Novel 9-Alkyl- and 9-Alkylidene-Substituted 1α,25-Dihydroxyvitamin D3 Analogues: Synthesis and Biological Examinations

Article abstract of DOI:10.1021/acs.jmedchem.5b00795

Continuing the structure-activity relationship studies in the vitamin D area, we designed and synthesized novel C-9 substituted calcitriol analogues, possessing different nonpolar groups at this position. 9α-Methyl-1α,25-(OH)₂D₃, bot

Full text of DOI:10.1021/acs.jmedchem.5b00795

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Novel 9ꢀAlkylꢀ and 9ꢀAlkylideneꢀsubstituted 1α,25ꢀDihydroxyvitamin D3 Analogues:
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Synthesis and Biological Examinations
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Urszula Kulesza,† Lori A. Plum,‡ Hector F. DeLuca,‡ Antonio Mouriño,*,¶ and Rafal R.
Sicinski*,†
†Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
‡Department of Biochemistry, University of Wisconsin-Madison, 433 Babcock Drive, Madison, Wisconsin
53706, United States
¶Departamento de Química Orgánica, Laboratorio de Investigación Ignacio Ribas, Universidad
de Santiago de Compostela, 15782 Santiago de Compostela, Spain
AUTHOR INFORMATION
Corresponding Authors
antonio.mourino@usc.es
rasici@chem.uw.edu.pl
Keywords: Vitamin D analogues; Functionalization at Cꢀ9; 19ꢀNorvitamin D analogues; Vitamin
D receptor; Cellular differentiation; Transcriptional activity.
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ABSTRACT
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Continuing the structureꢀactivity relationship studies in the vitamin D area, we designed
and synthesized novel Cꢀ9 substituted calcitriol analogues, possessing different nonꢀpolar groups
at this position. 9αꢀMethylꢀ1α,25ꢀ(OH)2D3, both epimers of 9ꢀmethyleneꢀ10,19ꢀdihydroꢀ1α,25ꢀ
(OH)2D3 as well as the parent vitamin with the “reversed” triene system, 9ꢀmethyleneꢀ19ꢀnorꢀ
1α,25ꢀ(OH)2D3, were obtained from the previtamin D precursors, constructed by either Suzukiꢀ
Miyaura, Sonogashira or Stille couplings of the corresponding Aꢀ and C,Dꢀring fragments. An
alternative synthetic path, leading to the latter vitamin and its homologue with 9ꢀethylidene
group, involved formation of dienynes as precursors of the respective 19ꢀnorprevitamin D
compounds. 9βꢀMethylꢀ19ꢀnorꢀ1α,25ꢀ(OH)2D3 was prepared by homogeneous hydrogenation
with Wilkinson catalyst, and this analogue was found to be the most active in vitro. Moreover,
9αꢀmethylꢀ1α,25ꢀ(OH)2D3 and 9ꢀmethyleneꢀ19ꢀnorꢀ1α,25ꢀ(OH)2D3 showed some in vitro
activity, however, the in vivo assays indicated only weak calcemic potency of these compounds
in the intestinal calcium transport.
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INTRODUCTION
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1α,25ꢀDihydroxyvitamin D3 [calcitriol, 1α,25ꢀ(OH)2D3 (1), Figure 1], being the active
form of vitamin D3 in vivo in the regulation of calcium and phosphorous,1 is also active in many
other biological processes, for example, induction of cell differentiation and apoptosis as well as
inhibition of cell proliferation and angiogenesis.2 The genomic actions of the natural hormone 1
are mediated through the vitamin D receptor (VDR),3 a transcription factor and member of the
nuclear receptor superfamily,4 whose presence has been detected in numerous tissues.5 These
findings make calcitriol potentially useful in various biomedical applications, including treatment
of metabolic bone diseases, psoriasis, some kinds of cancers and immune disorders.6 However,
the broader clinical application of 1α,25ꢀ(OH)2D3 is limited by its strong calcemic potency.7 In an
effort to enhance selective biological actions and minimize undesired side effects, more than
3000 vitamin D analogues, modified in different parts of the molecule, have been developed in
the recent years, but only few of them have found clinical applications.8
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So far, only few vitamin D3 analogues functionalized at Cꢀ9 have been described in the
literature. The first 9ꢀsubstituted analogue of calcitriol with a different configuration of the
intercyclic diene moiety, (7Z)ꢀ9αꢀhydroxyvitamin D3, was synthesized by Mouriño and coꢀ
workers9 and later by Dauben and Greenfield.10 WittigꢀHorner coupling of 9αꢀbenzoyloxy or 9αꢀ
fluoro Grundmann ketone with the anion of the Aꢀring phospine oxide, due to the steric
interaction between Hꢀ6 and 9αꢀsubstituent, led to the compound characterized by 7Eꢀgeometry
of the newly formed double bond. In 2006, a series of 9αꢀalkylated derivatives of 19ꢀnorcalcitriol
(2) was prepared by Shimizu11 using WittigꢀHorner and Julia olefination approach but almost no
information was disclosed about the biological properties of these compounds. Very recently,
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Japanese chemists synthesized few 9αꢀalkylated and 9αꢀhydroxyalkylated 2ꢀsubstituted 19ꢀ
norcalcitriols and analyzed their crystal complexes with W282R mutant of rVDR.12
On the basis of the limited knowledge of the biological effect caused by modification at
Cꢀ9 position in the vitamin D3 molecule, and as an extension of the structureꢀactivity research
conducted in our laboratories, we designed new homologues of calcitriol and 19ꢀnorcalcitriol (4ꢀ
9) substituted with nonꢀpolar (alkyl and alkylidene) groups at Cꢀ9 (Figure 1). As the direct
precursors of these vitamin D analogues, the corresponding 9ꢀalkylated previtamin D compounds
10ꢀ12 (or their derivatives) were chosen. These, in turn, could be constructed from the synthons
described in the literature ꢀ the protected 25ꢀhydroxy Grundmann ketone 1313 and vinyl iodide
14,14 derived from vitamin D3,15 as well as two enynes 15 and 16, readily obtainable from quinic
acid16 and Sꢀcarvone,17 respectively.18
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RESULTS AND DISCUSSION
Chemistry. As we reported previously, the conjugated triene systems of vitamins 5, 7 and
8 were obtained by thermal sigmatropic [1,7]ꢀhydrogen shift from the same previtamin D
compound 11, whereas the dienyne precursor of the required previtamin was efficiently
constructed employing three alternative approaches: Sonogashira, Stille and stereoselective
dehydration of a tertiary 8βꢀalcohol by Burgess reagent.14 Here we also describe a new mild and
efficient convergent route to the previtamin D system that circumvents the troublesome
semihydrogenation of dienynes. The new method generates the previtamin D skeleton by Suzukiꢀ
Miyaura coupling19 between an alkenylꢀboronic ester and a vinyl iodide. For the conversion of
enyne 16 to the Aꢀring precursor, cisꢀalkenyl boronate 18 (Scheme 1), we applied the procedure
based on hydroboration of alkynes developed by Miyaura, employing pinacolborane in the
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presence of Rh(I)ꢀPiꢀPr3 complex and Et3N.20 Use of more than one equivalent of Et3N and
excess of alkyne with respect to the borane reagent were critical for achieving the high cis-
selectivity. Under these conditions, terminal alkyne 16 was transformed to the desired (Z)ꢀ1ꢀ
alkenyl boronate 18 (74% yield) along with its geometric isomer 17 (18% yield). The double
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bond stereochemistries in the reaction products were determined by analysis of their H NMR
data, namely, by comparison of the vicinal coupling constants of doublets derived from the
olefinic protons resonating at 5.51 and 7.45 ppm (J = 18 Hz) for the transꢀboronate 17, and at
5.40 and 6.89 ppm (J = 15 Hz) for the cisꢀisomer 18, respectively.
The vinyl boronate 18 was then coupled with the known vinyl iodide 1414 in the presence
of bis[triphenylphosphine]palladium(II) chloride and 2M aqueous solution of K3PO4,21 to give
directly the desired previtamin D compound 19. Although the desired previtamin D analogue was
obtained in high yield, the side product of the Aꢀring homocoupling reaction was also observed.
Finally, the previtamin 19 was easily converted, by subsequent deprotection of hydroxyl groups
and thermolysis, into the target vitamin D compounds 5, 7 and 8, which were purified by HPLC.
To examine the synergetic biological effect of Cꢀring substitution of the vitamin D
analogue with 9ꢀalkylidene group and 19ꢀnor modification of its ring A, compounds 6 and 9 were
prepared using enyne 15 as the Aꢀring building block.18 Thus, the vinyl iodide 14 was coupled
with either the enyne 15 or its tributyltin derivative 20 (Scheme 2) using Sonogashira or Stille
reactions. The yields of the dienyne 21 prepared by both methods were high, although it was
necessary to use a 5ꢀfold molar excess of the starting enyne 15 in the case of Sonogashira
coupling due to the concomitant homocoupling process. Partial hydrogenation of the triple bond
of dienyne 21 using Lindlar catalyst poisoned with quinoline afforded 9ꢀmethylꢀ19ꢀnorprevitamin
D compound 22 in 91% yield. In an alternative attempt, Pdꢀcatalyzed Suzuki coupling of iodide
14 with (Z)ꢀalkenyl boronate 24, prepared from enyne precursor 15, led also directly to the
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previtamin 22. The presence of methyl group attached to the terminal Cꢀ9 of the conjugated
hexatriene moiety made such previtamin D analogue capable of equilibration with the
corresponding vitamin D isomer 23. Final deprotection of hydroxyl groups in this product
provided the desired 19ꢀnorcalcitriol analogue 6, possessing an ‘unnatural’ triene system.
The synthesis of 9ꢀethylideneꢀ19ꢀnorcalcitriol (9) began from the αꢀalkylation of protected
25ꢀhydroxy Grundmann ketone performed analogously as described for the preparation of 26.14
Thus, αꢀethylation of 13 was achieved by treatment with LDA in the presence of DMPU,
followed by addition of EtI to afford the single product 27 in 66% yield (Scheme 3). Attempts to
transform the resulting ketone 27 into the corresponding vinyl iodide were unsuccessful. For the
construction of dienynes 30 and 31, the precursors of 19ꢀnorcalcitriol analogues 6 and 9, we
applied the procedure based on the addition of an acetylide ion (generated from 15 by nꢀ
butyllithium) to the alkylated Grundmann ketones 26 and 27 in the presence of cerium
trichloride. Stereoselective dehydration of the resulted propargylic alcohols 28 and 29 with
Burgess reagent afforded the desired dienynes 30 (95%) and 31 (96%).14 Further conversion of
dienynes 30 and 31 to the respective 9ꢀalkylidene analogues of 19ꢀnorcalcitriol 6 and 9 with an
‘unnatural’ triene system was accomplished as described above. The thermal isomerization
process of previtamin 33 led to the vitamin analogue 35 as a single isomer with Eꢀconfiguration
of the ethylidene group as assigned by NOE experiment. On irradiation of the vinylic proton
signal from the 9ꢀethylidene substituent (CH3CH=) at δ 5.33 ppm, 2% enhancement was
observed for the olefinic Hꢀ6 signal at 6.37 ppm. A similar preferential formation of more stable
geometrical isomers of vitamin D analogues during the reversible thermal isomerization process
was observed in the series of 19ꢀfunctionalized compounds.22 The UV spectra of compounds 6
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and 9, possessing the ‘reversed’ triene chromophore in their structures, were similar to the
spectrum of 1α,25ꢀdihydroxyvitamin D3 (1).
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The successful preparation of the vitamin D analogues with 9αꢀmethyl and 9ꢀalkylidene
groups encouraged us to extend the studies on the effect of nonꢀpolar substituents at Cꢀ9 on the
biological activity of these compounds, and to synthesize also the 9βꢀalkylated vitamin D
compound 4. We expected that introduction of such an equatorial substituent at Cꢀ9, could
increase the steric strain between this group and C(6)ꢀH and influence hydrophobic interactions
between the ligand and the amino acids in the ligand binding pocket of the VDR.
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As we previously established,14 9βꢀmethylꢀcalcitriol was not formed during the thermal
rearrangement of 1α,25ꢀdihydroxyꢀ9ꢀmethylprevitamin D3 (11). Other methods of direct
synthesis of the vitamin D triene system (for instance, WittigꢀHorner approach) would require
9βꢀalkylated Grundmann ketone derivatives as precursors of the CDꢀring building blocks. As
previously observed, alkylation of the Grundmann ketone led exclusively to 9αꢀsubstituted
products and, moreover, formation of products with the reversed configuration of the intercyclic
diene moiety could be expected in the WittigꢀHorner coupling.9,10 We decided to prepare such a
vitamin D analogue by selective reduction of 9ꢀexomethylene group. Such chemoselectivity was
achieved by the homogeneous hydrogenation of 9ꢀmethyleneꢀ19ꢀnorcalcitriol (6), carried out in
the presence of tris(triphenylphosphine)rhodium(I) chloride (Wilkinson catalyst). The desired 9βꢀ
methylꢀ19ꢀnorcalcitriol (4) was formed in 56% yield as single diene product (Scheme 4), which
was successfully separated from the unreacted starting material (36%) by reversedꢀphase HPLC.
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The structure of compound 4 was assigned on the basis of its H NMR spectra. Spin decoupling
experiment performed with the multiplet derived from the methine proton at Cꢀ9 (2.29 ppm),
allowed measuring its vicinal coupling constants (9.1 and 2.8 Hz) with the neighboring protons at
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Cꢀ11. Their values, being in agreement with these resulting from molecular modeling
(HyperChem and PCModel), indicated an axial disposition of Hꢀ9 and, consequently, equatorial
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βꢀorientation of the introduced methyl group.23 Also, a comparison of the H NMR spectrum of
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the vitamin analogue 4 with the respective spectral data of its 9ꢀepimer 3, as reported in the
Japanese patent,11 also supported our configurational assignment. Interestingly, the UV spectrum
of the vitamin D compound 4 exhibits three absorption maxima (λmax 244, 252 and 261 nm),
characteristic of the planar 5,7ꢀdiene chromophore present in 19ꢀnorvitamin D compounds. This
finding seems to indicate that, despite severe interaction of equatorial 9βꢀmethyl group with the
vinyl hydrogen at Cꢀ6, there is no significant deviation of the intercyclic C(5)=C(6)ꢀC(7)=C(8)
moiety from planarity. Further confirmation of the ascribed structure of the synthesized
compound 4, including its 9Sꢀconfiguration and the geometry of molecule, was obtained by Xꢀray
crystallographic analysis. Thus, Xꢀray diffraction data indicate that vitamin 4 crystallizes with its
ring A fixed in the βꢀchair conformation,24 and the ring C exists in the chair conformation with an
equatorially oriented 9βꢀmethyl group (Figure 2). The torsion angle of the intercyclic 5,7ꢀdiene
moiety was found to be 168o, that explained the UV absorption data of the examined compound.
Biological Evaluation. Biological in vitro activities of the novel vitamin D analogues are
compared to the natural hormone and summarized in Table 1.25 In the first assay, the synthesized
derivatives of 1α,25ꢀ(OH)2D3 were tested for their ability to bind the fullꢀlength recombinant rat
vitamin D receptor. The competition binding assays revealed that the 9αꢀmethyl group in 5
significantly reduced VDR binding ability by 200ꢀfold from that of the natural hormone. Among
the compounds with a “reversed” triene system, the best binder was the 9ꢀmethyleneꢀ19ꢀ
norcalcitriol (6) characterized by similar binding activity as 5. Both Aꢀring methylated
derivatives 7 and 8, as well as homologue 9 with 9ꢀethylidene substituent, were practically
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devoid of binding activity. Interestingly, 19ꢀnorvitamin D compound 4 with 9βꢀmethyl
substituent exhibited affinity for VDR similar to 1α,25ꢀ(OH)2D3 and this analogue was also found
to be very active in inducing differentiation of HLꢀ60 cells and in transcriptional activity.
Analogue 6 had weak HLꢀ60 cell differentiating potency whereas activities of other tested
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compounds were reduced by ca. four orders of magnitude from that of 1
α,25ꢀ(OH)2D3. With a
notable exception for 4, the analogues also exhibited low activities in the transcriptional assay,
being 200ꢀ300 times (5, 6 and 8) less active than 1
of magnitude (7 and 9).
α,25ꢀ(OH)2D3 or decreased by ca. four orders
Rather surprisingly, the results of the in vivo tests clearly demonstrated that 9βꢀmethylꢀ19ꢀ
norcalcitriol (4), being the most active in vitro, was characterized by a weak calcemic potency. Its
activity in intestinal calcium transport was lower than that of calcitriol itself, and the analogue
showed only marginal activity in bone calcium mobilization when tested at very large doses
(Figure 3). 9αꢀMethylꢀcalcitriol (5) showed some intestinal calcium transport activity, being
inactive in mobilizing calcium from bone (Figure 4).
When tested in vivo, compounds possessing 9ꢀalkylidene substituents gave no response in
bone calcium mobilization (Figure 4). Analogue 6 showed very low intestinal calcium transport,
while a slightly increased intestinal activity was observed for compound 9 possessing 9ꢀ
ethylidene group.
Studies on docking the analogues 4ꢀ6 into the VDR. Binding studies of the newly
synthesized 9ꢀsubstituted calcitriol analogues showed that only vitamins 4ꢀ6 exerted significant
affinity for the VDR. To gain better understanding of this important biological function, we
performed docking simulations of these compounds to the ligand binding pocket (LBP) of the
VDR using Gold (5.1 version) software. When analyzing the calculated complexes, we
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considered the energy criterion, formation of hydrogen bonds and the parallel orientation of the
ligand intercyclic 5,7ꢀdiene moiety in respect to the aromatic rings of Trpꢀ286. This unique
tryptophan residue plays an essential role in anchoring the ligand in the binding pocket and
transcription of genes controlled by VDR, which was demonstrated by mutation26 and NMR27
experiments.
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The analysis of docking experiments shows that 9βꢀmethylꢀ19ꢀnorcalcitriol (4), 9αꢀ
methylꢀcalcitriol (5) and 9ꢀmethyleneꢀ19ꢀnorcalcitriol (6) anchor the VDR cavity in an analogous
fashion to 1α,25ꢀ(OH)2D3 in the crystalline VDR(LBD)ꢀ1 complex28 (1DB1) (Figure 5aꢀc) and
they similarly interact with the receptor. All six hydrogen bonds present in the crystalline
VDR(LBD)ꢀ1 complex28 were also found in these modeled complexes with the synthetic ligands.
However, in the case of complexes with analogues 5 and 6, the interactions between their 25ꢀ
hydroxyl groups and Hisꢀ305 and Hisꢀ397 were much weaker than for the natural hormone.
Moreover, inspection of the complex VDR(LBD)ꢀ6 indicated a poor contact between Serꢀ278 and
the ligand’s 3βꢀhydroxyl (3.35 Å).
In the analyzed complexes, the intercyclic diene moieties of all analogues were
approximately parallel to the Trpꢀ286 rings. However, in the VDR(LBD)ꢀ4 complex, the
tryptophan indole ring was positioned little closer to the ligand’s 5,7ꢀdiene fragment (3.79 Å)
than in the crystalline complex VDR(LBD)ꢀ1 (3.88 Å). The distance between Trpꢀ286 and
C(7)=C(8) bond of analogue 6 was larger, and in VDR(LBD)ꢀ5 exceeded 4 Å. Evidently, the
presence of the 9αꢀmethyl substituent in the compound 5 causes that the secoꢀB ring of this
analogue is significantly moved away from the tryptophan moiety. Thus, interestingly, it seems
that the observed differences between the compared complexes can be potentially explained by
considering the distances of the modeled ligands from Trpꢀ286.
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CONCLUSIONS
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CꢀRing modification of calcitriol and 19ꢀnorcalcitriol derivatives represented an
interesting target for our continued structureꢀactivity studies on 1α,25ꢀ(OH)2D3 analogues. An
attractive goal for the planned synthetic efforts in this area seemed to be vitamin D analogues
possessing nonꢀpolar groups at the rather unexplored position Cꢀ9. Such 9ꢀsubstitution could
potentially enhance interactions of the novel compounds with hydrophobic residues in the ligand
binding pocket of the vitamin D receptor. Also introduction of 9ꢀalkylidene (methylene and
ethylidene) substituents to the 19ꢀnorcalcitriol skeleton seemed to be an interesting endeavor,
because it could lead to 1α,25ꢀ(OH)2D3 analogues with the ‘reversed’ triene system. In the
present work, we described an efficient syntheses of such compounds and evaluated their
biological activity. It was established that compounds with a nonꢀnatural triene system are
characterized by significantly decreased biological in vitro and in vivo activity. In strong contrast
to the 9αꢀalkylated calcitriol 5, analogue 4 representing the first 9βꢀsubstituted vitamin D
compound described in the literature, was found as active as calcitriol in the in vitro tests.
However, despite its strong binding affinity to the VDR and high transcriptional activity, this
analogue was devoid of bone calcium mobilization ability and poorly supported intestinal
calcium transport. Further studies on this interesting compound should be undertaken. Thus, it
would be highly desirable to examine the crystal structure of its complex with VDR(LBD) and
establish which conformational changes of the receptor are responsible for the decreased
calcemic activity of the ligand.
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EXPERIMENTAL SECTION
Chemistry. Melting points (uncorrected) were determined on a SMP10 Stuart Scientific
capillary melting point apparatus. Optical rotations were measured in chloroform using a Perkinꢀ
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Elmer model 343 polarimeter at 24 °C. Ultraviolet (UV) absorption spectra were obtained on a
Shimadzu UVꢀ1800 UV spectrophotometer in absolute ethanol. Nuclear magnetic resonance
spectra were recorded in CDCl3 solutions using Varian Unity plus (200 MHz), Bruker WMꢀ250,
Varian Inova (400 MHz), Varian Unity Inova 500 and Bruker DMXꢀ500 or Varian 700 Active
Shield (700 MHz) instruments. Chemical shifts (δ) are reported in parts per million relative to
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CH3Si (δ 0.00) or solvent signal as an internal standard. COSY, NOESY, HMBC and HMQC
spectra, as well as spin decoupling, DEPT 90, DEPT 135 and differential NOE experiments were
1
13
1
used to assign particular signals in the H and C NMR spectra. Signals in H NMR spectra are
described using the following abbreviations: s ꢀ singlet, d ꢀ doublet, t ꢀ triplet, q – quartet, m –
multiplet, br – broad, narr – narrow. Highꢀresolution mass spectra were recorded on LCT (TOF)
or Mass Quattro LC spectrometers using electrospray ionization (ESI) technique.
Reactions were usually carried out with magnetic stirring. All reactions involving
moistureꢀ or oxygenꢀsensitive compounds were carried out under dry argon atmosphere. Reaction
temperatures refer to external bath temperatures. Tetrahydrofuran was distilled from
Na/benzophenone; dichloromethane and toluene were distilled from P2O5, whereas pyridine,
diisopropylamine, diethylamine and triethylamine were distilled from CaH2. The organic extracts
were dried over anhydrous MgSO4 or Na2SO4, filtered and concentrated using a rotary evaporator
at a water aspirator pressure (20ꢀ30 mm Hg). Reactions were monitored by thinꢀlayer
chromatography (TLC) using aluminumꢀbacked MERCK 60 silica gel plates (0.2 mm thickness).
The chromatograms were visualized first with ultraviolet light (254 nm) and then by immersion
in a ceriumꢀmolybdenum solution [10 g Ce(SO4)2 × 4 H2O, 25 g phosphomolybdic acid, 60 mL
H2SO4 and 940 mL H2O] or pꢀanisaldehyde solution (5 mL H2SO4, 1.5 mL glacial HOAc, 3.7 mL
pꢀanisaldehyde, 135 mL H2O) followed by heating. Flash column chromatography was
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performed with MERCK 60 (230ꢀ400 mesh) silica gel and Cꢀ18 reversed phase silica gel. Highꢀ
performance liquid chromatography (HPLC) purifications were performed on Waters Associates
liquid chromatograph equipped with a Model 486 tunable absorbance detector and refractive
index detector (R 401) and Shimadzu UFLS liquid chromatograph equipped with SPDꢀ20A
tunable absorbance detector.
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The purity of final compounds was determined by HPLC, and they were judged at least
99% pure. Two HPLC columns (9.4 mm × 25 cm ZorbaxꢀSil and Zorbax RXꢀC18) were used as
indicated in Table 2 (Supporting Information). The purity and identity of the synthesized
1
vitamins were additionally confirmed by inspection of their H NMR and highꢀresolution mass
spectra.
2ꢀ{(E)ꢀ2’ꢀ[(3”S,5”R)ꢀ3”,5”ꢀbisꢀ[(tertꢀbutyldimethylsilyl)oxy]ꢀ2”ꢀmethylꢀcyclohexꢀ1”ꢀenyl]ꢀ
vinyl}ꢀ4,4,5,5ꢀtetramethylꢀ[1,3,2]ꢀdioxaborolane (17) and 2ꢀ{(Z)ꢀ2’ꢀ[(3”S,5”R)ꢀ3”,5”ꢀBisꢀ
[(tertꢀbutyldimethylsilyl)oxy]ꢀ2”ꢀmethylꢀcyclohexꢀ1”ꢀenyl]ꢀvinyl}ꢀ4,4,5,5ꢀtetramethylꢀ
[1,3,2]ꢀdioxaborolane (18). Et3N (564 ꢀL, 4.0 mmol), iꢀPr3P (1 drop), [RhCl2(cod)]2 (6 mg,
0.012 mmol) and 4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane (1 M in THF, 0.80 mL, 0.80 mmol)
were dissolved in cyclohexane (4 mL). After 30 min a solution of the enyne 16 (400 mg, 1.052
mmol) in cyclohexane (4 mL) was added via cannula and the reaction mixture was stirred at
room temperature for 1 h. The solvent was removed under reduced pressure to give a residue
which was applied to a silica SepꢀPak (10 g). Elution with hexane/Et2O (99.5:0.5) provided
unreacted substrate 16 (200 mg) and a mixture of isomeric boronates (252 mg). Final separation
of the boronates 17 and 18 was achieved by HPLC (10 mm × 25 cm Phenomenex Luna Silica
column, 4 mL/min) using a hexane/ethyl acetate (97:3) solvent system. The Eꢀisomer 17 was
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collected at Rv 34 mL (48 mg, 9%; 18% considering the recovered substrate) and the Zꢀisomer 18
at Rv 28 mL (198 mg, 37%; 74% considering the recovered substrate).
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1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ25ꢀhydroxyꢀ9ꢀmethylꢀprevitamin D3 tertꢀbutyldimethylsilyl
ether (19). An aqueous solution of K3PO4 (2M; 1.36 mL) and PdCl2(PPh3)2 (4 mg, 4.8 ꢁmol)
were added to a solution of the boronate 18 (198 mg, 0.39 mmol) and iodide 14 (40 mg, 0.1
mmol) in THF (10 mL). The reaction mixture was stirred vigorously for 2 h and then extracted
with Et2O. The combined organic layers were dried (MgSO4) and concentrated. The residue was
purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) afforded the
previtamin 19 (51 mg, 78%) as a colorless oil.
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1α,25ꢀDihydroxyꢀ9ꢀmethylꢀprevitamin D3 (11). Tetrabutylammonium fluoride (1.0 M in THF;
1.6 mL, 1.6 mmol) was added to a solution of protected previtamin D compound 19 (51 mg, 0.08
mmol) in dry THF (4 mL) at room temperature. The reaction mixture was stirred overnight and
quenched by addition of brine, extracted with EtOAc, dried (MgSO4) and concentrated. The
residue was purified by flash chromatography on silica gel. Elution with EtOAc gave the
deprotected previtamin 11 (31 mg, 91%) as a colorless oil.
1α,25ꢀDihydroxyꢀ9αꢀmethylꢀvitamin
D3
(5),
1α,25ꢀdihydroxyꢀ9ꢀmethyleneꢀ10(S),19ꢀ
dihydrovitamin D3 (7) and 1α,25ꢀdihydroxyꢀ9ꢀmethyleneꢀ10(R),19ꢀdihydrovitamin D3 (8). A
solution of the previtamin 11 (31 mg, 0.073 mmol) in isooctane (18 mL) was refluxed under
argon in the darkness for 14 h and then concentrated. The isomeric compounds were separated by
HPLC (9.4 mm × 25 cm Eclipse XDBꢀC18 column, 4 mL/min) using a methanol/water (86:14)
solvent system. The 10αꢀmethyl analogue 8 was collected at Rv 40 mL (4.6 mg, 15%), 10βꢀ
methyl vitamin 7 at Rv 43 mL (8.7 mg, 28%), 9αꢀmethyl analogue 5 at Rv 46 mL (1.9 mg, 6%)
and the unreacted previtamin 11 at Rv 55 mL (12.4 mg, 40%). Taking into account the recovered
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substrate, the yields of the isolated products 5, 7 and 8 were 10, 46 and 25%, respectively, based
on recovered starting material.
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(3S,5R)ꢀ3,5ꢀBisꢀ[(tertꢀbutyldimethylsilyl)oxy]ꢀ1ꢀtributylstannylethynylꢀcyclohexene (20). nꢀ
Hexyllithium (2.3 M in hexane; 0.18 mL, 0.41 mmol) was slowly added to a stirred solution of
the enyne 15 (138 mg, 0.38 mmol) in dry THF (3 mL) at ꢀ78 ºC. After 1 h, freshly distilled triꢀnꢀ
butyltin chloride (0.13 mL, 0.45 mmol) was added, and the mixture was stirred at ꢀ78 ºC for 15
min and then at room temperature for 2 h. The reaction was quenched by addition of water. The
mixture was diluted with Et2O and washed with saturated NaHCO3. The organic phase was dried
(MgSO4) and concentrated. The residue was purified by flash chromatography on Cꢀ18 reversed
phase silica gel. Elution with acetonitrile gave the compound 20 (190 mg, 77%).
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1α,3βꢀBis[(tertꢀbutyldimethylsilyl)oxy]ꢀ9ꢀmethylꢀ25ꢀhydroxyꢀ19ꢀnorꢀ9,10ꢀsecocholestaꢀ
5(10),8ꢀdienꢀ6ꢀyne (21).
(a) A catalytic amount of Pd(PPh3)4 (5 mg, 4 ꢁmol) and LiCl (16 mg, 0.38 mmol; dried under
vacuum) were successively added to a solution of compound 20 (36 mg, 0.054 mmol) and iodide
14 (20 mg, 0.050 mmol) in dry THF (3 mL). The resulting mixture was refluxed in the darkness
for 3 h, poured into water and extracted with Et2O. The combined organic extracts were dried
(MgSO4) and concentrated. The residue was purified by flash chromatography on silica gel.
Elution with hexane/Et2O (98:5) gave the dienyne 21 (29 mg, 83%) as a colorless oil.
(b) CuI (6 mg, 0.02 mmol) and Pd(PPh3)2Cl2 (6 mg, 0.006 mmol) were successively added to a
solution of the iodide 14 (10 mg, 0.025 mmol) and the enyne 15 (37 mg, 0.1 mmol) in dry Et2NH
(0.2 mL) at 0 ºC. The mixture (protected from light) was stirred at this temperature for 1 h and
then at room temperature for 3 h. Saturated solution of NH4Cl was added and the mixture was
extracted with Et2O. The combined organic layers were dried (MgSO4) and concentrated to give
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an oily residue which was purified by flash chromatography on silica gel. Elution with
5
6
hexane/Et2O (95:5) afforded the dienyne 21 (15 mg, 92%) as a colorless oil.
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1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ25ꢀhydroxyꢀ9ꢀmethylꢀ19ꢀnorprevitamin
D3
tertꢀ
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butyldimethylsilyl ether (22).
(a) Lindlar catalyst (20 mg) and a 0.5% (v/v) solution of quinoline in hexane (38 ꢀL) were
successively added to a solution of the dienyne 21 (15 mg, 0.023 mmol) in hexane (7 mL). The
mixture was stirred under a hydrogen atmosphere for 4 h until all starting material disappeared
(TLC control). The reaction mixture was filtered through Celite and the filtrate was concentrated
under vacuum. The residue was purified by flash chromatography on silica gel. Elution with
hexane/Et2O (95:5) furnished the previtamin 22 (13.5 mg, 91%) as a colorless oil.
(b) An aqueous solution of K3PO4 (2 M, 0.7 mL) and PdCl2(PPh3)2 (2 mg, 2.5 ꢀmol) were
successively added to a solution of the boronate 24 (33 mg, 0.067 mmol; prepared as described
below) and iodide 14 (18 mg, 0.049 mmol) in dry THF (3 mL). The mixture was stirred
vigorously at room temperature for 2 h and then extracted with Et2O. The combined organic
layers were dried (MgSO4) and concentrated in vacuo to give a residue which was purified by
flash chromatography on silica gel. Elution with hexane/Et2O (99:1) gave the previtamin 22 (25
mg, 79%).
1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ25ꢀhydroxyꢀ9ꢀmethyleneꢀ19ꢀnorvitamin
D3
tertꢀ
butyldimethylsilyl ether (23). A solution of the previtamin 22 (13.5 mg, 0.021 mmol) in
isooctane (7 mL) was refluxed in the darkness for 6.5 h and then concentrated under vacuum. The
resulting product was purified by flash chromatography on silica gel. Elution with hexane/Et2O
(95:5) gave the protected vitamin 23 (13.5 mg, 100%) as a colorless oil.
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1α,25ꢀDihydroxyꢀ9ꢀmethyleneꢀ19ꢀnorvitamin D3 (6). Tetrabutylammonium fluoride (1.0 M in
THF; 1.05 mL, 1.05 mmol) was added to a solution of the protected vitamin D analogue 23 (13.5
mg, 0.021 mmol) in dry THF (3 mL) at room temperature. The reaction mixture was stirred
overnight and quenched by the addition of brine, extracted with EtOAc, dried (MgSO4) and
concentrated in vacuum. The residue was purified by HPLC [(10 mm × 25 cm Phenomenex Luna
Silica column, 4 mL/min, hexane/2ꢀpropanol (8:2)]. The vitamin D compound 6 was collected at
Rv 67 mL. Final purification of the product was achieved by reversedꢀphase HPLC [(9.4 mm ×
25 cm Eclipse XDBꢀC18 column, 4 mL/min, methanol/water (88:12)]. The 9ꢀmethyleneꢀ19ꢀ
norcalcitriol (6) was collected at Rv 28 mL (7.5 mg, 88%).
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2ꢀ{(Z)ꢀ2’ꢀ[(3”S,5”R)ꢀ3”,5”ꢀbisꢀ[(tertꢀbutyldimethylsilyl)oxy]ꢀcyclohexꢀ1”ꢀenyl]ꢀvinyl}ꢀ
4,4,5,5ꢀtetramethylꢀ[1,3,2]ꢀdioxaborolane (24) and 2ꢀ{(E)ꢀ2’ꢀ[(3”S,5”R)ꢀ3”,5”ꢀbisꢀ[(tertꢀ
butyldimethylsilyl)oxy]ꢀcyclohexꢀ1”ꢀenyl]ꢀvinyl}ꢀ4,4,5,5ꢀtetramethylꢀ[1,3,2]ꢀdioxaborolane
(25). Dry Et3N (290 ꢀL, 2.1 mmol), iꢀPr3P (1 drop), [RhCl2(cod)]2 (3 mg, 0.006 mmol) and
4,4,5,5ꢀtetramethylꢀ1,3,2ꢀdioxaborolane (1 M in THF, 0.42 mL, 0.42 mmol) were successively
dissolved in cyclohexane (1 mL). After 30 min, a solution of the enyne 15 (200 mg, 0.545 mmol)
in cyclohexane (2 mL) was added via cannula and the resulting mixture was stirred for 1 h. The
solvent was removed under vacuum to give a residue which was applied to silica SepꢀPak (5 g).
Elution with hexane/Et2O (99.5:0.5) provided the unreacted substrate 15 (44%) and a mixture of
isomeric boronates. Final separation of the boronates 24 and 25 was achieved by HPLC [(10 mm
× 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/ethyl acetate (98.5:1.5)]. The Eꢀ
isomer 25 was collected at Rv 55 mL (8%; 15% based on recovered substrate) and the Zꢀisomer
24 at Rv 60 mL (37%; 66% based on recovered substrate).
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(1R,3aR,5R,7aR)ꢀ1ꢀ[(R)ꢀ1’,5’ꢀDimethylꢀ5’ꢀ[(triethylsilyl)oxy]ꢀhexyl]ꢀ5ꢀethylꢀ7aꢀmethylꢀ
octahydroꢀindenꢀ4ꢀone (27). A solution of the ketone 13 (500 mg, 1.27 mmol) in dry THF (7
mL) was added dropwise to a solution of LDA (2.0 M in THF/heptane/ethylbenzene; 1.27 mL,
2.53 mmol) in dry THF (7 mL) under argon at ꢀ78 °C, followed by addition of DMPU (0.12 mL,
1.01 µmol). The solution was warmed to ꢀ30 °C and stirred at this temperature for 1 h. Then it
was cooled to ꢀ78 °C and ethyl iodide (0.41 mL, 5.09 mmol) was added. The reaction mixture
was allowed to reach 10 °C during 1 h, stirred at this temperature for 5 h and then quenched by
addition of water. The product was extracted with Et2O, the combined organic extracts were
washed with brine, dried (MgSO4) and concentrated in vacuo. The oily residue was purified by
flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the ethylated ketone 27
(354 mg, 66%) as a colorless oil.
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1α,3βꢀBis[(tertꢀbutyldimethylsilyl)oxy]ꢀ9αꢀmethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorꢀ9,10ꢀ
secocholestꢀ5(10)ꢀenꢀ6ꢀynꢀ8βꢀol (28). nꢀButyllithium (1.6 M in hexane; 165 µL, 0.264 mmol)
was added dropwise to a stirred solution of the enyne 15 (88 mg, 0.24 mmol) in anhydrous THF
(1.5 mL) at ꢀ78 ºC. After 5 min, CeCl3 (65 mg, 0.264 mmol) was added and the resulting
suspension was stirred at ꢀ78 ºC for 30 min. A solution of ketone 26 (88 mg, 0.216 mmol) in dry
THF (1.5 mL) was added. The mixture was stirred for 15 min and warmed to room temperature.
The reaction was quenched after 1 h by addition of saturated NH4Cl. The resulting mixture was
extracted with Et2O and the combined organic layers were washed with brine, dried (MgSO4) and
concentrated in vacuo. The residue was purified by flash chromatography on silica gel. Elution
with hexane/Et2O (98:2) gave the enynol 28 (112 mg, 60%) as a colorless oil.
1α,3βꢀBis[(tertꢀbutyldimethylsilyl)oxy]ꢀ9αꢀethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorꢀ9,10ꢀ
secocholestꢀ5(10)ꢀenꢀ6ꢀynꢀ8βꢀol (29). Following the procedure described above for preparation
of 28, the alcohol 29 was obtained by coupling of the ketone 27 with the anion of the enyne 15,
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performed analogously to the process described above for the preparation of 28. The product was
purified by flash chromatography on silica gel. Elution with hexane/Et2O (98:2) gave the enynol
29 (68%) as a colorless oil.
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1α,3βꢀBis[(tertꢀbutyldimethylsilyl)oxy]ꢀ9ꢀmethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorꢀ9,10ꢀ
secocholestaꢀ5(10),8ꢀdienꢀ6ꢀyne (30). A solution of alcohol 28 (112 mg, 0.144 mmol) in dry
toluene
(5
mL)
was
added
dropwise
to
a
solution
of
methyl
Nꢀ
(triethylammoniumsulfonyl)carbamate (69 mg, 0.289 mmol) in dry toluene (5 mL). The reaction
mixture was heated at 50 ºC under stirring for 30 min. The mixture was poured into brine and
extracted with Et2O. The combined organic layers were dried (MgSO4) and evaporated to give a
residue, which was purified by flash chromatography on silica gel. Elution with hexane/Et2O
(98:2) gave the dienyne 30 (134 mg, 95%) as a colorless oil.
1α,3βꢀBis[(tertꢀbutyldimethylsilyl)oxy]ꢀ9ꢀethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorꢀ9,10ꢀ
secocholestaꢀ5(10),8ꢀdienꢀ6ꢀyne (31). Dehydration of the alcohol 29 with methyl Nꢀ
(triethylammoniumsulfonyl)carbamate was performed as described above for the preparation of
30. The product was purified by flash chromatography on silica gel. Elution with hexane/Et2O
(98:2) gave the dienyne 31 (96%) as a colorless oil.
1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ9ꢀmethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorprevitamin D3 tertꢀ
butyldimethylsilyl ether (32). The previtamin 32 was obtained by semihydrogenation of the
dienyne 30, as described above for the conversion of 21 to 22. The product was purified by flash
chromatography on silica gel. Elution with hexane furnished the previtamin 32 (89% yield) as a
colorless oil.
1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ9ꢀethylꢀ25ꢀ[(triethylsilyl)oxy]ꢀ19ꢀnorprevitamin D3 tertꢀ
butyldimethylsilyl ether (33). The previtamin 33 was obtained by hydrogenation of the dienyne
31, performed analogously to the process described above for the conversion of 21 to 22.
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Purification of the product by flash chromatography (hexane) on silica gel gave the previtamin 33
(78% yield) as a colorless oil.
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1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ25ꢀ[(triethylsilyl)oxy]ꢀ9ꢀmethyleneꢀ19ꢀnorvitamin D3 tertꢀ
butyldimethylsilyl ether (34). A solution of the previtamin 32 (119 mg, 0.157 mmol) in
isooctane (50 mL), protected from light, was refluxed for 6 h and then concentrated. The
resulting product was purified by flash chromatography on silica gel. Elution with hexane
furnished the protected vitamin 34 (119 mg, 100%) as a colorless oil.
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21
22
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(9E)ꢀ1αꢀ[(tertꢀButyldimethylsilyl)oxy]ꢀ25ꢀ[(triethylsilyl)oxy]ꢀ9ꢀethylideneꢀ19ꢀnorvitamin D3
tertꢀbutyldimethylsilyl ether (35). A solution of the previtamin 33 (99 mg, 0.128 mmol) in
isooctane (40 mL), protected from light, was refluxed for 7 h and then concentrated. The
resulting product was purified by flash chromatography (hexane) on silica gel to give the
protected vitamin 35 (99 mg, 100%) as a colorless oil.
1α,25ꢀDihydroxyꢀ9ꢀmethyleneꢀ19ꢀnorvitamin D3 (6). The silyl protecting groups in the vitamin
D compound 34 were removed by treatment with tetrabutylammonium fluoride, following the
same experimental procedure as described above for vitamin 23. HPLC purification afforded the
vitamin D analogue 6 in 89% yield.
(9E)ꢀ1α,25ꢀDihydroxyꢀ9ꢀethylideneꢀ19ꢀnorvitamin D3 (9). Removal of silyl protecting groups
in 35 by treatment with TBAF, following the same procedure as for the diprotected vitamin D
compound 23, afforded a product that was purified by HPLC [(10 mm × 25 cm Phenomenex
Luna Silica column, 4 mL/min, hexane/2ꢀpropanol (8:2)]. The vitamin D compound 9 was
collected at Rv 54 mL. Final purification of the product was achieved by reversedꢀphase HPLC
[(9.4 mm × 25 cm Eclipse XDBꢀC18 column, 4 mL/min, methanol/water (85:15)]. 9ꢀEthylideneꢀ
19ꢀnorcalcitriol (9) was collected at Rv 54 mL (90%).
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1α,25ꢀDihydroxyꢀ9βꢀmethylꢀ19ꢀnorvitamin D3 (4). Tris(triphenylphosphine)ꢀrhodium(I)
chloride (31 mg, 0.034 mmol) was added to dry benzene (freshly distilled from CaH2; 20 mL)
presaturated with hydrogen. The mixture was stirred under H2 at room temperature until
homogeneous solution was formed (ca. 30 min). A solution of the vitamin compound 6 (14 mg,
0.034 mmol) in benzene (5 mL) was then added and the reaction mixture was allowed to proceed
under a continuous stream of H2 for 3 h. Benzene was removed under vacuum and the residue
was purified by HPLC [(10 mm × 25 cm Phenomenex Luna Silica column, 4 mL/min, hexane/2ꢀ
propanol (8:2)]. The vitamin D compound 4 was collected at Rv 36 mL. Final purification of the
product was achieved by reversedꢀphase HPLC [(9.4 mm × 25 cm Eclipse XDBꢀC18 column, 4
mL/min, methanol/water (85:15)]. 9βꢀMethylꢀ19ꢀnorcalcitriol (4) was collected at Rv 49 mL (8
mg, 56%).
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Biological Studies
1. In vitro Studies.
Measurement of VDR Binding Activity. Measurement of binding ability of the
synthesized vitamin D analogues to the VDR was carried out using purified fullꢀlength rat
recombinant receptor.29 The protein was diluted in TEDK50 (50 mM Tris, 1.5 mM EDTA, pH 7.4,
5 mM DTT, 150 mM KCl) with 0.1% Chaps detergent. The radiolabeled ligand [3Hꢀ1α,25ꢀ
(OH)2D3, ~ 159 Ci/mmol] was added in ethanol at a final concentration of 1 nM. The
radiolabeled and unlabeled ligands were added to 100 ꢁL of the diluted protein at a final ethanol
concentration of ≤ 10%, then mixed and incubated overnight on ice to reach binding equilibrium.
The following day, 100 ꢁL of hydroxylapatite slurry (50%) was added to each tube and mixed at
10ꢀmin intervals for 30 min. The hydroxylapatite was collected by centrifugation and then
washed three times with Tris–EDTA buffer (50 mM Tris, 1.5 mM EDTA, pH 7.4) containing
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0.5% Triton Xꢀ100. After the final wash, the pellets were transferred to scintillation vials
containing 4 mL of Biosafe II scintillation cocktail, mixed and placed in a scintillation counter.
Total binding was determined from the tubes containing only the radiolabeled ligand. The
experiments were carried out in duplicate on two to three different occasions. The Ki values are
derived from doseꢀresponse curves and represent the inhibition constant when radiolabeled
1α,25ꢀ(OH)2D3 is present at 1 nM and a Kd of 0.2 nM is used.
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Measurement of Cellular Differentiation. Human promyelocytic leukemia (HLꢀ60)
cells were grown in RPMIꢀ1640 medium containing 10% fetal bovine serum at 37 °C in the
presence of 5% CO2. HLꢀ60 cells were plated at 1.2 × 105 cells/plate. Eighteen hours after
plating, cells in duplicate were treated with the tested compound so that the final concentration of
ethanol was less than 0.2%. Four days later, the cells were harvested and a nitro blue tetrazolium
(NBT) reduction assay was performed. The percentage of differentiated cells was determined by
counting a total of 200 cells and recording the number that contained intracellular blackꢀblue
formazan deposits.30 The ED50 values are derived from the doseꢀresponse curves and represent
the tested compound concentration capable of inducing 50% maturation. The experiment was
repeated 2 to 3 times, and the results are reported as the mean. Verification of differentiation to
monocytic cells was determined by measuring phagocytic activity (data not shown).
Measurement of Transcriptional Activity. The ability of the vitamin D compounds to
induce transcription in rat osteosarcoma cells was established using 24ꢀhydroxylase (CYPꢀ24)
luciferase reporter gene system. Transcriptional activity was measured in ROS 17/2.8 (bone) cells
that were stably transfected with a 24ꢀhydroxylase (24OHase) gene promoter upstream of a
luciferase reporter gene.31 Cells were subjected to a range of doses. Sixteen hours after dosing,
the cells were harvested and luciferase activities were measured using a luminometer. The ED50
values are derived from doseꢀresponse curves and represent the analogue concentration capable
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of increasing the luciferase activity by 50%. Each experiment was performed in duplicate two to
three separate times.
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2. In vivo Studies.
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Bone calcium mobilization and intestinal calcium transport. Male, weanling Spragueꢀ
Dawley rats were purchased from Harlan (Indianapolis, IN). The animals were group housed and
placed on Diet 11 (0.47% Ca) + AEK oil for one week followed by Diet 11 (0.02% Ca) + AEK
oil for 3 weeks. The rats were then switched to a diet containing 0.47% Ca3221 for one week,
followed by two weeks on a diet containing 0.02% Ca. Dose administration began during the last
week on 0.02% Ca diet. Four consecutive intraperitoneal doses were given approximately 24
hours apart. Twenty four hours after the last dose, blood was collected from the severed neck and
the concentration of serum calcium determined as a measure of bone calcium mobilization. The
first 10 cm of the intestine was also collected for the intestinal calcium transport analysis using
the everted gut sac method.29
All animals were managed in accordance with University of Wisconsin standards and protocols
for animal care and use. Our experiments were approved by the College of Agricultural and Life
Sciences Institutional Animal Care and Use Committee.
Molecular Modeling. The synthesized analogues 4, 5 and 6 were docked into vitamin D
receptor using Gold (release 5.1) software package. The Builder module, available in the
InsightII suite of programs,33 allowed us to construct new analogues by modifying the structure
of 1α,25ꢀ(OH)2D3 extracted from crystalline VDR(LBD)ꢀ1 complex (PDB Code: 1DB1). The
new structures were energyꢀminimized (cvff potentials) with the Discover module in InsightII. In
the next step, the minimized structures of the new ligands 4, 5 and 6 (in all cases with their Aꢀ
ring assuming the βꢀconformation with equatorially oriented 1αꢀhydroxyl groups) were docked
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into the VDR(LBP). For final consideration, only the lowest energy complexes were selected,
possessing tryptophan oriented parallel to the ligand’s 5,7ꢀdiene moiety.
Crystallographic studies
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Crystal data for 9βꢀmethylꢀ19ꢀnorcalcitriol (4). C27H46O3, M = 417.63, T = 100 K,
orthorhombic, space group P212121, Z = 4, a = 6.5288(8), b = 19.093(2), c = 19.969(2) Å, αβγ =
2
90°, V = 2489.2(5) Å3, Dx = 1.114 g·cmꢀ3, 4381 unique data (2θmax = 25.000), 2429 with F0 >
2
2σ (F0 ), R = 0.0935, Rw = 0.2518, S = 1.049.
Structure determination. The structure of the vitamin 4 was determined in a singleꢀ
crystal Xꢀray diffraction measurement on a Kuma KM4CCD κꢀaxis diffractometer with graphiteꢀ
monochromated Mo Kα radiation (λ = 0.71073 A, 50.0 kV, 40.0 mA) at 100 K, using the Oxford
Cryostream cooling device. The 1369 frames were measured with scan width of 1.00o and
counting time of 60 s. Indexing, integration and scaling were performed with original KUMA
Diffraction software.34 The 11866 reflections were measured and merged to 8607 unique
reflections, of which 4226 were rejected during the refinement. The analytical absorption
correction was applied in the scaling procedure. The structure solution and refinement were
performed using merged reflections.
The structure was solved by direct approach35 using the SHELXSꢀ97 program and refined
with the SHELXLꢀ97.36 The refinement was based on F2 for all reflections except those with
negative intensities. Weighted R factors wR and all goodnessꢀofꢀfit S values were based on F2,
whereas conventional R factors were based on the amplitudes, with F set to zero for negative F2.
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The F0 > 2σ(F0 ) criterion was applied only for R factors calculation and was not relevant to the
choice of reflections for the refinement. The R factor based on F2 is about twice as large as the
one based on F. The hydrogen atoms were located in idealized geometrical positions. Scattering
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factors were taken from Tables 4.2.6.8 and 6.1.1.4 from the International Tables for
Crystallography.37
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Crystallographic data for the structure reported in this paper have been deposited at the
Cambridge Crystallographic Data Centre with the deposition number CCDCꢀ1413876.
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ASSOCIATED CONTENT
Supporting Information Available: Purity criteria of the newly synthesized vitamin D
1
analogues 4, 6 and 9, their H and 13C NMR spectra, and spectral data of all the synthesized
compounds, figures with either competitive binding curves or doseꢀresponse curves derived from
cellular differentiation and transcriptional assays of the vitamin D analogue 4. CCDCꢀ1413876
contains the supplementary crystallographic data for this article. These data can be obtained free
of
charge
from
the
Cambridge
Crystallographic
Data
Centre
via
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www.ccdc.cam.ac.uk/data_request/cif. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Prof. Antonio Mouriño. Telephone: +34 981 563100 ext. 14254. Fax: +34 983 595012. Eꢀmail:
antonio.mourino@usc.es
*Prof. Rafal R. Sicinski. Telephone: +48 22 8220211 ext. 216. Fax: +48 22 8225996. Eꢀmail:
rasici@chem.uw.edu.pl
.
.
ACKNOWLEDGMENTS
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U.K. thanks the Foundation for Polish Science; the MPD Program has been coꢀfinanced by the
EU European Regional Development Fund. H.F.D and L.A.P. thank the Wisconsin Alumni
Research Foundation for financial support. A.M. thanks the Spanish Ministry of Education and
Innovation (SAF2010ꢀ15291) and Xunta de Galicia (project GPC2014/001) for financial support.
The authors gratefully acknowledge William Blaser and Erin Gudmundson from University of
WisconsinꢀMadison (USA) for their excellent technical assistance.
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ABBREVIATION USED
1α,25ꢀ(OH)2D3, 1α,25ꢀdihydroxyvitamin D3; VDR, vitamin D receptor.
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REFERENCES
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system by thermal sigmatropic rearrangements. J. Org. Chem. 2013, 78, 1444ꢀ1450.
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from vitamin D3. Bioorg. Med. Chem. Lett. 1995, 5, 159ꢀ162.
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Sarandeses, L. A.; Mascareñas, J. L.; Castedo, L.; Mouriño, A. Synthesis of 1,25ꢀdihydroxyꢀ
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(a) Castedo, L.; Mascareñas, J. L.; Mouriño, A. An improved synthesis of 1,25ꢀ
dihydroxyvitamin D synthons. Tetrahedron Lett. 1987, 28, 2099ꢀ2102. (b) Aurrecoechea, J. M.;
Okamura, W. H. A short, enantiospecific synthesis of the 1αꢀhydroxyvitamin D enyne Aꢀring
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analogues: stereoselective preparation of 6βꢀvitamin D vinylallenes and a concise enynol
synthesis for preparing the Aꢀring. J. Org. Chem. 1989, 54, 4072ꢀ4083.
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Part of this work has previously been communicated: Kulesza U.; Mouriño A.; Plum L. A.;
DeLuca H. F.; Sicinski R. R. Synthesis of novel 19ꢀnorvitamin D3 analogs with unnatural triene
system. J. Steroid Biochem. Mol. Biol. 2013, 136, 23ꢀ26.
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(a) Ohꢀe, T.; Miyaura, N.; Suzuki, A. Palladiumꢀcatalyzed crossꢀcoupling reaction of aryl or
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Ohmura, T.; Yamamoto, Y.; Miyaura, N. Rhodiumꢀ or iridiumꢀcatalyzed transꢀhydroboration
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of terminal alkynes, giving (Z)ꢀ1ꢀalkenylboron compounds. J. Am. Chem. Soc. 2000, 122, 4990ꢀ
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4991.
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Gogoi, P.; Sigüeiro, R.; Eduardo, S.; Mouriño, A. An expeditious route to 1α,25ꢀ
dihydroxyvitamin D3 and its analogues by an aqueous tandem palladiumꢀcatalyzed Aꢀring closure
and Suzuki coupling to the C/D unit. Chem. Eur. J. 2010, 16, 1432ꢀ1435.
22 Sicinski, R. R. Photochemical and thermal isomerizations of Cꢀ19 functionalized previtamin D
analogs in the androstane series. Acta Chim. Hung. 1992, 129, 191ꢀ200.
23
Huitric, A. C.; Carr, J. B.; Trager, W. F.; Nist, B. J. Configurational and conformational
analysis: axialꢀaxial and axialꢀequatorial coupling constants in sixꢀmembered ring compounds.
Tetrahedron 1963, 19, 2145ꢀ2151.
24
(a) Wing, R. M.; Okamura, W. H.; Sine, S. M.; Norman, A. W. Vitamin D in solution:
conformations of vitamin D3, 1α,25ꢀdihydroxyvitamin D3, and dihydrotachysterol D3. Science
1974, 186, 939ꢀ941. (b) Bouillon, R.; Okamura, W. H.; Norman, A. W. Structureꢀfunction
relationship in the vitamin D endocrine system. Endocr. Rev. 1995, 16, 200ꢀ257.
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In vitro studies (VDR binding, HLꢀ60 differentiation and transcriptional assay) as well as
measurement of intestinal calcium transport and bone calcium mobilization were performed as
previously described: Glebocka, A.; Sicinski, R.R.; Plum, L.A.; ClagettꢀDame, M.; DeLuca, H.F.
J. Med. Chem. 2006, 49, 2909ꢀ2920.
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Yamada, S.; Yamamoto, K. Ligand recognition by vitamin D receptor: Total alanine scanning
mutational analysis of the residues lining the ligand binding pocket of vitamin D receptor. Curr.
Top. Med. Chem. 2006, 6, 1255ꢀ1265.
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