,25-dihydroxy vitamin D(3) and analogues

The 1 alpha-hydroxy A-ring phosphine oxide 1, a useful building block for vitamin D analogues, was synthesized from (S)-carvone in nine synthetic operations and a single chromatographic purification in 25% overall yield. The synthesis features two novel efficient synthetic transformations: the Criegee rearrangement of alpha-methoxy hydroperoxyacetate 10 in methanol to obtain directly the desired secondary 3 beta-alcohol 11 and the highly chemo- and stereoselective isomerization of dieneoxide ester (E)-7 to the 1 alpha-allylic alcohol with an exocyclic double bond (E)-8. Further insight into the selectivity control of the latter rearrangement was obtained from the reactions of (Z)-epimeric substrates. The new synthetic approach leading to the 1 alpha-hydroxy epimers complements our previously reported synthesis of the corresponding 1 beta-epimers, thus producing all stereoisomers of these versatile building blocks efficiently from carvone.     

Synthesis of (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3: compounds to probe vitamin D conformation in receptor complex by 19F-NMR

To study the interaction of vitamin D with its receptor by 19F-NMR, (5Z,10Z)- and (5Z,10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 were synthesized starting from vitamin D2 via electrophilic fluorination of vitamin D-SO2 adducts as the key step. Regio- and stereoselective electrophilic fluorination at C(19) of vitamin D-SO2 adducts was achieved under the conditions using (PhSO2)2NF and bulky bases. The stereochemistry of the addition and elimination of SO2 of various vitamin D derivatives was studied in detail. SO2 causes Z-E isomerization of the 5,6-double bond of vitamin D and adds to the resulting (5E)-isomer from the sterically less hindered side opposite to the substituent at C(1). Elimination of SO2 from 19-substituted vitamin D-SO2 adducts proceeded exclusively in a suprafacial manner with respect to the diene part under either thermal or reductive conditions. Dye-sensitized photochemical isomerization of 19-fluorovitamin D derivatives was studied in detail. The rapid isomerization at the 5,6-double bond was followed by the slow isomerization at the 10,19-double bond to yield the (5E,10Z)-isomer (by nomenclature of the 1-OH derivatives) as the major product. (10Z)- and (10E)-19-Fluorovitamin Ds were also interconverted thermally probably via the corresponding previtamin D by 1,7-sigmatropic isomerization.