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.     

(10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3: an improved synthesis via 19-nor-10-oxo-vitamin D

An efficient synthetic route to (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3 was developed. The key feature of this pathway is the introduction of a 19-fluoromethylene group to a (5E)-19-nor-10-oxo-vitamin D derivative. The 10-oxo-compound was obtained via a 1,3-dipolar cycloaddition reaction of (5E)-1alpha,25-dihydroxyvitamin D with in situ generated nitrile oxide followed by ring cleavage of the formed isoxazoline moiety with molybdenum hexacarbonyl. Conversion of the keto group of (5E)-19-nor-10-oxo-vitamin D to the E and Z fluoromethylene group was achieved through a two-step sequence involving a reaction of lithiofluoromethyl phenyl sulfone followed by the reductive desulfonylation of the alpha-fluoro-beta-hydroxy sulfone. The dye-sensitized photoisomerization of the (5E)-19-fluorovitamin D afforded the desired (5Z)-19-fluorovitamin D derivatives, (10Z)- and (10E)-19-fluoro-1alpha,25-dihydroxyvitamin D3.     

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 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.     

Synthesis,biological activity,and conformational analysis of CD-ring modified trans-decalin 1 alpha,25-dihydroxyvitamin D analogs

A novel series of analogs of 1,25-dihydroxyvitamin D3, the hormonally active metabolite of vitamin D3, characterised by the presence of a trans-fused decalin CD-ring system, possesses surprising biological activities in combination with specific structural modifications in the flexible parts of the molecule, when compared with the natural hydrindane derivatives. (1) A large difference in biological activity is observed between the 20-epimeric trans-decalin analogs that follows a pattern opposite to what is usually observed for the natural ring size. (2) Several trans-decalin analogs that are modified in the seco-B-ring region, including previtamin derivatives, possess a pronounced vitamin D-like activity, whereas the corresponding hydrindane derivatives are inactive. The molecular origin of this behavior is still under study.     

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.     

Catalytic asymmetric synthesis and anticancer effects of the novel non-calcemic analog of vitamin D, 2α-fluoro-19-nor-22-oxa-1α,25-dihydroxyvitamin D3 in metastatic lung carcinoma

1α,25-Dihydroxyvitamin D₃ (1α,25-D₃) has potent antiproliferative and anti-invasive properties in vitro in cancer cells. However, the major limitation to its clinical use is that it causes hypercalcemia. Therefore, vitamin D analogs with potent cell regulatory effects but with weaker calcemic effects than 1α,25-D₃ are required. Among them, 22-oxa-1α,25-D₃ and 19-nor-1α,25-D₃ have anti-cancer effects with relatively low calcemic effects. Modifications at the C-2α position of the A-ring also produced analogs with a unique biological profile. Not only the side-chain but also the A-ring modification thus generates a unique analog with potent cell regulatory effects and low calcemic activity as well. We report here that the hybrid 1α,25-D₃ analog, synthesized via the highly regio- and stereo-selective ring opening 2α-fluorination and catalytic asymmetric carbonyl-ene cyclization, with 2α-fluoro, 19-nor, and 22-oxa modification exhibits unique cell regulatory activities against the development of metastatic lung carcinoma.     

Cal-B-catalyzed alkoxycarbonylation of a-ring stereoisomeric synthons of 1alpha,25-dihydroxyvitamin D3 and 1alpha,25-dihydroxy-19-nor-previtamin D3: a comparative study. first regioselective chemoenzymatic synthesis of 19-nor-A-ring carbonates

A comparative study of alkoxycarbonylation processes of both 19-nor-A-ring and A-ring stereoisomers of 1alpha,25-dihydroxyvitamin D3 analogues catalyzed by Candida antarctica lipase B (CAL-B) has been described. The presence of the methyl group in the A-ring at C-2, as in 3-6, has a determining role in the regioselectivity of the biocatalysis, mainly allowing the hydroxyl group at C-5 position to react. For the 19-nor-A-ring stereoisomers 7-10, which lack the C-2 methyl group, the configurations at C-3 and C-5 have a high influence in the selectivity exhibited by CAL-B. Thus, each couple of enantiomers showed opposing regioselectivities depending on the C-3 configuration. When C-3 possesses an (S)-configuration, enzymatic alkoxycarbonylations took place at the C-5-(R) or C-5-(S) hydroxyl groups. However, if the chiral centers at C-3 are (R), CAL-B alkoxycarbonylated the C-3-(R) hydroxyl group independently of the configuration at C-5. The corresponding carbonates are useful A-ring precursors of 1alpha,25-dihydroxyvitamin D3 analogues, selectively modified at the C-1 or C-3 positions. In addition, an improved synthesis of cis A-ring synthons 5 and 6 is described using a Mitsunobu methodology.     

Sensitive analysis of 1alpha,25-dihydroxyvitamin D3 in biological fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric assay using 5% bovine serum albumin as the calibration matrix has been developed for the quantitative analysis of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in biological fluids. The analyte was extracted from the matrix after protein precipitation using an automated solid-phase extraction procedure involving both a reversed-phase and normal-phase procedure on a single C18 cartridge. The analytical chromatography was performed using a Symmetry C8 50 x 2.1 mm, 3.5 microm column. The mobile phase was a linear gradient from 75 to 99% methanol with a constant concentration of 2 mM ammonium acetate. 1alpha,25(OH)2D3 and the internal standard [2H6]1alpha,25(OH)2D3 were detected by using MS-MS. The ion source was operated in the positive electrospray ionisation mode. The assay is specific, sensitive, and has a capacity of more than 100 samples per day, with a limit of quantitation of 20 pg ml(-1) for a 1.0-ml sample aliquot. The assay has been used for the analysis of 1alpha,25(OH)2D3 in serum from rats and pigs simultaneously with the analysis of the vitamin D analog seocalcitol.     

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).     

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.     

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.     

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.