Design and efficient synthesis of 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D3 Analogues, including 2-epi-ED-71 and their 20-epimers with HL-60 cell differentiation activity

A concise and efficient synthetic approach to 2 alpha-(omega-hydroxyalkoxy)-1 alpha,25-dihydroxyvitamin D(3) (4a-c), including 2-epi-ED-71, was developed starting from D-glucose as a chiral template for the construction of the 2 alpha-modified A-ring precursors (11a-c). It was found that the best ligand for the bovine thymus vitamin D receptor (VDR) in this series is 4b, which has 1.8 times greater binding affinity for the bovine thymus VDR than that of the natural hormone 1. Interestingly, potency in the induction of HL-60 cell differentiation for 4a-c was almost the same or weaker than that of 1 despite the strong binding affinity for the VDR. Next, we were interested in the "double modification"of 1 based on 4a-c with C20-epimerization, affording 2 alpha-(omega-hydroxyalkoxy)-20-epi-1 alpha,25-dihydroxyvitamin D(3) (20-epi-4a-c). All three 2 alpha-substituted 20-epi analogues of 1 (20-epi-4a-c) exhibited stronger binding affinities for the VDR, and their conformations in the ligand binding domain of VDR were analyzed by molecular modeling. Double-modified analogues of 20-epi-4a-c showed marked HL-60 cell differentiation activity, and 20-epi-4a possesses an activity 58-fold higher than that of the natural hormone 1.     

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).     

Synthesis of a 1α-C-methyl analogue of 25-hydroxyvitamin D3: interaction with a mutant vitamin D receptor Arg274Leu

Vitamin D₃ analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1α-OH of 1α,25-dihydroxyvitamin D₃ to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective synthesis of the A-ring part of the novel vitamin D analogue, 2α-(3-hydroxypropyl)-1α-methyl-25-hydroxyvitamin D₃ (4), from d-galactose was accomplished with the key steps of the introduction of the methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than the natural hormone 1α,25-dihydroxyvitamin D₃ (1).     

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]     

Synthesis of monoacyl A-ring precursors of 1alpha,25-dihydroxyvitamin D3 through selective enzymatic hydrolysis

An efficient synthesis of monoacylated 1alpha,25-dihydroxyvitamin D3 A-ring precursors 15, 16, 18, and 19 has been described through an enzymatic hydrolysis process. Candida antarctica A lipase (CAL-A) hydrolyzes the C-5 acetate ester in trans stereoisomers 9 and 13, with complete and high selectivity, respectively. In the case of cis isomers 11 and 14, Chromobacterium viscosum lipase (CVL) is the enzyme of choice, exhibiting opposite selectivity for these two enantiomers. This lipase selectively catalyzes the hydrolysis at the C-3 acetate in diester 11 and at C-5 position in diester 14. It is noteworthy that through a hydrolysis reaction CAL-A and CVL allow the synthesis of the four A-ring monoacetylated precursors of 1alpha,25-dihydroxyvitamin D3, precursors which are complementary to those obtained by the enzymatic acylation process. In addition, with excellent yield CVL selectively hydrolyzes the C-3 chloroacetate ester instead of the C-5 acetate in diester 22, a key intermediate in the synthesis of new A-ring modified 1alpha,25-dihydroxyvitamin D3 analogues.     

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.     

A concise and efficient route to 2alpha-(omega-hydroxyalkoxy)-1alpha,25-dihydroxyvi tam in D3: remarkably high affinity to vitamin D receptor

[reaction: see text]A convenient and potentially valuable synthetic approach to the novel 2alpha-functionalized 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] derivatives (1a-c), which are the C2-epimer of ED-71 and its analogues, has been developed. The C2alpha-modified ring A precursors (1,7-enynes 16, n = 0, 1, and 2) were constructed stereoselectively starting from D-glucose in high yield. In the synthesized 2alpha-(omega-hydroxyalkoxy)-1alpha,25(OH)2D3 derivatives, 1a and 1b showed a greater binding affinity to vitamin D receptor (VDR), up to 1.8 times that of the native hormone.     

New convergent synthesis of 1alpha,25-dihydroxyvitamin D3 and its analogues by Suzuki-Miyaura coupling between A-ring and C,D-ring parts

A new convergent method for the synthesis of 1alpha,25-dihydroxyvitamin D(3) and its analogues has been developed that involves efficient preparation of the A-ring part 1a, (Z)-(3S,5R)-1-bromomethylene-3,5-bis(tert-butyldimethylsilyloxy)-2-methylenecyclohexane, starting from epichlorohydrin (4) and its Suzuki-Miyaura coupling reaction with the C,D-ring part 12. Thus, (R)-4 was converted to (3S,5R)-5-(tert-butyldimethylsilyloxy)-8-(trimethylsilyl)-oct-1-en-7-yn-3-ol (3a) through a ten-step reaction sequence in 49% overall yield. Compound 3a thus obtained was treated with a Ti(O-i-Pr)(4)/2 i-PrMgCl reagent and then with NBS to afford (Z)-(1S,2S,5R)-2-bromomethyl-3-[bromo(trimethylsilyl)methylene]-5-(tert-butyldimethylsilyloxy)cyclohexanol (10a) in 51% yield, from which 1a was obtained in 87% yield by sequential treatment with TBSCl/imidazole, DBU, and Cs(2)CO(3). The resulting A-ring intermediate 1a was reacted with alkenylboronate 12 in the presence of a PdCl(2)(dppf) catalyst to furnish 1alpha,25-dihydroxyvitamin D(3) in 82% yield after protodesilylation. Similarly, all of the other three possible stereoisomers of A-ring parts 1b, 1c, and 1d were prepared, from which 1-epi-, 3-epi-, and 1,3-di-epi-1alpha,25-dihydroxyvitamin D(3) were synthesized by coupling with 12 in excellent yield, respectively. Starting from 1a and 1c, des-C,D-1alpha,25-dihydroxyvitamin D(3) analogues, retiferol 13 and its 3-epi derivative, were also prepared, respectively.     

Synthesis of vitamin D(3) and calcitriol dimers as potential chemical inducers of vitamin D receptor dimerization

The design and synthesis of vitamin D(3) dimers 2 and 3 and 1 alpha, 25-dihydroxyvitamin D(3) (calcitriol) dimers 4 and 5 are described. The dimers were designed with a view to doubly binding the vitamin D receptor (VDR) and inducing the receptor homodimerization. In the dimers the units are linked through the C-11 position in ring C by an alkyl side chain of six or 10 carbon atoms, far from the hydroxy groups responsible for the VDR binding. The linker is formed by olefin metathesis of an olefinic side chain at the C-11 position introduced by stereoselective cuprate addition. The synthesis, which is both short and convergent, uses the Wittig-Horner approach to construct the vitamin D triene system and allows the preparation of dimers with a linker of modulated length with the purpose of optimizing the vitamin D(3)-VDR interaction.     

Novel nonsecosteroidal vitamin D mimics exert VDR-modulating activities with less calcium mobilization than 1,25-dihydroxyvitamin D3.

BACKGROUND: The secosteroid 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) acts through the vitamin D receptor (VDR) to elicit many activities that make it a promising drug candidate for the treatment of a number of diseases, including cancer and psoriasis. Clinical use of 1,25(OH)2D3 has been limited by hypercalcemia elicited by pharmacologically effective doses. We hypothesized that structurally distinct, nonsecosteroidal mimics of 1,25(OH)2D3 might have different activity profiles from vitamin D analogs, and set out to discover such compounds by screening small-molecule libraries. RESULTS: A bis-phenyl derivative was found to activate VDR in a transactivation screening assay. Additional related compounds were synthesized that mimicked various activities of 1,25(OH)2D3, including growth inhibition of cancer cells and keratinocytes, as well as induction of leukemic cell differentiation. In contrast to 1, 25(OH)2D3, these synthetic compounds did not demonstrate appreciable binding to serum vitamin D binding protein, a property that is correlated with fewer calcium effects in vivo. Two mimics tested in mice showed greater induction of a VDR target gene with less elevation of serum calcium than 1,25(OH)2D3. CONCLUSIONS: These novel VDR modulators may have potential as therapeutics for cancer, leukemia and psoriasis with less calcium mobilization side effects than are associated with secosteroidal 1,25(OH)2D3 analogs.     

Synthesis of C-2 substituted vitamin D derivatives having ringed side chains and their biological evaluation, especially biological effect on bone by modification at the C-2 position

In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and demonstrated the SAR of the C-2 position of VD(3). Furthermore, by using the OVX model, we found that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported compound, 2α-(3-hydroxypropoxy)-1α,25-dihydroxyvitamin D(3)1d, and this derivative shows a possibility that calcemic activity is less than 1d in vivo.     

Efficient synthesis of 2-modified 1alpha,25-dihydroxy-19-norvitamin D3 with Julia olefination: high potency in induction of differentiation on HL-60 cells

Six novel 2-substituted analogues of 1alpha,25-dihydroxy-19-norvitamin D(3), 6a,b-8a,b, were efficiently synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared as 4-alkylated (3R,5R)-3,5-dihydroxycyclohexanones 12-15 from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The new type of the CD-ring coupling partner 23 was synthesized from 25-hydroxy Grundmann's ketone 19 to apply to the modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were 47-62%. After the separation of the diastereomers based on C2 stereochemistry, the structure (2alpha or 2beta) was determined by (1)H NMR experiments and compared to DeLuca's 2-methyl- and 2-ethyl-1alpha,25-dihydroxy-19-norvitamin D(3). Thus, the synthesized 2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D(3) (8a) showed almost the same potency in binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone 1, while its beta-isomer 8b had only a 3% affinity. Both 2alpha-allyl- and 2alpha-propyl-1alpha,25-dihydroxy-19-norvitamin D(3) (6a and 7a) and their 2beta-analogues (6b and 7b) possessed a weak affinity for the VDR. The strong VDR ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1, and interestingly, even the weaker ligand 8b showed a 6.7-fold higher potency in the cell differentiation activity than that of 1.     

Synthesis of an optically active intermediate for A-ring of 19-nor- 1α,25-dihydroxyvitamin D3

An optically active intermediate 5 for A-ring of 19-nor-1a,25-dihydroxyvitamin D3 2 has been synthesized in five steps, starting from readily available, inexpensive D( )-xylose 6 with good yield.     

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).     

Enantioselective synthesis of A key A-ring intermediate for the preparation of 1α,25-dihydroxyvitamin D(3)

A novel approach to the key A-ring α, β-unsaturated aldehyde 1, an important intermediate for the preparation of 1α,25-dihydroxyvitamin D(3), has been developed. The strategy started from the inexpensive starting material (R)-carvone with an ene reaction serving as the key step toward the potential synthesis of vitamin D(3) analogues bearing the modification at the C-2 position.     

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).     

Rational design of vitamin D3 analogues which selectively restore activity to a vitamin D receptor mutant associated with rickets

[formula: see text] Vitamin D3-resistant rickets (VDRR) is associated with mutations to the Vitamin D receptor (VDR) which effect ligand-dependent transactivation. Some VDRR associated mutants directly disrupt ligand binding. Using the reported VDR-1,25-dihydroxy vitamin D3 (1,25(OH)2D3) cocrystal structure, three 1,25(OH)2D3 analogues were designed to uniquely complement the rickets associated mutant VDR(Arg274-->Leu). The three analogues were 17 to 286 times more potent than 1,25(OH)2D3 with the mutant in cell-based assays and did not substantially activate cellular calcium influx.