Synthesis and biological evaluation of 1α,25-dihydroxyvitamin D 3 analogues with a long side chain at C12 and short C17 side chains

Article abstract of DOI:10.1021/jm3008272

Structure-guided optimization was used to design new analogues of 1α,25-dihydroxyvitamin D₃ bearing the main side chain at C12 and a shorter second hydroxylated chain at C17. The new compounds 5a-c were efficiently synthesized from ketone 9 (wh

Full text of DOI:10.1021/jm3008272

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of 1α,25-Dihydroxyvitamin D3
Analogues with a Long Side Chain at C12 and Short C17 Side Chains†
Diego M. Carballa,‡ Samuel Seoane,§ Flavia Zacconi,‡,# Xenxo Perez,‡ Antonio Rumbo,‡
́
Silvia Alvarez-Díaz,∥,▽ María Jesus Larriba,∥ Roman Perez-Fernandez,§ Alberto Munoz,∥
́
́
́
́
̃
Miguel Maestro,⊥ Antonio Mourino,‡ and Mercedes Torneiro*,‡
̃
‡Departamento de Química Organ
Compostela, Spain
́
ica y Unidad Asociada al CSIC, Universidad de Santiago de Compostela, 15782 Santiago de
§Departamento de FisiologíaCentro de Investigacion
́ ́
en Medicina Molecular y Enfermedades Cronicas (CIMUS), Universidad de
Santiago de Compostela, 15782 Santiago de Compostela, Spain
∥Instituto de Investigaciones Biomed
⊥Departamento de Química Fundamental, Universidad de A Coruna, 15071 A Coruna, Spain.
́
icas “Alberto Sols”, CSICUniversidad Auton
́
oma de Madrid, 28029 Madrid, Spain
̃
̃
S
* Supporting Information
ABSTRACT: Structure-guided optimization was used to design
new analogues of 1α,25-dihydroxyvitamin D3 bearing the main
side chain at C12 and a shorter second hydroxylated chain at C17.
The new compounds 5a−c were efficiently synthesized from
ketone 9 (which is readily accessible from the Inhoffen−Lythgoe
diol) with overall yields of 15%, 6%, and 3% for 5a, 5b, and 5c,
respectively. The triene system was introduced by the Pd-
catalyzed tandem cyclization−Suzuki coupling method. The new
analogues were assayed against human colon and breast cancer
cell lines and in mice. All new vitamin D3 analogues bound less
strongly to the VDR than 1α,25-dihydroxyvitamin D3 but had similar antiproliferative, pro-differentiating, and transcriptional
activity as the native hormone. In vivo, the three analogues had markedly low calcemic effects.
activation of gene transcription.1b,7 In addition, 1α,25-(OH)2D3
INTRODUCTION
■
exerts rapid, transcription-independent (nongenomic) regula-
tory actions on ion channels, kinases, phosphatases, and
phospholipases that are mediated by VDR and perhaps other
still not well-characterized receptors.8 Recent findings show
that 1,25D mediates nongenomic effects via binding to the
alternative ligand-binding pocket, a ligand binding site of the
VDR overlapping the well-characterized genomic pocket.9
A key for understanding the molecular mode of action of the
hormone and hence for rational design of analogues was the
elucidation by Moras et al. in 2000 of the crystal structure of
the ligand binding domain (LBD) of the human VDR forming
a complex with its natural ligand 1,25D.10 Since then, around
50 structures of agonist or superagonist calcitriol analogues
complexed with human, rat, or zebrafish VDR LBDs have been
determined.11
We recently designed and synthesized new active analogues
of 1α,25-(OH)2D3 on the basis of simulation studies of their
docking in the human VDR(LBD).12 In silico, all these new
analogues bind significantly to the Moras VDR(LBD).13
Among these vitamin D analogues exhibiting interesting
biological profile are compounds 2a−c,14 which have
The active form of vitamin D3, the hormone 1α,25-
dihydroxyvitamin D3 (1α,25-(OH)2D3, 1,25D, 1, also known
as calcitriol, Figure 1) participates in numerous biological
processes. In addition to its classical role in mineral
homeostasis and bone mineralization, this hormone promotes
cell differentiation, inhibits proliferation and is involved in the
regulation of the immune system, among other activities.1
Because of these “nonclassical” actions, calcitriol and its
analogues have attracted considerable interest as potential
drugs for the treatment of hyperproliferative diseases and
immune disorders.2 Nevertheless, their clinical application is
severely hampered by their side effects: potent hypercalcemia
and increased bone resorption.3 The challenge to medicinal
chemists is to develop analogues with selective properties.4
Calcitriol exerts most of its biological functions by regulating
the transcription of target genes through interaction with a
specific nuclear receptor, the vitamin D receptor (VDR).5 The
VDR is a transcription factor that binds to the gene promoter
region as a heterodimer with the retinoid X receptor (RXR).
Binding to calcitriol or other agonist ligands induces a
conformational change of the VDR, in which helix 12 closes
the ligand-binding pocket by a mouse-trap-like mechanism.6
This conformational shift promotes the release of corepressor
proteins and the recruitment of coactivator proteins, leading to
Received: June 12, 2012
© XXXX American Chemical Society
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Figure 1. Structure of 1α,25-dihydroxyvitamin D3 and analogues 2−5.
Figure 2. Predicted structures of the complexes of analogues 5a (left), 5b (center), and 5c (right) with the Moras VDR(LBD). Ligand conformation
and interactions with Leu309 and with hydrogen-bond forming residues are shown. Distances are measured in angstroms. Nitrogens are depicted in
blue, oxygens in red, and carbons in green (analogues 5), yellow (Leu309), or gray (hydrogen bond forming residues). Hydrogens are omitted.
substituents at C12 and compounds 3a−c12c in which the side
chain has been moved to C12 (Figure 1). In particular, the
VDR binding affinity of 2c is 4.4 times higher than that of
1,25D, and the affinities of analogues 3a−c for the VDR are
60−71% as compared with the natural hormone 1,25D (100%),
analogue 3b being the most active in transactivation studies
(20% compared with 1,25D). We have accordingly used 3b as
the starting point for the development of new analogues in this
series with improved biological potency. Inspired by the
interesting biological profile of Gemini, a 1,25D analogue with
two hydroxylated side chains (4, Figure 1),15 and related
compounds,16 we developed the new analogues 5a−c (Figure
1), which like 3b bear a 7C-long side chain at C12 and also
have a second shorter hydroxylated chain at C17. We describe
here their design on the basis of docking studies, their
syntheses, and their biological evaluation.
the 3b side chain with the protein are similar to those of 1,25D
except with Leu309 and decreased interactions with His305 and
His397.12c The observation of a free space around position
C17, originated by the removal of the natural side chain in 3b,
led us to envisage that the agonist conformation of the ligand−
VDR complex might receive additional stabilization from the
attachment of a second small side chain at C17. Structural
studies of several superagonist analogues of 1,25D in complex
with the VDR have shown their superagonism to be due to
additional stabilization of helix H12 and stronger interactions
with transcription coactivators.17,18 Accordingly, we modified
3b as analogues 5a−c, in which C17 bears the Inhoffen−
Lythgoe diol side chain fragment or a higher homologue. In
contrast to 3b, the three new analogues have a 21-methyl group
as 1,25D for interaction with Leu309, which plays a key role in
ligand-mediated protein folding and therefore in the main-
tenance of the active conformation of the VDR,19 and they each
also hold an extra hydroxyl group for additional polar contacts
with the receptor. Docking calculations show that analogues
5a−c form stronger complexes with Moras VDR(LBD) than
the natural ligand 1,25D in the order: 5b > 5c > 5a. In all the
complexes (Figure 2), the hydrophobic interaction of the 21-
methyl group with the side chain of the Leu309 amino acid
residue is restored, the ring A hydroxyl groups form hydrogen
bonds with the same amino acid residues as the natural
ligand,10 and His305 is within hydrogen bonding distance of
RESULTS AND DISCUSSION
■
Design. The new vitamin D3 analogues 5a−c were designed
on the basis of computational docking studies using the LBD
derived from the X-ray crystal structure of Moras VDR(LBD)-
1,25D complex and the biological activity of analogues 3. In
previous docking studies, we found that the rings and triene
system of analogue 3b adopt the same conformation as the
natural hormone 1,25D in the binding pocket. Interactions of
B
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the hydroxyl group of the longer side chain. Only in the
complex with 5b, the His397 forms a hydrogen bond with the
hydroxyl group of the longer C12 side chain and it also forms a
hydrogen bond with the hydroxyl group of the shorter (C17)
side chain. Thus 5b interacts best in terms of both energy and
the number of hydrogen bonds with the receptor. It is worth
noting that this theoretical hydrogen bonding pattern of
analogue 5b is similar to that observed in the crystallographic
structures of Gemini20 and the superagonist Gemini7217e
analogues, in complex with a wild-type zebrafish VDR(LBD).
We have previously found that 1,25D analogues that bind
significantly in silico are also biologically active.12 The
promising docking results for 5a−c accordingly encouraged
us to pursue their synthesis to further study the structure−
activity relationships of vitamin D analogues.
alcohol 17, which was converted to ketone 19 by catalytic
hydrogenation and pyridinium dichromate oxidation (82% over
the three steps). Ketone 19 was transformed into the
enoltriflate 21 in 84% yield by deprotonation with LDA and
trapping of the resulting enolate with Comins triflimide (20).
The desired triol 8a was finally prepared in 82% yield by a
three-step sequence: palladium-catalyzed coupling of 21 with
alkyne 22,14 catalytic hydrogenation of the resulting enyne 23,
and deprotection of 24 (13 steps from 9, 44% overall yield).
The structure and stereochemistry of triol 8a was confirmed by
X-ray crystallography (see Supporting Information).
With the parent triol 8a at hand, we proceeded to the
preparation of triols 8b and 8c, precursors of analogues 5b and
5c, respectively. Elongation of the residual side chain at C17 of
8a (Scheme 3) to 8b was accomplished in 46% yield by a three-
step sequence: selective tosylation of the primary hydroxyl
group, SN2 displacement of the tosylate 25a with potassium
cyanide, and reduction of the nitrile 26a with diisobutylalumi-
num hydride. A second homologation following the same
procedure provided triol 8c in 46% yield via tosylate 25b and
nitrile 26b.24
Synthesis. Our strategy for the synthesis of analogues 5a−c
was based on the convergent method recently developed in our
group (Scheme 1).21 In this approach, the triene system is
Scheme 1. Retrosynthetic Analysis of Analogues 5a, 5b, and
5c
Completion of the synthesis of analogues 5a−c is illustrated
in Scheme 4. Selective silylation of the primary hydroxyl group
of triols 8a−c followed by oxidation of the secondary hydroxyl
group afforded ketones 28a−c, which were converted to the
desired alkenyl bromides 29a−c by Wittig reaction with ylide
Ph3PCHBr.25 The upper boronates 6a−c were prepared by
Miyaura borylation26 of 29a−c with bis(pinacolato)diboron in
the presence of [1,1′-bis(diphenylphosphino)ferrocene]-
dicloro-palladium(II)−dichloromethane complex as catalyst
and tricyclohexylphosphine as ligand. Finally, the triene system
was installed by treatment of 6a−c with an equimolar amount
of enol triflate 721 in the presence of catalytic PdCl2(PPh3)2 and
K3PO4 in H2O/THF. Removal of the protecting groups gave
the desired analogues 5a−c in good yields (overall yields from
8a−c: 34% for 5a, 27% for 5b, and 29% for 5c).
Biological Assays. The biological activity of analogues 5a,
5b, and 5c was first evaluated in intact human SW480-ADH
colon cancer cells. All three compounds have a prodifferentiat-
ing action on these cells comparable to that of 1,25D, inducing
at 10−7 M the formation of compact epithelioid islands of
highly adherent cells (Figure 3). This effect was partially
evident at 10−8 M in the case of 1,25D but not of the three
analogues. Accordingly, the three compounds induced the
expression of E-cadherin, the key intercellular adhesion protein
in epithelial cells that behaves as an invasion suppressor,
similarly to 1,25D at 10−7 M although with less potency at 10−9
M (Figure 4A).27 As phosphatidylinositol-5-phosphate 4-kinase
type II β (PIP4K2B)-mediated PI(4,5)P2 signaling has been
reported to be important to E-cadherin induction by 1,25D in
colon cancer cells,28 we studied the expression of this enzyme.
However, the cellular level of PIP4K2B RNA did not change
following treatment with 1,25D or analogues 5a−c in SW480-
ADH cells as measured by quantitative RT-PCR (Supporting
Information Figure 2S).
efficiently constructed by a stereoselective Pd-catalyzed intra-
molecular cyclization of an enol triflate (A-ring precursor 7)
followed by a Suzuki−Miyaura coupling of the resulting Pd(II)-
intermediate with an alkenyl boronic ester (CD−side chain
fragments 6a−c). Boronates 6 would be accessed by
manipulation of the secondary hydroxyl group of triols 8a−c.
Triol 8a, precursor of the elongated triols 8b−c, would be
prepared from the Inhoffen−Lythgoe diol (10) employing
methodology developed in our laboratories.12c,14,22
The key triol intermediate 8a was prepared from the
Inhoffen−Lythgoe diol (10) through the known unsaturated
ketone 923 (Scheme 2). Reduction of ketone 9 with DIBAL-H
followed by protection of the resulting allylic alcohol 11 with
tert-butyldimethylsilyl chloride provided the silyl ether 12,
which upon epoxidation from the less hindered face with m-
chloroperbenzoic acid gave epoxide 13 (82% over the three
steps). Opening of epoxide 13 with lithium diethylamide
followed by hydroxyl-directed epoxidation of the resulting
allylic alcohol 14 furnished the epoxide 15, which was
converted to mesylate 16 (91% over the three steps).
Treatment of 16 with sodium naphthalene provided allylic
As for E-cadherin, the three analogues increased the level of
the tumor suppressor protein Cystatin D29 with slightly less
potency than 1α,25-(OH)2D3 at low concentrations (Figure
4A). We also studied the effect of the analogues 5a−c on the
expression of c-MYC oncogene, a strong inducer of cell
proliferation that is repressed by 1,25D by direct and indirect
mechanisms in several cell types.27,30 Again, all three
C
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a
Scheme 2. Synthesis of Triol 8a
a
Reagents and conditions: (a) DIBAL-H, THF, −78 °C; (b) TBSCl, Im, DMF; (c) m-CPBA, CH2Cl2, 0 °C; (d) LiNEt2, HMPA, Et2O; (e) m-
CPBA, CH2Cl2, 0 °C; (f) MsCl, Et3N, CH2Cl2, −20 °C; (g) Na/naphthalene, THF, 0 °C; (h) H2, 10% Pd−C, EtOAc; (i) PDC, CH2Cl2; (j) LDA,
THF, −78 °C; 20, −78 °C → 0 °C; (k) 22, PdCl2(Ph3P)2, CuI, Et2NH; (l) H2, 10% Pd−C, EtOAc; (m) TBAF, THF, Δ. SiTBSSi(t-Bu)Me2.
a
a
Scheme 3. Synthesis of Triols 8b and 8c
Scheme 4. Synthesis of Analogues 5a−c
a
Reagents and conditions: (a) TsCl, Py, 4 °C; (b) KCN, DMSO, 90
°C; (c) DIBAL-H, CH2Cl2, −5 °C, HCl 3 M/Et2O, DIBAL-H, THF,
−78 °C.
compounds showed comparable gene regulatory activity than
the natural hormone (Figure 4A).
a
Reagents and conditions: (a) TBSCl, Im, DMF; (b) PDC, CH2Cl2;
(c) (Ph3PCH2Br)Br, KOt-Bu, toluene, ultrasounds, −17 → 0 °C; then
28; (d) PdCl2(dppf)·CH2Cl2, PCy3, Pin2B2, KOAc, DMSO, 80 °C; (e)
7, PdCl2(PPh3)2, K3PO4, THF, H2O; (f) TBAF, THF.
Cell proliferation was evaluated in the MCF-7 breast cancer
cells using the MTT assay. Compound 5a at 10−7
M
significantly (P < 0.001) decreased cell proliferation, similarly
to 1,25D, with respect to untreated cells. Compounds 5b and
5c were also able to statistically (P < 0.05) decrease cell
proliferation in relation to control cells (Figure 4B).
Together, the data of the biological evaluation of the
analogues show that 5a−c share the mechanism of action of
1α,25-(OH)2D3 in human cancer cells, albeit with slightly less
potency at low concentrations. The upregulation of E-cadherin
and Cystatin D expression and the prodifferentiation and
antiproliferative effects indicate a protective action against
cancer.
natural hormone 1. The VDR binding affinity of compounds
5a−c is approximately 10 times less than that of the 1,25D
(Figure 5A).
The ability of the analogues to induce transcriptional
activation of a vitamin D target gene was evaluated by
transfecting MCF-7 cells with the pCYP24A1-Luc vector. The
compounds 5a−c and the natural hormone 1 induced a strong
dose-dependent activation of the VDRE, as measured by
luciferase activity. However, all analogues were less potent to
activate the reporter gene transcription than 1,25D (Figure 5B).
We next tested by competitive binding assay31 the biological
ability of the analogues 5a−c to bind VDR, as compared to the
D
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analogues 5a, 5b, and 5c showed reduced VDR binding
compared to hormone 1, in vitro biological assays demon-
strated that all compounds have pro-differentiating, antiproli-
ferative, and transcriptional actions comparable to that of
1α,25-(OH)2D3. Interestingly, the three analogues showed
markedly lower calcemic effects in vivo than the natural
hormone 1. The antiproliferative properties of compounds 5a−
c coupled to its very low calcemic effects make these new
vitamin D analogues of potential clinical interest.
EXPERIMENTAL SECTION
■
Docking Procedure for Structure-Guided Design. The
docking process for the ligands 5a−c was similar to the one previously
described for other vitamin D analogues.12f The binding affinity was
determined by the difference in energy between the complex and its
components (protein and ligand) for the top 200 poses of every
ligand. The calculated values (kcal/mol) of the binding energy
promediated for the 200 better-bound conformations of each ligand
were −93.2 for the natural hormone 1, −96.7 for analogue 5a, −105.6
for analogue 5b, and −104.2 for analogue 5c.
Chemistry. General Methods and Materials. All reactions
involving oxygen or moisture sensitive compounds were carried out
under argon (L-50) atmosphere. Reaction temperatures refer to
external bath temperatures. All solvents were distilled under argon
immediately prior to use. THF and Et2O were distilled from Na/
benzophenone, toluene was distilled from Na, CH2Cl2 was distilled
from P2O5, and pyridine, Et3N, Et2NH, and i-Pr2NH were distilled
from CaH2. DMF was stored over activated 4 Å molecular sieves.
DMSO and HMPA were distilled from CaH2 and stored over activated
4 Å molecular sieves. Acetone−dry ice baths were used for reactions at
low temperature. Alternatively, acetone baths were cooled with a
CRYOCOOL immersion cooler, provided with a temperature
regulator. Sonications were carried out in a 120−240 W, 35 kHz
ultrasonic cleaning bath. Organic extracts were dried over anhydrous
Na2SO4, filtered, and concentrated using rotary evaporator at aspirator
pressure (20−30 mmHg). Reactions were monitored by thin-layer
chromatography (TLC) using aluminum-backed MERCK 60 silica gel
plates (0.2 mm thickness). After visualization under ultraviolet light at
254 nm, the plates were developed by immersion in a solution
containing either a mixture of p-anisaldehyde (2.5%), acetic acid (1%),
and sulfuric acid (3.4%) in 95% ethanol or a solution of ceric
ammonium nitrate (0.5 g) and ammonium molybdate (4.8 g) in H2O
(100 mL) and H2SO4 (5.6 mL) followed by heating with a hot gun.
Flash column chromatography was performed with Merk silica gel
(230−400 mesh). NMR spectra were recorded in CDCl3 or methanol-
d4 solutions on a Bruker AMX 500 MHz, Varian Inova 400 MHz and
Bruker DPX 250 MHz. Chemical shifts are reported on the δ scale
(ppm) downfield from tetramethylsilane (δ = 0.0 ppm) using the
residual solvent signal at δ = 7.26 ppm (1H, CDCl3), δ = 3.31 ppm
(1H, q, methanol-d4), δ = 77.0 ppm (13C, t, CDCl3), or δ = 49.0 ppm
(13C, hp, methanol-d4) as internal standard; coupling constants are
reported in Hz. Distortionless enhancement by polarization transfer
(DEPT) was used to assign carbon types. Melting points (mp) were
Figure 3. Activity of analogues 5a, 5b, and 5c in human colon cancer
cells. Phase-contrast micrographs showing the induction by analogues
5a−c of a differentiated adhesive epithelial phenotype in human
SW480-ADH colon cancer cells. The cells were treated with analogues
5a−c or 1,25D at the indicated concentrations or with vehicle for 48 h.
A representative experiment is shown. Bar, 15 μm.
Calcium serum levels were evaluated in mice injected
intraperitoneally with compounds 5a−c, as well as with the
natural ligand 1 (Figure 6). Our data indicated that
administration of analogues 5a, 5b, and 5c to mice did not
significantly raise calcium levels, as compared with vehicle-
treated mice. However, the natural hormone significantly (P <
0.001) increases calcemia, in comparison with control mice. No
significant changes in body weight were observed in mice after
three weeks of treatment with compounds 1 or 5.
When comparing the docking ranking of the analogues with
the measured VDR binding and transcriptional and calcemic
potencies, a general tendency is not observed (Table 1).
Compound 5b, which shows the best in silico binding, is also
the analogue with the higher in vitro affinity for the VDR, but it
shows the lower transcriptional potency and the highest
calcemic activity. Although the analogues 5a−c are only slightly
less potent than the natural hormone 1 in the transactivation
assays, they showed markedly lower calcemic effects in vivo.
CONCLUSIONS
■
On the basis of the biological profile of 1α,25-(OH)2D3
analogues with susbtituents at C12 (2a−c and 3a−c) and
docking simulations in the binding domain of the nuclear
vitamin D receptor, we have designed three new analogues
(5a−c) of 1α,25-(OH)2D3 bearing the side chain at C12 and a
second short hydroxylated chain of different length at C17 that
replaces the natural side chain. These compounds were readily
synthesized from the Inhofen−Lythgoe diol via the α,β-
unsaturated ketone 9 in 15% (19 steps, 5a), 6% (22 steps,
5b), and 3% (25 steps, 5c) global yields from 9. The triene
system was efficiently introduced by Pd(II)-catalyzed coupling
of the enol-triflate 7 (A-ring fragment) with boronates 6a−c
(CD−side chain fragments). The new analogues 5 are more
potent in terms of biological activity than the parent analogues
3 lacking substitution at C17. Thus, in spite of the fact that
measured in a Buchi apparatus and are uncorrected. Infrared (IR)
̈
spectra were recorded on a Bruker spectrometer, model IFS-66 V FT-
IR. Low (MS) and high resolution mass spectra (HRMS) were
performed in a Micromas Instruments Autospec spectrometer for (CI)
and (FAB) while (ESI-TOF) was performed in Bruker-Microtof
spectrometer. Elementary analysis (EA) were recorded on element
analyzer FISONS, model EA 1108. Optical rotations were measured at
25 °C on a Jasco, model DIP-370. UV spectra were recorded on a HP
spectrophotometer, model 8452A. HPLC purifications were per-
formed on a Shimadzu preparative liquid chromatograph, model LC-
8A, equipped with a UV absorbance detector using a HPLC
Phenomenex-Luna silica column (⌀ 250 mm × 10 mm). Analogues
5a−c have a purity of >95% (HPLC).
Chemicals. CuI was purified following Kauffman’s indications.32
Pyridinium dichromate (PDC) was prepared following Corey’s
E
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Figure 4. Activity of analogues 5a, 5b, and 5c in human colon and breast cancer cells. (A) Regulation of target genes. Western blot analysis of the
induction of E-cadherin and Cystatin D and of the repression of c-MYC proteins at 48 h of treatment of SW480-ADH cells with each compound
(10−9 to 10−7 M). Actin was used as loading control. Numbers correspond to mean values of fold-change obtained in two experiments. (B) Cell
proliferation in human MCF-7 breast adenocarcinoma cells. Cells were treated with vehicle or analogues 5a, 5b, and 5c or 1,25D at concentrations of
10−8, 10−7, or 5 × 10−7 M for 48 h, and then a MTT assay was carried out. Each point represents the average of three individual experiments. Error
bars represent standard deviation (SD). * P < 0.05, ***P < 0.001.
procedure.33 N-(2-Pyridyl)triflimide and (Ph3PCH2Br)Br were pre-
pared as reported.34,35 m-Chloroperbenzoic acid (m-CPBA) was
purified according to Perrin’s indications.36
mmol) in DMF (35 mL). After 48 h, the reaction was quenched by
addition of saturated NaCl (30 mL). The aqueous layer was extracted
with hexanes (3 × 15 mL). The combined organic layer was dried,
filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 3 cm × 8 cm, hexanes) to afford 12 [1.648
g, 3.76 mmol, 96%, Rf = 0.95 (10% EtOAc−hexanes), colorless oil,
22-(tert-Butyldimethylsilyloxy)-de-A,B-23,24-dinorchol-9-en-8β-
ol (11). A solution of diisobutyl-aluminum hydride in CH2Cl2 (9 mL, 1
M, 9 mmol) was added dropwise to a solution of 923 (1.939 g, 6.01
mmol) in THF (30 mL) at −78 °C. The reaction mixture was allowed
to warm to rt. After 30 min, the reaction was quenched by slowly
addition of saturated NaCl at 0 °C. The aqueous layer was extracted
with EtOAc (3 × 25 mL). The combined organic layer was dried,
filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 4 cm × 10 cm, 4% EtOAc−hexanes) to
afford 11 [1.808 g, 5.57 mmol, 93%, Rf = 0.40 (20% EtOAc−hexanes),
colorless oil, [α]D = +101.5 (c = 1.0, CHCl3)]. 1H NMR (CDCl3, 250
MHz): 5.91−5.68 (2H, m, H-9 and H-11), 4.11 (1H, broad s, H-8),
3.58 (1H, dd, J1 = 3.5, J2 = 9.6, H-22), 3.28 (1H, dd, J1 = 7.2, J2 = 9.6,
H-22), 0.95 (3H, d, J = 6.4, H-21), 0.87 (9H, s, Me3C-Si), 0.81 (3H, s,
H-18), 0.01 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz): 129.6 (CH),
128.3 (CH), 67.6 (CH2, C-22), 66.2 (CH, C-8), 52.9 (CH), 50.0
(CH), 41.8 (CH2), 39.8 (C, C-13), 38.4 (CH), 26.8 (CH2), 25.9 (3 ×
CH3, Me3C-Si), 21.2 (CH2), 18.2 (C, C-Si), 16.5 (CH3), 13.4 (CH3),
−5.5 (2 × CH3, Me2Si). IR (film, cm−1): 3380 (νO−H), 3022 (νC−H),
2957 (νC−H), 2929 (νC−H). MS ([ESI-TOF]+, m/z, %): 347 ([M +
Na]+, 100), 307 ([M − OH]+, 6). HRMS: [ESI-TOF]+, calcd for
[C19H36O2SiNa]+, 347.2377; found, 347.2354.
1
[α]D = +143.9 (c = 1.9, CHCl3)]. H NMR (CDCl3, 250 MHz):
5.84−5.64 (2H, m, H-9 and H-11), 4.15 (1H, broad s, H-8), 3.64 (1H,
dd, J1 = 2.9, J2 = 9.4, H-22), 3.35 (1H, dd, J1 = 7.1, J2 = 9.4, H-22), 1.03
(3H, d, J = 6.5, H-21), 0.95 (9H, s, Me3C-Si), 0.94 (9H, s, Me3C-Si),
0.88 (3H, s, H-18), 0.14−0.03 (12H, m, 2 × Me2Si). 13C NMR
(CDCl3, 63 MHz): 129.1 (CH), 128.4 (CH), 67.8 (CH2, C-22), 66.3
(CH, C-8), 53.3 (CH), 50.7 (CH), 42.4 (CH2), 40.2 (C, C-13), 38.7
(CH), 27.2 (CH2), 26.1 (3 × CH3, Me3C-Si), 25.9 (3 × CH3, Me3C-
Si), 22.0 (CH2), 18.4 (C, C-Si), 18.1 (C, C-Si), 16.8 (CH3), 13.6
(CH3), −4.3 (CH3, MeSi), −5.0 (CH3, MeSi), −5.3 (CH3, MeSi),
−5.3 (CH3, MeSi). IR (film, cm−1): 3022 (νC−H), 2957 (νC−H), 2929
(νC−H). MS ([ESI-TOF]+, m/z, %): 461 ([M + Na]+, 4), 381 ([M − t-
Bu]+, 41). HRMS: [ESI-TOF]+, calcd for [C25H50O2Si2Na]+,
461.3242; found, 461.3256.
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinor-9α,11α-
epoxycholane (13). m-Chloroperbenzoic acid (4.05 g, 23.49 mmol)
was added in three portions (each 15 min) to a solution of 12 (3.68 g,
8.39 mmol) in CH2Cl2 (80 mL) at 0 °C. The mixture was stirred in
the dark at 0 °C for 1.5 h and then allowed to warm to rt for 2.5 h. The
reaction was quenched at 0 °C by slow addition of saturated Na2S2O3
(50 mL). The aqueous layer was extracted with CH2Cl2 (3 × 50 mL).
The combined organic layer was washed with saturated NaHCO3 (2 ×
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorchol-9-
ene (12). Imidazole (876 mg, 12.87 mmol) and TBSCl (1.47 g, 9.75
mmol) were successively added to a solution of 11 (1.266 g, 3.90
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s, H-18), 0.09 (3H, s, MeSi), 0.04 (3H, s, MeSi), 0.01 (6H, s, Me2Si).
13C NMR (CDCl3, 125 MHz): 67.5 (CH2, C-22), 67.2 (CH, C-8),
56.3 (CH, C-9), 52.7 (CH), 52.3 (CH, C-11), 44.6 (CH), 40.0 (C, C-
13), 40.0 (CH2, C-12), 38.3 (CH, C-20), 26.9 (CH2), 25.9 (3 × CH3,
Me3C-Si), 25.8 (3 × CH3, Me3C-Si), 21.1 (CH2), 18.3 (C, C-Si), 18.0
(C, C-Si), 16.7 (CH3, C-21), 16.1 (CH3, C-18), −4.7 (CH3, MeSi),
−5.2 (CH3, MeSi), −5.4 (CH3, MeSi), −5.5 (CH3, MeSi). IR (neat,
cm−1): 2956 (νC−H), 2929 (νC−H). MS ([ESI-TOF]+, m/z, %): 477
([M + Na]+, 65), 455 ([M − H2O − TBSOH]+, 14). EA: calculated
for [C25H50O3Si2], C (66.02), H (11.08); found, C (65.94), H (11.37).
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorchol-11-
en-9α-ol (14). A solution of lithium diethylamide was prepared by
dropwise addition of a solution of n-BuLi in hexanes (18.7 mL, 2.25 M,
42.0 mmol) to diethylamine (5.1 mL, 49.0 mmol) at −40 °C. The
white semisolid slurry formed was melted at rt, then cooled to −40 °C
and dissolved in Et2O (22.5 mL). After warm to rt, a solution of 13
(3.18 g, 6.99 mmol) in Et2O (50 mL) and HMPA (8.5 mL, 49.0
mmol) were successively added via cannula. After 15 h, the reaction
was quenched by the slowly addition of a few drops of saturated
NH4Cl and aqueous HCl (100 mL, 2%). The aqueous layer was
extracted with MTBE (3 × 50 mL). The combined organic layer was
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 6 cm × 12 cm, 2% EtOAc−hexanes) to afford
14 [3.06 g, 6.73 mmol, 96%, Rf = 0.33 (10% EtOAc−hexanes), white
solid, mp 95−97 °C (EtOAc−hexanes), [α]D = −4.5 (c = 0.7,
CHCl3)]. 1H NMR (CDCl3, 250 MHz): 6.42 (1H, d, J = 10.1, H-12),
5.56 (1H, dd, J1 = 3.6, J2 = 10.1, H-11), 4.06−3.92 (2H, m, H-8 and H-
9), 3.58 (1H, dd, J1 = 3.2, J2 = 9.6, H-22), 3.28 (1H, dd, J1 = 7.1, J2 =
9.6, H-22), 1.07 (3H, d, J = 6.4, H-21), 0.96 (3H, s, H-18), 0.88 (18H,
overlapped s, 2 × Me3C-Si), 0.08 (3H, s, MeSi), 0.04 (3H, s, MeSi),
0.02 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz): 142.2 (CH), 125.7
(CH), 75.0 (CH), 72.8 (CH), 67.5 (CH2, C-22), 48.1 (2 × CH), 43.2
(C, C-13), 38.9 (CH), 28.1 (CH2), 25.9 (3 × CH3, Me3C-Si), 25.8 (3
× CH3, Me3C-Si), 21.9 (CH2), 18.3 (C, C-Si), 18.0 (C, C−Si), 17.0
(CH3), 16.4 (CH3), −4.9 (CH3, MeSi), −5.2 (CH3, MeSi), −5.4 (2 ×
CH3, Me2Si). IR (neat, cm−1): 3315 (νO−H), 2955 (νC−H), 2929
(νC−H). MS ([ESI-TOF]+, m/z, %): 477 ([M + Na]+, 100), 437 ([M
− OH]+, 48). EA: calcd for [C25H50O3Si2], C (66.02), H (11.08);
found, C (65.59), H (11.23).
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinor-
11α,12α-epoxycholan-9α-ol (15). m-Chloroperbenzoic acid (1.374 g,
7.96 mmol) was added in three portions (each 30 min) to a solution of
14 (1.81 g, 3.98 mmol) in CH2Cl2 (40 mL) at 0 °C. The cooling bath
was removed, and the mixture was stirred in the dark for 6 h. The
reaction was quenched at 0 °C by the slowly addition of saturated
Na2S2O3 (30 mL). The aqueous layer was extracted with CH2Cl2 (3 ×
50 mL). The combined organic layer was washed with saturated
NaHCO3 (2 × 30 mL), saturated NaCl (30 mL), dried, filtered, and
concentrated. The residue was purified by flash chromatography (SiO2,
4 cm × 8 cm, 5% EtOAc−hexanes) to afford 15 [1.799 g, 3.82 mmol,
96%, Rf = 0.53 (10% EtOAc−hexanes), white solid, mp 115−118 °C
(EtOAc−hexanes), [α]D = +7.8 (c = 1.0, CHCl3)]. 1H NMR (CDCl3,
500 MHz): 3.89 (1H, d, J = 4.1, H-9), 3.62−3.58 (2H, m, H-8 and H-
22), 3.53 (1H, d, J = 4.1, H-12), 3.37 (1H, t, J = 4.2, H-11), 3.28 (1H,
dd, J1 = 7.4, J2 = 9.5, H-22), 1.09 (3H, d, J = 6.3, H-21), 1.00 (3H, s,
H-18), 0.89 (9H, s, Me3C-Si), 0.88 (9H, s, Me3C-Si), 0.07 (3H, s,
MeSi), 0.03 (6H, s, Me2Si), 0.02 (3H, s, MeSi). 13C NMR (CDCl3,
125 MHz): 74.1 (CH, C-8), 72.0 (CH, C-9), 67.4 (CH2, C-22), 62.7
(CH, C-12), 54.5 (CH, C-11), 47.4 (CH), 42.0 (C, C-13), 41.4 (CH),
38.3 (CH), 26.3 (CH2), 25.9 (3 × CH3, Me3C-Si), 25.8 (3 × CH3,
Me3C-Si), 21.4 (CH2), 18.3 (C, C-Si), 17.9 (C, C-Si), 17.0 (CH3, C-
21), 13.7 (CH3, C-18), −4.9 (CH3, MeSi), −5.2 (CH3, MeSi), −5.4
(CH3, MeSi), −5.4 (CH3, MeSi). IR (neat, cm−1): 3345 (νO−H), 2956
(νC−H), 2929 (νC−H). MS ([ESI-TOF]+, m/z, %): 493 ([M + Na]+,
100), 453 ([M − OH]+, 7). HRMS: [ESI-TOF]+, calcd for
[C25H50O4Si2Na]+, 493.3140; found, 493.3147.
Figure 5. Vitamin D receptor binding and transactivation activities.
(A) Competitive binding of 1α,25-(OH)2D3 (1) and the synthesized
vitamin D3 analogues 5a−c to the full-length human VDR. The
experiments were carried out in duplicate on two different occasions.
IC50 values are derived from dose−response curves and represent the
measure of 50% inhibition of polarization of a compound. (B) MCF-7
cells were transfected with a 24-hydroxylase gene reporter vector and
then treated with 10−11 to 10−6 M concentrations of 1,25D or
analogues 5a−c for 48 h. The luciferase activity was then measured.
The EC50 values are derived from dose−response curves and represent
the analogue concentration capable of increasing the luciferase activity
by 50%. All of the experiments were carried out in duplicate on at least
two different occasions. Error bars represent standard deviation (SD).
Figure 6. Calcium levels in mice treated with the natural hormone
1,25D and compounds 5a−c. Six mice per group were treated with 0.3
μg/kg of compounds 5, 1,25D, or vehicle every other day during 3
weeks, and calcium levels were measured on day 21. Error bars
represent standard deviation (SD). ***P < 0.001.
30 mL), dried, filtrated, and concentrated. The residue was purified by
flash chromatography (SiO2, 6 cm × 8 cm, hexanes) to afford 13 [3.65
g, 8.02 mmol, 96%, Rf = 0.75 (6% EtOAc−hexanes), white solid, mp
1
47−49 °C (EtOAc−hexanes), [α]D = +42.0 (c = 1.4, CHCl3)]. H
NMR (CDCl3, 500 MHz): 4.20 (1H, broad d, J = 2.2, H-8), 3.54 (1H,
dd, J1 = 3.2, J2 = 9.6, H-22), 3.25 (1H, dd, J1 = 7.1, J2 = 9.6, H-22), 3.16
(1H, dd, J1 = 3.3, J2 = 5.8, H-11), 3.07 (1H, d, J = 3.3, H-9), 2.13 (1H,
dd, J1 = 5.8, J2 = 15.0, H-12), 1.49−1.40 (1H, m, H-20), 0.95 (3H, d, J
= 6.6, H-21), 0.90 (9H, s, Me3C-Si), 0.87 (9H, s, Me3C-Si), 0.83 (3H,
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinor-
11α,12α-epoxycholan-9α-yl methanesulfonate (16). Dry triethyl-
amine (1.138 mL, 8.16 mmol) was added to a solution of 15 (1.923 g,
4.08 mmol) in CH2Cl2 (25 mL) at −20 °C. After 15 min,
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Table 1. VDR Binding Properties, Transcriptional Activities, and Calcemic Effects of the Natural Hormone 1α,25-(OH)2D3 (1),
a
and 5a, 5b, and 5c Vitamin D Analogues
VDR binding
IC50 (M)
transcriptional activity
EC50(M) %
calcemic activity
%
in silico binding energy
kcal/mol
compd
%
1
−93.2
−96.7
−105.6
−104.2
1.24 × 10−9
2.09 × 10−8
2.73 × 10−8
1.57 × 10−8
100
17
3.28 × 10−9
8.19 × 10−9
9.04 × 10−9
5.74 × 10−9
100
40
100
0
5a
5b
5c
22
36
18
1
13
57
a
The in silico binding energy refers to the calculated energy difference between the 1/5−VDR complex and the free protein and ligand 1/5 and is
promediated for the 200 better-bound conformations of the ligand. The VDR binding and transcriptional activity is expressed as percentage activity
at IC50 or EC50, respectively, in comparison with 1α,25-(OH)2D3 (1, 100% activity). Calcemic activity is expressed as percentage of the calcium
increase in mice treated with the different compounds in relation with untreated mice, considering 100% 1α,25-(OH)2D3 treated-mice.
methanesulfonyl chloride (635 μL, 8.16 mmol) was added. The
mixture was stirred for 1 h. The reaction was quenched by the slow
addition of H2O (25 mL). The aqueous layer was extracted with
CH2Cl2 (3 × 30 mL). The combined organic layer was dried, filtered,
and concentrated to give 16 [2.22 g, 4.04 mmol, 99%, Rf = 0.53 (10%
EtOAc−hexanes), white solid, mp 99−101 °C (EtOAc−hexanes),
[α]D = −20.9 (c = 1.1, CHCl3)] that was used in the next reaction
washed with EtOAc (3 × 15 mL), and the resulting filtrate was
concentrated to give 18 [1.237 g, 2.71 mmol, 98%, Rf = 0.78 (15%
EtOAc−hexanes), white solid, mp 51−54 °C (EtOAc−hexanes), [α]D
= +46.1 (c = 1.4, CHCl3)], which was used in the next reaction
1
without further purification. H NMR (CDCl3, 250 MHz): 3.96 (2H,
overlapped broad s, H-8 and H-12), 3.58 (1H, dd, J1 = 3.8, J2 = 9.6, H-
22), 3.28 (1H, dd, J1 = 7.1, J2 = 9.6, H-22), 1.01 (3H, d, J = 6.4, H-21),
0.95 (3H, s, H-18), 0.88 (18H, overlapped s, 2 × Me3C-Si), 0.02 (6H,
s, Me2Si), 0.00 (3H, s, MeSi), −0.01 (3H, s, MeSi). 13C NMR (CDCl3,
63 MHz): 73.1 (CH), 69.2 (CH), 67.6 (CH2, C-22), 45.9 (C, C-13),
45.0 (CH), 44.2 (CH), 38.2 (CH), 28.2 (CH2), 25.9 (CH2), 25.9 (3 ×
CH3, Me3C-Si), 25.8 (3 × CH3, Me3C-Si), 24.5 (CH2), 22.2 (CH2),
18.3 (C, C-Si), 17.9 (C, C-Si), 15.7 (CH3), 14.7 (CH3), −4.9 (CH3,
MeSi), −5.2 (CH3, MeSi), −5.4 (2 × CH3, Me2Si). IR (neat, cm−1):
3403 (νO−H), 2956 (νC−H), 2929 (νC−H). MS ([ESI-TOF]+, m/z, %):
479 ([M + Na]+, 100), 439 ([M − OH]+, 13). HRMS: [ESI-TOF]+,
calcd for [C25H52O3Si2Na]+, 479.3347; found, 479.3343.
1
without further purification. H NMR (CDCl3, 250 MHz): 4.93 (1H,
d, J = 3.8, H-9), 3.82 (1H, broad d, J = 2.3, H-8), 3.60 (1H, dd, J1 =
2.0, J2 = 9.4, H-22), 3.54−3.42 (2H, m, H-11 and H-12), 3.28 (1H, dd,
J1 = 6.7, J2 = 9.4, H-22), 3.14 (3H, s, MeS), 1.09 (3H, d, J = 5.6, H-21),
1.01 (3H, s, H-18), 0.89 (9H, s, Me3C-Si), 0.88 (9H, s, Me3C-Si), 0.12
(3H, s, MeSi), 0.03 (9H, overlapped s, Me2Si and MeSi). 13C NMR
(CDCl3, 63 MHz): 81.9 (CH, C-9), 71.8 (CH, C-8), 67.3 (CH2, C-
22), 61.1 (CH), 51.2 (CH), 47.4 (CH), 42.2 (CH), 41.7 (C, C-13),
39.3 (CH3, MeS), 38.2 (CH), 26.2 (CH2), 25.9 (3 × CH3, Me3C-Si),
25.7 (3 × CH3, Me3C-Si), 21.1 (CH2), 18.3 (C, C-Si), 17.9 (C, C-Si),
17.0 (CH3), 13.8 (CH3), −4.8 (2 × CH3, Me2Si), −5.4 (2 × CH3,
Me2Si). IR (neat, cm−1): 2956 (νC−H), 2929 (νC−H), 1208 (νOSO).
MS ([ESI-TOF]+, m/z, %): 571 ([M + Na]+, 100), 475 ([M − O − t-
Bu]+, 15). HRMS: [ESI-TOF]+, calcd for [C26H52O6SSi2Na]+,
571.2915; found, 571.2921.
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorcholan-
12-one (19). Pyridinium dichromate (2.547 g, 6.77 mmol) was added
to a solution of 18 (1.237 g, 2.71 mmol) in CH2Cl2 (40 mL). The
reaction mixture, protected from light, was stirred for 36 h. The
mixture was filtered through a layer of silica gel. The solids were
washed with MTBE (3 × 40 mL), and the resulting filtrate was
concentrated. The residue was purified by flash chromatography (SiO2,
3 cm × 10 cm, 2% EtOAc−hexanes) to afford 19 [1.114 g, 2.45 mmol,
90%, Rf = 0.66 (10% EtOAc−hexanes), colorless oil, [α]D = +88.8 (c =
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorchol-9-
en-12α-ol (17). A solution of naphthalene (5.23 g, 40.8 mmol) in dry
THF (13 mL) was added via cannula to freshly cut sodium pieces (938
mg, 40.8 mmol). After 18 h, the resulting deep-blue mixture was added
via cannula to a solution of 16 (2.22 g, 4.04 mmol) in THF (25 mL) at
0 °C. The mixture was stirred for 3 h at 0 °C. The reaction was
quenched by slowly addition of H2O (50 mL). The aqueous layer was
extracted with EtOAc (3 × 60 mL). The combined organic layer was
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 6 cm × 10 cm, 3% EtOAc−hexanes) to afford
17 [1.715 g, 3.78 mmol, 93%, Rf = 0.45 (15% EtOAc−hexanes), white
solid, mp 75−78 °C (EtOAc−hexanes), [α]D = +177.9 (c = 0.6,
1
1.6, CHCl3)]. H NMR (CDCl3, 250 MHz): 4.02 (1H, broad d, J =
1.8, H-8), 3.61 (1H, dd, J1 = 3.5, J2 = 9.5, H-22), 3.30 (1H, dd, J1 = 7.8,
J2 = 9.5, H-22), 3.19−2.93 (1H, m, H-11), 1.31 (3H, s, H-18), 0.95−
0.89 (12H, m, H-21 and Me3C−Si), 0.88 (9H, s, Me3C-Si), 0.06 (3H,
s, MeSi), 0.04 (3H, s, MeSi), 0.02 (6H, s, Me2Si). 13C NMR (CDCl3,
63 MHz): 215.6 (C, C-12), 68.2 (CH, C-8), 67.7 (CH2, C-22), 57.2
(C, C-13), 57.0 (CH), 43.9 (CH), 39.1 (CH), 37.8 (CH2), 34.9
(CH2), 26.5 (CH2), 26.0 (3 × CH3, Me3C-Si), 25.8 (3 × CH3, Me3C-
Si), 22.9 (CH2), 18.3 (C, C-Si), 18.0 (C, C-Si), 17.4 (CH3), 13.3
(CH3), −4.8 (CH3, MeSi), −5.1 (CH3, MeSi), −5.4 (2 × CH3,
Me2Si). IR (film, cm−1): 2955 (νC−H), 2929 (νC−H), 2857 (νC−H),
1712 (νCO). MS ([CI]+, m/z, %): 455 ([M + H]+, 81), 439 ([M −
Me]+, 75), 397 ([M − t-Bu], 60), 323 ([M − TBSO]+, 100). HRMS:
[CI]+, calcd for [C25H51O2Si2]+, 455.3377; found, 455.3365.
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorchol-11-
en-12-yl trifluoromethanesulfonate (21). A solution of lithium
diisopropylamide was prepared by dropwise addition of solution of
n-BuLi in hexanes (1.62 mL, 2.27 M, 3.67 mmol) to diisopropylamine
(397 mg, 3.92 mmol) at −78 °C. The mixture was stirred at rt until
formation of a semisolid slurry. The slurry was cooled to −78 °C and
dissolved in THF (16 mL). A solution of 19 (1.114 g, 2.45 mmol) in
THF (30 mL) was added via cannula. The mixture was stirred at rt for
4 h and then cooled to −78 °C. A solution of triflimide 20 (1.924 g,
4.90 mmol) in THF (20 mL) was added. The mixture was allowed to
warm to rt overnight. The reaction was quenched by addition of
saturated NaCl (40 mL). The aqueous layer was extracted with MTBE
(3 × 40 mL), and the combined organic layer was dried, filtered, and
concentrated. The residue was purified by flash chromatography (SiO2,
1
CHCl3)]. H NMR (CDCl3, 250 MHz): 5.91−5.83 (2H, m, H-9 and
H-11), 4.07 (1H, t, J = 3.9, H-8), 4.00 (1H, d, J = 4.5, H-12), 3.63
(1H, dd, J1 = 4.3, J2 = 9.8, H-22), 3.28 (1H, dd, J1 = 7.4, J2 = 9.8, H-
22), 1.02 (3H, d, J = 6.6, H-21), 0.89 (9H, s, Me3Si-C), 0.87 (9H, s,
Me3C-Si), 0.04 (9H, overlapped s, MeSi and Me2Si), 0.02 (3H, s,
MeSi). 13C NMR (CDCl3, 63 MHz): 132.0 (CH), 129.2 (CH), 71.7
(CH), 67.8 (CH2, C-22), 65.7 (CH), 44.9 (C, C-13), 44.1 (CH), 43.4
(CH), 38.3 (CH), 26.1 (CH2), 26.0 (3 × CH3, Me3C−Si), 25.8 (3 ×
CH3, Me3C-Si), 21.0 (CH2), 18.4 (C, C-Si), 18.0 (C, C-Si), 15.4
(CH3), 13.9 (CH3), −4.4 (CH3, MeSi), −5.2 (CH3, MeSi), −5.3 (2 ×
CH3, Me2Si). IR (neat, cm−1): 3318 (νO−H), 2956 (νC−H), 2929
(νC−H). MS ([ESI-TOF]+, m/z, %): 477 ([M + Na]+, 100), 437 ([M
− OH]+, 4). HRMS: [ESI-TOF]+, calcd for [C25H50O3Si2Na]+,
477.3191; found, 477.3193.
8β,22-Bis(tert-butyldimethylsilyloxy)-de-A,B-23,24-dinorcholan-
12α-ol (18). Pd/C (38 mg, 10%) was added to a solution of 17 (1.249
g, 2.75 mmol) in EtOAc (50 mL). The mixture was degassed under
reduced pressure and refilled with H2 for three times. The reaction
mixture was stirred under H2 atmosphere (balloon pressure) for 48 h.
The mixture was filtered through a layer of silica gel. The solids were
H
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3 cm × 8 cm, 20% CH2Cl2−hexanes and 10% EtOAc−hexanes) to
afford 21 [1.208 g, 2.06 mmol, 84%, Rf = 0.69 (5% EtOAc−hexanes),
(νC−H), 2930 (νC−H), 2857 (νC−H). MS ([CI]+, m/z, %): 583 ([M +
H]+, 4), 565 ([M − OH]+, 20), 451 ([M − TBSO]+, 3), 433 ([M −
OH − TBSOH]+, 34), 301 ([M − OH − 2TBSOH]+, 100). HRMS:
[CI]+, calcd for [C34H71O3Si2]+, 583.4942; found, 583.4916.
1
colorless oil, [α]D = +25.6 (c = 1.2, CHCl3)]. H NMR (CDCl3, 250
MHz): 5.53 (1H, t, J = 3.9, H-11), 4.12 (1H, broad d, J = 6.1, H-8),
3.66 (1H, dd, J1 = 4.1, J2 = 9.7, H-22), 3.41 (1H, dd, J1 = 7.0, J2 = 9.7,
H-22), 2.66 (1H, ddd, J1 = 3.7, J2 = 6.2, J3 = 19.0, H-9), 2.32 (1H, dd,
J1 = 4.2, J2 = 19.0, H-9), 1.26 (3H, s, H-18), 1.04 (3H, d, J = 6.9, H-
21), 0.90 (18H, overlapped s, 2 × Me3C-Si), 0.06 (3H, s, MeSi), 0.04
(9H, overlapped s, MeSi and Me2Si). 13C NMR (CDCl3, 63 MHz):
157.3 (C, C-12), 118.4 (C, q, J = 319, CF3), 112.6 (CH, C-11), 66.5
(CH2, C-22), 65.8 (CH, C-8), 53.8 (CH), 50.7 (CH), 45.9 (C, C-13),
36.4 (CH2), 34.9 (CH), 25.9 (3 × CH3, Me3C-Si), 25.7 (3 × CH3,
Me3C-Si), 23.3 (CH2), 22.2 (CH2), 19.5 (CH3), 18.2 (C, C-Si), 17.9
(C, C-Si), 15.8 (CH3), −4.8 (CH3, MeSi), −5.2 (CH3, MeSi), −5.5 (2
× CH3, Me2Si). IR (film, cm−1): 2956 (νC−H), 2931 (νC−H), 2886
(νC−H), 2859 (νC−H), 1666 (νCC). MS ([CI]+, m/z, %): 587 ([M +
H]+, 18), 455 ([M − TBSO]+, 20), 305 ([M − TBSO − TfOH]+, 86).
HRMS: [CI]+, calcd for [C26H50O5F3SSi2]+, 587.2870; found,
587.2864.
12β-(7-Hydroxy-7-methyloctyl)-de-A,B-23,24-dinorcholan-8β,22-
diol (8a). A solution of TBAF in THF (8.25 mL, 1 M, 8.25 mmol) was
added to a solution of 24 (320 mg, 0.548 mmol) in THF (10 mL).
The reaction mixture was heated at 55 °C for 7 days. The reaction was
quenched by addition of saturated NH4Cl (30 mL). Aqueous layer was
extracted with EtOAc (3 × 20 mL). The combined organic layer was
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 2 cm × 15 cm, 40% EtOAc−hexanes) to afford
8a [172 mg, 0.485 mmol, 89%, Rf = 0.30 (70% EtOAc−hexanes),
white solid, mp 114−118 °C (Et2O−hexanes), [α]D = +30.6 (c = 1.7,
CHCl3)]. 1H NMR (CDCl3, 250 MHz): 4.01 (1H, broad d, J = 2.6, H-
8), 3.76 (1H, dd, J1 = 3.9, J2 = 10.3, H-22), 3.36 (1H, dd, J1 = 8.2, J2 =
10.3, H-22), 1.19 (6H, s, Me2C-OH), 1.03 (3H, d, J = 6.9, H-21), 0.86
(3H, s, H-18). 13C NMR (CDCl3, 63 MHz): 70.9 (C, C-OH), 68.7
(CH, C-8), 66.3 (CH2, C-22), 56.1 (CH), 53.2 (CH), 49.3 (CH), 45.2
(C, C-13), 43.8 (CH2), 34.9 (CH), 33.9 (CH2), 31.2 (CH2), 30.1
(CH2), 29.9 (CH2), 29.0 (2 × CH3, Me2C-OH), 28.1 (CH2), 24.2
(CH2), 23.4 (CH2), 22.2 (CH2), 21.1 (CH2), 20.3 (CH3), 10.8 (CH3).
IR (film, cm−1): 3384 (νO−H), 2932 (νC−H), 2862 (νC−H). MS ([CI]+,
m/z, %): 337 ([M − OH]+, 8), 319 ([M − OH − H2O]+, 77), 301
([M − OH − 2H2O]+, 100). HRMS: [CI]+, calcd for [C22H39O]+,
319.3001; found, 319.3008.
8β,22-Bis(tert-butyldimethylsilyloxy)-12-(7-hydroxy-7-methyloct-
1-ynyl)-de-A,B-23,24-dinorchol-11-ene (23). CuI (60 mg, 0.31
mmol) and PdCl2(PPh3)2 (60 mg, 0.08 mmol) were successively
added to a solution of 21 (1.00 g, 1.71 mmol) and 2214 (1.20 g, 8.56
mmol) in Et2NH (100 mL) at 0 °C. The reaction mixture was stirred
for 1 h at 0 °C and 12 h at rt. The reaction was quenched by slowly
addition of saturated NH4Cl (100 mL). Aqueous layer was extracted
with MTBE (3 × 50 mL). The combined organic layer was dried,
filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 4.5 cm × 9 cm, 10% EtOAc−hexanes) to
afford 23 [925 mg, 1.60 mmol, 94%, Rf = 0.62 (30% EtOAc−hexanes),
22-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-23,24-dinorcholan-8β-ol (27a). Imidazole (65 mg, 0.958
mmol) and TBSCl (116 mg, 0.766 mmol) were successively added
to a solution of 8a (170 mg, 0.479 mmol) in DMF (7 mL) at 0 °C.
After 10 h at rt, the reaction was quenched by addition of saturated
NaCl (35 mL). The aqueous layer was extracted with EtOAc (3 × 20
mL). The combined organic layer was dried, filtered, and
concentrated. The residue was purified by flash chromatography
(SiO2, 2 cm × 8 cm, 30% EtOAc−hexanes) to afford 27a [206 mg,
0.44 mmol, 92%, Rf = 0.60 (60% EtOAc−hexanes), colorless oil, [α]D
1
colorless oil, [α]D = +28.2 (c = 1.1, CHCl3)]. H NMR (CDCl3, 250
MHz): 5.80−5.64 (1H, m, H-11), 4.08 (1H, broad d, J = 6.0, H-8),
3.70 (1H, dd, J1 = 3.1, J2 = 9.1, H-22), 3.30 (1H, t, J = 9.1, H-22), 1.18
(6H, s, Me2C-OH), 1.06 (3H, s, H-18), 1.00 (3H, d, J = 6.8, H-21),
0.86 (9H, s, Me3C-Si), 0.84 (9H, s, Me3C-Si), 0.01 (6H, s, Me2Si),
−0.02 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz): 132.1 (C, C-12),
131.9 (CH, C-11), 89.5 (C), 80.1 (C), 70.8 (C, C-OH), 66.6 (CH, C-
8), 66.3 (CH2, C-22), 51.5 (CH), 51.5 (CH), 44.8 (C, C-13), 43.3
(CH2), 37.1 (CH2), 33.8 (CH), 29.1 (2 × CH3, Me2C-OH), 25.9 (3 ×
CH3, Me3C-Si), 25.7 (3 × CH3, Me3C-Si), 23.8 (CH2), 21.8 (CH2),
21.5 (CH2), 20.2 (CH3), 19.4 (2 × CH2), 18.1 (C, C-Si), 17.9 (C, C−
Si), 17.1 (CH3), −4.8 (CH3, MeSi), −5.2 (CH3, MeSi), −5.3 (CH3,
MeSi), −5.3 (CH3, MeSi). IR (film, cm−1): 3368 (νO−H), 2955 (νC−H),
2930 (νC−H), 2884 (νC−H), 2858 (νC−H). MS ([CI]+, m/z, %): 577
([M + H]+, 9), 559 ([M − OH]+, 28), 445 ([M − TBSO]+, 14), 427
([M − OH − TBSOH]+, 51), 295 ([M − OH − 2TBSOH]+, 100).
HRMS: [CI]+, calcd for [C34H65O3Si2]+, 577.4472; found, 577.4468.
8β,22-Bis(tert-butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloc-
tyl)-de-A,B-23,24-dinorcholane (24). Pd/C (240 mg, 10%) was added
to a solution of enyne 23 (1.89 g, 3.27 mmol) in EtOAc (75 mL). The
mixture was degassed under reduced pressure and refilled with H2 for
three times. The reaction mixture was stirred under H2 atmosphere
(balloon pressure) for 72 h. The mixture was filtered through a layer of
silica gel. The solids were washed with EtOAc (3 × 20 mL), and the
resulting filtrate was concentrated. The residue was purified by flash
chromatography (SiO2, 4 cm × 8 cm, 4% EtOAc−hexanes) to afford
24 [1.88 g, 3.22 mmol, 98%, Rf = 0.70 (30% EtOAc−hexanes),
1
= +25.8 (c = 0.6, CHCl3)]. H NMR (CDCl3, 250 MHz): 3.98 (1H,
broad d, J = 1.2, H-8), 3.65 (1H, dd, J1 = 4.3, J2 = 9.6, H-22), 3.32 (1H,
dd, J1 = 7.7, J2 = 9.6, H-22), 1.18 (6H, s, Me2C-OH), 0.96 (3H, d, J =
6.8, H-21), 0.86 (9H, s, Me3C-Si), 0.83 (3H, s, H-18), 0.05 (6H, s,
Me2Si). 13C NMR (CDCl3, 63 MHz): 70.9 (C, C-OH), 68.9 (CH, C-
8), 66.5 (CH2, C-22), 56.3 (CH), 53.3 (CH), 49.2 (CH), 45.2 (C, C-
13), 44.0 (CH2), 34.8 (CH), 34.0 (CH2), 31.3 (CH2), 30.2 (CH2),
30.0 (CH2), 29.1 (2 × CH3, Me2C-OH), 28.2 (CH2), 25.9 (3 × CH3,
Me3C-Si), 24.3 (CH2), 23.5 (CH2), 22.1 (CH2), 21.1 (CH2), 20.5
(CH3), 18.2 (C, C-Si), 10.8 (CH3), −5.4 (2 × CH3, Me2Si). IR (film,
cm−1): 3399 (νO−H), 2930 (νC−H), 2858 (νC−H). MS ([FAB]+, m/z,
%): 469 ([M + H]+, 63), 451 ([M − OH]+, 52). HRMS: [FAB]+,
calcd for [C28H57O3Si]+, 469.4077; found, 469.4086.
22-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-23,24-dinorcholan-8-one (28a). Pyridinium dichromate (400
mg, 1.069 mmol) was added to a solution of 27a (167 mg, 0.356
mmol) in CH2Cl2 (10 mL). The reaction mixture was protected from
the light and stirred for 2 h at rt. The mixture was filtered through a
layer of silica gel. The solids were washed with MTBE (3 × 40 mL),
and the resulting filtrate was concentrated. The residue was purified by
flash chromatography (SiO2, 3 cm × 6 cm, 30% EtOAc−hexanes) to
afford ketone 28a [150 mg, 0.321 mmol, 89%, Rf = 0.48 (40% EtOAc−
hexanes), colorless oil]. 1H NMR (CDCl3, 250 MHz): 3.60 (1H, dd, J1
= 4.5, J2 = 9.7, H-22), 3.32 (1H, dd, J1 = 7.6, J2 = 9.7, H-22), 1.16 (6H,
s, Me2C−OH), 0.97 (3H, d, J = 6.9, H-21), 0.84 (9H, s, Me3C-Si),
0.56 (3H, s, H-18), −0.02 (6H, s, Me2Si). 13C NMR (CDCl3, 63
MHz): 212.4 (C, C-8), 70.8 (C, C-OH), 66.0 (CH2, C-22), 61.8
(CH), 56.0 (CH), 52.2 (C, C-13), 48.3 (CH), 43.8 (CH2), 40.3
(CH2), 35.2 (CH), 30.7 (CH2), 30.1 (CH2), 29.8 (CH2), 29.6 (CH2),
29.1 (2 × CH3, Me2C-OH), 28.2 (CH2), 25.8 (3 × CH3, Me3C-Si),
24.2 (CH2), 21.6 (CH2), 20.1 (CH3), 18.7 (CH2), 18.2 (C, C-Si), 9.8
(CH3), −5.4 (2 × CH3, Me2Si). IR (CHCl3, cm−1): 3459 (νO−H),
2956 (νC−H), 2930 (νC−H), 2884 (νC−H), 1717 (νCO). MS ([CI]+, m/
1
colorless oil, [α]D = +37.4 (c = 1.0, CHCl3)]. H NMR (CDCl3, 250
MHz): 3.92 (1H, broad d, J = 1.7, H-8), 3.69 (1H, dd, J1 = 4.2, J2 =
9.5, H-22), 3.32 (1H, dd, J1 = 8.4, J2 = 9.5, H-22), 1.19 (6H, s, Me2C-
OH), 0.96 (3H, d, J = 6.7, H-21), 0.88 (9H, s, Me3C-Si), 0.87 (9H, s,
Me3C-Si), 0.81 (3H, s, H-18), 0.02 (6H, s, Me2Si), −0.01 (3H, s,
MeSi), −0.02 (3H, s, MeSi). 13C NMR (CDCl3, 63 MHz): 71.0 (C, C-
OH), 69.0 (CH, C-8), 66.5 (CH2, C-22), 56.6 (CH), 53.8 (CH), 49.4
(CH), 45.5 (C, C-13), 44.0 (CH2), 34.9 (CH2), 34.9 (CH), 31.4
(CH2), 30.2 (CH2), 30.1 (CH2), 29.2 (2 × CH3, Me2C-OH), 28.3
(CH2), 26.0 (3 × CH3, Me3C-Si), 25.8 (3 × CH3, Me3C-Si), 24.4
(CH2), 23.7 (CH2), 22.5 (CH2), 21.2 (CH2), 20.5 (CH3), 18.2 (C,
C−Si), 18.0 (C, C-Si), 11.0 (CH3), −4.8 (CH3, MeSi), −5.1 (CH3,
MeSi), −5.3 (2 × CH3, Me2Si). IR (film, cm−1): 3362 (νO−H), 2954
I
dx.doi.org/10.1021/jm3008272 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
z, %): 349 ([M − OH]+, 20), 317 ([M − OH − TBSOH]+, 100).
HRMS: [CI]+, calcd for [C28H53O2Si]+, 349.3815; found, 349.3821.
(E)-8-(Bromomethylene)-22-(tert-butyldimethylsilyloxy)-12β-(7-
hydroxy-7-methyloctyl)-de-A,B-24,23-dinorcholane (29a). A suspen-
sion of (Ph3PCH2Br)Br (1.121 g, 2.57 mmol) in toluene (18 mL) was
prepared by sonication for 30 min. After cooling at −17 °C, a solution
of KOt-Bu in THF (2.5 mL, 1 M, 2.5 mmol) was added and the
resulting mixture was stirred for 3 h. A solution of ketone 28a (150
mg, 0.32 mmol) in toluene (12 mL) previously cooled to 0 °C was
added via cannula. The mixture was stirred for 2 h at −17 °C and 3 h
at rt. The reaction was quenched by addition of saturated NH4Cl (1
mL), and the mixture was filtered through a layer of silica gel. The
solids were washed with EtOAc (3 × 15 mL), and the filtrate was
concentrated. The residue was purified by flash chromatography (SiO2,
3 cm × 8 cm, 30% EtOAc−hexanes) to afford 29a [121 mg, 0.22
mmol, 69%, Rf = 0.50 (20% EtOAc−hexanes), colorless oil]. 1H NMR
(CDCl3, 250 MHz): 5.63 (1H, s, H-7), 3.67 (1H, dd, J1 = 4.2, J2 = 9.6,
H-22), 3.44−3.30 (1H, m, H-22), 1.20 (6H, s, Me2C-OH), 0.99 (3H,
d, J = 6.9, H-21), 0.88 (9H, s, Me3C-Si), 0.50 (3H, s, H-18), 0.02 (6H,
s, Me2Si). 13C NMR (CDCl3, 63 MHz): 145.3 (C, C-8), 97.6 (CH, C-
7), 71.4 (C, C-OH), 66.5 (CH2, C-22), 56.9 (CH), 55.6 (CH), 49.4
(CH), 49.1 (C, C-13), 44.4 (CH2), 36.3 (CH), 31.6 (CH2), 31.6
(CH2), 30.6 (CH2), 30.4 (CH2), 29.6 (2 × CH3, Me2C-OH), 28.8
(CH2), 28.6 (CH2), 26.4 (3 × CH3, Me3C-Si), 24.8 (CH2), 22.2
(CH2), 22.0 (CH2), 20.5 (CH3), 18.7 (C, C-Si), 9.8 (CH3), −4.9 (2 ×
CH3, Me2Si). IR (film, cm−1): 3369 (νO−H), 3085 (ν=C−H), 2953
(νC−H), 2930 (νC−H), 2857 (νC−H), 1632 (νCC). MS ([CI]+, m/z,
%): 525 ([M − OH]+, 3), 393 ([M − OH − TBSOH]+, 31), 313 ([M
− C6H18BrO2Si]+, 100). HRMS: [CI]+, calcd for [C29H54BrOSi]+,
525.3127; found, 525.3134.
was extracted with EtOAc (3 × 15 mL). The combined organic layer
was dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 2 cm × 10 cm, 60−95% EtOAc−hexanes) to
give 5a [50 mg, 0.102 mmol, 86%, Rf = 0.18 (90% EtOAc−hexanes),
white solid, mp 85−88 °C (Et2O−hexanes), [α]D = −25.6 (c = 0.9,
EtOH 96%)]. Further purification by HPLC (Phenomenex-LUNA 5 μ
Silica(2) column, 10 mm × 250 mm, 20% i-PrOH−hexanes) gave an
analytically pure (>95%) sample which was used for biological assays.
1H NMR (CD3OD, 500 MHz): 6.31 (1H, d, J = 11.2, H-6), 6.09 (1H,
d, J = 11.2, H-7), 5.29 (1H, s, H-19), 4.90 (1H, s, H-19), 4.36 (1H, t, J
= 5.9, H-1), 4.16−4.10 (1H, m, H-3), 3.76 (1H, dd, J1 = 3.4, J2 = 10.4,
H-22), 3.30−3.24 (1H, m, H-22), 2.87 (1H, dd, J1 = 2.9, J2 = 13.9, H-
9), 2.52 (1H, dd, J1 = 3.4, J2 = 13.4, H-4), 2.26 (1H, dd, J1 = 6.8, J2 =
13.4, H-4), 2.13−2.04 (1H, m, H-20), 1.17 (6H, s, Me2C-OH), 1.06
(3H, d, J = 6.8, H-21), 0.54 (3H, s, H-18). 13C NMR (CD3OD, 125
MHz): 149.8 (C, C-10), 142.1 (C, C-8), 135.7 (C, C-5), 124.9 (CH,
C-6), 118.8 (CH, C-7), 112.0 (CH2, C-19), 71.4 (C, C-OH), 71.4
(CH, C-1), 67.4 (CH, C-3), 66.4 (CH2, C-22), 58.2 (CH), 57.1 (CH),
51.4 (CH), 50.3 (C, C-13), 46.1 (CH2, C-4), 44.9 (CH2), 43.7 (CH2,
C-2), 37.5 (CH), 32.5 (CH2), 31.4 (CH2), 31.1 (CH2), 30.5 (CH2),
30.0 (CH2, C-9), 29.4 (CH2), 29.2 (CH3, MeC-OH), 29.1 (CH3,
MeC-OH), 25.4 (CH2), 23.3 (CH2), 22.4 (CH2), 20.6 (CH3, C-21),
10.1 (CH3, C-18). IR (KBr, cm−1): 3403 (νO−H), 2930 (νC−H), 2861
(νC−H), 1635 (νCC). MS ([ESI-TOF]+, m/z, %): 511 ([M + Na]+,
100), 453 ([M − OH − H2O]+, 28). HRMS: [ESI-TOF]+, calcd for
[C31H52O4Na]+, 511.3758; found, 511.3764. UV (96% EtOH): λmax
264 nm, λmin = 230 nm.
=
8β-Hydroxy-12β-(7-hydroxy-7-methyloctyl)-de-A,B-23,24-di-
norcholan-22-yl p-toluenesulfonate (25a). p-Toluenesulfonyl chlor-
ide (138 mg, 0.724 mmol) was added to a solution of 8a (214 mg,
0.604 mmol) in pyridine (8 mL) at 0 °C. The mixture at 0 °C was
stirred for 2 h and then was kept at 4 °C for 22 h. The reaction was
quenched by addition of ice and saturated NaCl (10 mL). The
aqueous layer was extracted with MTBE (3 × 20 mL), and the
combined organic layer was washed with aqueous HCl (3 × 25 mL,
5%), saturated NaHCO3 (3 × 25 mL), and saturated NaCl (2 × 20
mL), dried, filtered, and concentrated. The residue was purified by
flash chromatography (SiO2, 3 cm × 10 cm, 30−60% EtOAc−
hexanes) to afford 25a [258 mg, 0.507 mmol, 84%, Rf = 0.58 (70%
(E)-22-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloc-
tyl)-8-[(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene]-de-
A,B-24,23-dinorcholane (6a). PCy3 (4 mg, 0.013 mmol) and
PdCl2(dppf)·CH2Cl2 (5 mg, 0.006 mmol) were dissolved in DMSO
(2 mL), and the mixture was stirred for 25 min. A solution of 29a (118
mg, 0.22 mmol) in DMSO (2 mL), KOAc (64 mg, 0.65 mmol), and
Pin2B2 (110 mg, 0.43 mmol) were successively added. The mixture
was heated to 80 °C for 3 h and then cooled to rt. The reaction was
quenched by addition of H2O (15 mL). The aqueous layer was
extracted with EtOAc (4 × 20 mL). The combined organic layer was
dried, filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 3 cm × 10 cm, 5−10% EtOAc−hexanes) to
afford 6a [74 mg, 0.15 mmol, 70%, Rf = 0.40 (20% EtOAc−hexanes),
1
EtOAc−hexanes), colorless oil, [α]D = +28.4 (c = 0.5, CHCl3)]. H
NMR (CDCl3, 250 MHz): 7.76 (2H, d, J = 8.1, H-Ar), 7.32 (2H, d, J =
8.1, H-Ar), 4.10 (1H, dd, J1 = 3.7, J2 = 9.1, H-22), 3.97 (1H, broad d, J
= 2.0, H-8), 3.75 (1H, t, J = 9.1, H-22), 2.43 (3H, s, Me-Ar), 1.19 (6H,
s, Me2C-OH), 0.96 (3H, d, J = 6.8, H-21), 0.70 (3H, s, H-18). 13C
NMR (CDCl3, 63 MHz): 144.6 (C, Ar-SO2), 133.0 (C), 129.7 (2 ×
CH, Ar-H), 127.8 (2 × CH, Ar-H), 74.4 (CH2, C-22), 71.0 (C, C-
OH), 68.6 (CH, C-8), 55.3 (CH), 53.0 (CH), 49.2 (CH), 45.2 (C, C-
13), 43.9 (CH2), 33.9 (CH2), 32.1 (CH), 31.2 (CH2), 30.1 (CH2),
29.9 (CH2), 29.1 (2 × CH3, Me2C-OH), 28.1 (CH2), 24.2 (CH2),
23.3 (CH2), 22.0 (CH2), 21.6 (CH3, Me-Ar), 21.1 (CH2), 20.1 (CH3),
10.7 (CH3). IR (film, cm−1): 3549 (νO−H), 3433 (νO−H), 2930 (νC−H),
2858 (νC−H), 1176 (νOSO). MS ([ESI-TOF]+, m/z, %): 531 ([M +
Na]+, 100), 491 ([M − OH]+, 22). HRMS: [ESI-TOF]+, calcd for
[C29H48O5SNa]+, 531.3106; found, 531.3115.
1
colorless oil, [α]D = +44.7 (c = 0.2, CHCl3)]. H NMR (CDCl3, 250
MHz): 4.90 (1H, s, H-7), 3.69 (1H, dd, J1 = 4.1, J2 = 9.6, H-22), 3.35
(1H, dd, J1 = 8.3, J2 = 9.6, H-22), 1.25 (12H, s, 2 × Me2C-OB), 1.20
(6H, s, Me2C-OH), 0.99 (3H, d, J = 6.9, H-21), 0.88 (9H, s, Me3C-Si),
0.50 (3H, s, H-18), 0.02 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz):
166.5 (C, C-8), 82.5 (2 × C, 2 × C-OB), 71.0 (C, C-OH), 66.2 (CH2,
C-22), 58.5 (CH), 56.2 (CH), 49.5 (CH), 49.3 (CH, C-13), 43.9
(CH2), 36.0 (CH), 33.2 (CH2), 31.3 (CH2), 30.2 (2 × CH2), 30.0
(CH2), 29.2 (2 × CH3, Me2C-OH), 28.3 (CH2), 26.0 (3 × CH3,
Me3C-Si), 24.9 (2 × CH3, Me2C-OB), 24.8 (2 × CH3, Me2C-OB),
24.3 (CH2), 22.1(CH2), 21.4 (CH2), 20.1 (CH3), 18.3 (C, C-Si), 9.7
(CH3), −5.3 (2 × CH3, Me2Si). IR (film, cm−1): 3385 (νO−H), 2954
(νC−H), 2930 (νC−H), 2857 (νC−H), 1640 (νCC). MS ([CI]+, m/z,
%): 573 ([M − OH]+, 10), 441 ([M − OH − TBSOH]+, 100), 315
([M − C12H28BO4Si]+, 20). HRMS: [CI]+, calcd for [C35H66BO3Si]+,
573.4874; found, 573.4878.
1α,22-Dihydroxy-12β-(7-hydroxy-7-methyloctyl)-23,24,25,26,27-
pentanorvitamin D3 (5a). An aqueous solution of K3PO4 (1.65 mL, 2
M) and PdCl2(PPh3)2 (4 mg, 0.006 mmol) were successively added to
a solution of 6a (70 mg, 0.118 mmol) and 721 (83 mg, 0.138 mmol) in
THF (4 mL). The reaction mixture protected from light was
vigorously stirred for 1 h. Then H2O (10 mL) was added, and the
aqueous layer was extracted with Et2O (3 × 15 mL). The combined
organic layer was dried, filtered, and concentrated. The residue was
dissolved in THF (7 mL) and a solution of TBAF in THF (835 μL, 1
M, 0.835 mmol) was added. After 6 h in the dark, the reaction was
quenched by addition of saturated NH4Cl (15 mL). The aqueous layer
8β-Hydroxy-12β-(7-hydroxy-7-methyloctyl)-de-A,B-23,24-di-
norcholane-22-carbonitrile (26a). Potassium cyanide (128 mg, 1.97
mmol) was added to a solution of 25a (500 mg, 0.983 mmol) in
DMSO (15 mL). The mixture was stirred at 90 °C for 2 h and then
cooled to rt. The reaction was quenched by addition of H2O (50 mL),
and the aqueous layer was extracted with EtOAc (4 × 40 mL). The
combined organic layer was dried, filtered, and concentrated. The
residue was purified by flash chromatography (SiO2, 3 cm × 5 cm, 20−
50% EtOAc−hexanes) to afford 26a [328 mg, 0.902 mmol, 92%, Rf =
0.50 (70% EtOAc−hexanes), colorless oil, [α]D = +25.9 (c = 0.2,
CHCl3)]. 1H NMR (CDCl3, 250 MHz): 4.01 (1H, broad d, J = 2.1, H-
8), 2.47 (1H, dd, J1 = 3.3, J2 = 16.1, H-22), 2.37−2.27 (1H, m, H-22),
1.20 (6H, s, Me2C-OH), 1.15 (3H, d, J = 6.7, H-21), 0.84 (3H, s, H-
18). 13C NMR (CDCl3, 63 MHz): 120.1 (C, CN), 71.0 (C, C-OH),
68.6 (CH, C-8), 55.8 (CH), 53.4 (CH), 49.2 (CH), 45.5 (C, C-13),
43.9 (CH2), 34.0 (CH2), 31.6 (CH2), 30.4 (CH), 30.2 (CH2), 30.0
J
dx.doi.org/10.1021/jm3008272 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(CH2), 29.2 (2 × CH3, Me2C-OH), 28.2 (CH2), 24.3 (CH2), 23.4
(CH2), 22.6 (CH3), 22.0 (CH2), 22.0 (CH2), 21.2 (CH2), 11.0 (CH3).
IR (film, cm−1): 3450 (νO−H), 2931 (νC−H), 2875 (νC−H), 2247
(νC≡N). MS ([CI]+, m/z, %): 346 ([M − OH]+, 52), 328 ([M − OH
− H2O]+, 100). HRMS: [CI]+, calcd for [C23H40NO]+, 346.3110;
found, 346.3110.
− OH − TBSOH]+, 94). HRMS: [CI]+, calcd for [C29H55O2Si]+,
463.3971; found, 463.3986.
(E)-8-(Bromomethylene)-23-(tert-butyldimethylsilyloxy)-12β-(7-
hydroxy-7-methyloctyl)-de-A,B-24-norcholane (29b). See 29a for
reaction procedure. Reagents: (Ph3PCH2Br)Br (689 mg, 1.58 mmol)
in toluene (18 mL), KOt-Bu in THF (1.56 mL, 1 M, 1.56 mmol), and
ketone 28b (95 mg, 0.19 mmol) in toluene (5 mL). Product: 29b [74
mg, 0.13 mmol, 67%, Rf = 0.43 (20% EtOAc−hexanes), colorless oil].
1H NMR (CDCl3, 250 MHz): 5.62 (1H, s, H-7), 3.76−3.49 (2H, m,
12β-(7-Hydroxy-7-methyloctyl)-de-A,B-24-norcholan-8β,23-diol
(8b). A solution of diisobutylaluminium hydride in CH2Cl2 (2.72 mL,
1 M, 2.72 mmol) was diluted in CH2Cl2 (3 mL) and cooled to −5 °C.
A solution of 26a (283 mg, 0.778 mmol) in CH2Cl2 (4 mL) was
added. The mixture was vigorously stirred at −15 °C for 2 h and then
allowed to warm to 0 °C for 1 h. A suspension of aqueous HCl (10
mL, 3 M) in Et2O (10 mL) was added, and the mixture was stirred for
2 h at 5 °C. The aqueous layer was extracted with CH2Cl2 (3 × 30
mL). The combined organic layer was washed with saturated NaCl (3
× 20 mL), dried, filtered, and concentrated. The residue was dissolved
in THF (10 mL). After cooling at −78 °C, a solution of
diisobutylaluminium hydride in CH2Cl2 (2.72 mL, 1 M, 2.72 mmol)
was added. After 2 h, the reaction was quenched by slowly addition of
aqueous HCl (20 mL, 10%) at 0 °C. The aqueous layer was extracted
with EtOAc (3 × 30 mL), and the combined organic layer was dried,
filtered, and concentrated. The residue was purified by flash
chromatography (SiO2, 3 cm × 7.5 cm, 40−50% EtOAc−hexanes)
to afford 8b [171 mg, 0.464 mmol, 60%, Rf = 0.30 (70% EtOAc−
H-23), 1.20 (6H, s, Me2C-OH), 0.91 (3H, d, J = 6.9, H-21), 0.88 (9H,
s, Me3C-Si), 0.51 (3H, s, H-18), 0.03 (6H, s, Me2Si). 13C NMR
(CDCl3, 63 MHz): 144.9 (C, C-8), 96.9 (CH, C-7), 71.0 (C, C-OH),
62.1 (CH2, C-23), 56.8 (CH), 55.8 (CH), 49.8 (CH), 48.9 (C, C-13),
44.0 (CH2), 36.2 (CH2), 31.1 (2 × CH2), 30.2 (CH2), 30.0 (CH2),
29.6 (CH), 29.2 (2 × CH3, Me2C-OH), 28.2 (CH2), 28.1 (CH2), 26.0
(3 × CH3, Me3C-Si), 24.3 (CH2), 22.5 (CH2), 22.0 (CH3), 21.4
(CH2), 18.3 (C, C-Si), 9.7 (CH3), −5.3 (2 × CH3, Me2Si). IR (film,
cm−1): 3369 (νO−H), 3084 (ν=C−H), 2954 (νC−H), 2930 (νC−H), 2858
(νC−H), 1632 (νCC). MS ([CI]+, m/z, %): 539 ([M− OH]+, 14), 407
([M − OH − TBSOH]+, 27), 327 ([M − C6H18BrO2Si]+, 100).
HRMS: [CI]+, calcd for [C30H56BrOSi]+, 539.3284; found, 539.3275.
(E)-23-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloc-
tyl)-8-[(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene]-de-
A,B-24-norcholane (6b). See 6a for reaction procedure. Reagents:
PCy3 (3 mg, 0.011 mmol) and PdCl2(dppf)·CH2Cl2 (5 mg, 0.005
mmol) in DMSO (2 mL), 29b (103 mg, 0.18 mmol) in DMSO (2
mL), KOAc (54 mg, 0.55 mmol), and Pin2B2 (91 mg, 0.36 mmol).
Product: 6b [79 mg, 0.13 mmol, 71%, Rf = 0.30 (10% EtOAc−
1
hexanes), white foam, [α]D = +24.9 (c = 0.7, CHCl3)]. H NMR
(CDCl3, 250 MHz): 3.98 (1H, broad s, H-8), 3.71−3.45 (2H, m, H-
23), 1.16 (6H, s, Me2C-OH), 0.89 (3H, d, J = 6.8, H-21), 0.83 (3H, s,
H-18). 13C NMR (CDCl3, 63 MHz): 71.0 (C, C-OH), 68.9 (CH, C-
8), 62.0 (CH2, C-23), 57.2 (CH), 53.7 (CH), 49.8 (CH), 45.4 (C, C-
13), 43.9 (CH2), 36.7 (CH2), 33.9 (CH2), 31.3 (CH2), 30.1 (CH2),
29.9 (CH2), 29.3 (CH), 29.1 (2 × CH3, Me2C-OH), 28.1 (CH2), 24.2
(CH2), 23.5 (CH2), 22.5 (CH2), 22.5 (CH3), 21.0 (CH2), 11.9 (CH3).
IR (film, cm−1): 3365 (νO−H), 2930 (νC−H), 2863 (νC−H). MS
([FAB]+, m/z, %): 351 ([M − OH]+, 70), 433 ([M − OH − H2O]+,
100). HRMS: [FAB]+, calcd for [C23H43O2]+, 351.3263; found,
351.3256.
1
hexanes), colorless oil, [α]D = +46.1 (c = 0.2, CHCl3)]. H NMR
(CDCl3, 250 MHz): 4.88 (1H, s, H-7), 3.74−3.41 (2H, m, H-23), 1.24
(12H, s, 2 × Me2C-OB), 1.19 (6H, s, Me2C-OH), 0.90 (3H, d, J = 6.9,
H-21), 0.87 (9H, s, Me3C-Si), 0.49 (3H, s, H-18), 0.02 (6H, s, Me2Si).
13C NMR (CDCl3, 63 MHz): 166.0 (C, C-8), 82.4 (2 × C, 2 × C-
OB), 71.0 (C, C-OH), 62.2 (CH2, C-23), 58.9 (CH), 56.8 (CH), 50.2
(CH), 49.5 (C, C-13), 43.9 (CH2), 36.2 (CH2), 33.2 (CH2), 31.2
(CH2), 30.2 (CH2), 30.0 (2 × CH2), 29.6 (CH), 29.2 (2 × CH3,
Me2C-OH), 28.2 (CH2), 26.0 (3 × CH3, Me3C-Si), 24.9 (2 × CH3,
Me2C-OB), 24.7 (2 × CH3, Me2C-OB), 24.3 (CH2), 22.7 (CH2), 22.0
(CH3), 21.2 (CH2), 18.3 (C, C−Si), 10.0 (CH3), −5.3 (2 × CH3,
Me2Si). IR (film, cm−1): 3390 (νO−H), 2955 (νC−H), 2930 (νC−H),
2858 (νC−H), 1640 (νCC). MS ([CI]+, m/z, %): 587 ([M − OH]+,
32), 455 ([M − OH − TBSOH]+, 100), 329 ([M − C12H28BO4Si]+,
25). HRMS: [CI]+, calcd for [C36H68BO3Si]+, 587.5028; found,
587.5031.
1α,23-Dihydroxy-12β-(7-hydroxy-7-methyloctyl)-24,25,26,27-tet-
ranorvitamin D3 (5b). See 5a for reaction procedure. Reagents:
aqueous solution of K3PO4 (1.6 mL, 2 M), PdCl2(PPh3)2 (4 mg, 0.006
mmol), 6b (68 mg, 0.112 mmol), 7 (79 mg, 0.132 mmol), and THF
(4 mL). Then THF (7 mL) and TBAF in THF (840 μL, 1 M, 0.840
mmol). Product: 5b [45 mg, 0.089 mmol, 80%, Rf = 0.20 (90%
EtOAc−hexanes), white solid, mp 87−91 °C (Et2O−hexanes), [α]D =
−30.0 (c = 1.2, EtOH 96%)]. 1H NMR (CDCl3, 400 MHz): 6.34 (1H,
d, J = 11.2, H-6), 6.00 (1H, d, J = 11.2, H-7), 5.31 (1H, s, H-19), 4.98
(1H, s, H-19), 4.41 (1H, dd, J1 = 4.3, J2 = 7.7, H-1), 4.26−4.15 (1H,
m, H-3), 3.69 (1H, ddd, J1 = 4.8, J2 = 8.8, J3 = 10.2, H-23), 3.58 (1H,
dt, J1 = 7.63, J2 = 10.2, H-23), 2.80 (1H, dd, J1 = 3.9, J2 = 13.4, H-9),
2.52 (1H, dd, J1 = 3.2, J2 = 13.3, H-4), 2.30 (1H, dd, J1 = 6.8, J2 = 13.3,
H-4), 1.20 (6H, s, Me2C-OH), 0.94 (3H, d, J = 6.8, H-21), 0.49 (3H, s,
H-18). 13C NMR (CDCl3, 100 MHz): 147.6 (C, C-10), 142.6 (C, C-
8), 133.0 (C, C-5), 124.9 (CH, C-6), 116.9 (CH, C-7), 111.7 (CH2,
C-19), 71.1 (C, C-OH), 70.7 (CH, C-1), 66.7 (CH, C-3), 62.2 (CH2,
C-23), 57.3 (CH), 56.7 (CH), 50.1 (CH), 49.3 (C, C-13), 45.2 (CH2,
C-4), 43.9 (CH2), 42.8 (CH2, C-2), 36.1 (CH2), 31.4 (CH2), 30.2
(CH2), 30.1 (CH), 29.9 (CH2), 29.3 (CH2), 29.2 (2 × CH3, Me2C-
OH), 29.0 (CH2, C-9), 28.2 (CH2), 24.3 (CH2), 22.6 (CH2), 22.1
(CH3, C-21), 21.5 (CH2), 10.0 (CH3, C-18). IR (KBr, cm−1): 3396
(νO−H), 2930 (νC−H), 2871 (νC−H), 1649 (νCC). MS ([ESI-TOF]+,
m/z, %): 525 ([M + Na]+, 100), 467 ([M − OH − H2O]+, 58).
HRMS: [ESI-TOF]+, calcd for [C32H54O4Na]+, 525.3914; found,
525.3913. UV (96% EtOH): λmax = 264 nm, λmin = 230 nm.
23-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-24-norcholan-8β-ol (27b). See 27a for reaction procedure.
Reagents: Imidazole (50 mg, 0.738 mmol), TBSCl (72 mg, 0.477
mmol), 8b (160 mg, 0.434 mmol), and DMF (10 mL). Product: 27b
[160 mg, 0.33 mmol, 76%, Rf = 0.78 (70% EtOAc−hexanes), colorless
1
oil, [α]D = +21.5 (c = 0.2, CHCl3)]. H NMR (CDCl3, 250 MHz):
3.98 (1H, broad s, H-8), 3.71−3.43 (2H, m, H-23), 1.15 (6H, s, Me2C-
OH), 0.92−0.74 (15H, m, H-18, H-21 and Me3C-Si), 0.00 (6H, s,
Me2Si). 13C NMR (CDCl3, 63 MHz): 70.8 (C, C-OH), 69.0 (CH, C-
8), 62.0 (CH2, C-23), 57.1 (CH), 53.7 (CH), 50.1 (CH), 45.5 (C, C-
13), 43.9 (CH2), 36.7 (CH2), 34.0 (CH2), 31.1 (CH2), 30.2 (CH2),
29.9 (CH2), 29.1 (2 × CH3, Me2C-OH), 28.8 (CH), 28.1 (CH2), 25.9
(3 × CH3, Me3C-Si), 24.3 (CH2), 23.3 (CH2), 22.7 (CH2), 22.3
(CH3), 20.8 (CH2), 18.2 (C-Si), 11.4 (CH3), −5.4 (2 × CH3, Me2Si).
IR (CHCl3, cm−1): 3402 (νO−H), 2929 (νC−H), 2858 (νC−H). MS
([CI]+, m/z, %): 465 ([M − OH]+, 19), 333 ([M − TBSO − H2O]+,
36), 315 ([M − TBSO − 2H2O]+, 100). HRMS: [CI]+, calcd for
[C29H57O2Si]+, 465.4128; found, 465.4124.
23-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-24-norcholan-8-one (28b). See 28a for reaction procedure.
Reagents: pyridinium dichromate (362 mg, 0.963 mmol), 27b (155
mg, 0.321 mmol), and CH2Cl2 (10 mL). Product: ketone 28b [146
mg, 0.304 mmol, 95%, Rf = 0.46 (40% EtOAc−hexanes), colorless oil].
1H NMR (CDCl3, 250 MHz): 3.67−3.43 (2H, m, H-23), 1.17 (6H, s,
Me2C-OH), 0.90 (3H, d, J = 6.9, H-21), 0.85 (9H, s, Me3C-Si), 0.58
(3H, s, H-18), 0.00 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz):
212.4 (C, C-8), 70.7 (C, C−OH), 62.2 (CH), 61.6 (CH2, C-23), 56.5
(CH), 52.5 (C, C-13), 49.1 (CH), 43.8 (CH2), 40.3 (CH2), 36.0
(CH2), 30.6 (CH2), 30.1 (CH2), 29.8 (CH2), 29.4 (CH2), 29.1 (2 ×
CH3, Me2C-OH), 28.7 (CH), 28.1 (CH2), 25.9 (3 × CH3, Me3C-Si),
24.2 (CH2), 21.8 (CH3), 21.1 (CH2), 19.4 (CH2), 18.1 (C, C-Si), 10.0
(CH3), −5.5 (2 × CH3, Me2Si). IR (CHCl3, cm−1): 3467 (νO−H),
2958 (νC−H), 2930 (νC−H), 2858 (νC−H), 1718 (νCO). MS ([CI]+, m/
z, %): 463 ([M − OH]+, 58), 405 ([M − OH − t-Bu]+, 48), 331 ([M
K
dx.doi.org/10.1021/jm3008272 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
8β-Hydroxy-12β-(7-hydroxy-7-methyloctyl)-de-A,B-24-norcho-
lan-23-yl p-toluenesulfonate (25b). See 25a for reaction procedure.
Reagents: p-toluenesulfonyl chloride (168 mg, 0.879 mmol), 8b (216
mg, 0.586 mmol), and pyridine (15 mL). Product: 25b [255 mg, 0.488
mmol, 83%, Rf = 0.60 (70% EtOAc−hexanes), colorless oil]. 1H NMR
(CDCl3, 250 MHz): 7.74 (2H, d, J = 8.2, H-Ar), 7.30 (2H, d, J = 8.2,
H-Ar), 4.18−3.87 (3H, m, H-8 and H-23), 2.40 (3H, s, Me-Ar), 1.16
(6H, s, Me2C-OH), 0.80 (3H, d, J = 6.8, H-21), 0.74 (3H, s, H-18).
13C NMR (CDCl3, 63 MHz): 144.5 (C, Ar-SO2), 133.0 (C), 129.7 (2
× CH, Ar-H), 127.7 (2 × CH, Ar-H), 70.8 (C, C-OH), 69.8 (CH2, C-
23), 68.7 (CH, C-8), 56.8 (CH), 53.5 (CH), 49.6 (CH), 45.4 (C, C-
13), 43.8 (CH2), 33.9 (CH2), 32.5 (CH2), 31.2 (CH2), 30.1 (CH2),
29.8 (CH2), 29.0 (2 × CH3, Me2C-OH), 28.9 (CH), 28.1 (CH2), 24.2
(CH2), 23.8 (CH2), 22.3 (CH2), 21.8 (CH3, Me-Ar), 21.5 (CH3), 20.8
(CH2), 11.2 (CH3). IR (film, cm−1): 3545 (νO−H), 3425 (νO−H), 2930
(νC−H), 2860 (νC−H), 1176 (νOSO). MS ([FAB]+, m/z, %): 545
([M + Na]+, 4), 487 ([M − OH − H2O]+, 25), 315 ([M − TsO −
2H2O]+, 31). HRMS: [FAB]+, calcd for [C30H50O5SNa]+, 545.3277;
found, 545.3271.
TBSO], 100). HRMS: [CI]+, calcd for [C30H59O2Si]+, 479.4284;
found, 479.4291.
24-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-cholan-8-one (28c). See 28a for reaction procedure. Reagents:
pyridinium dichromate (198 mg, 0.525 mmol), 27c (87 mg, 0.175
mmol), and CH2Cl2 (8 mL). Product: ketone 28c [80 mg, 0.162
mmol, 93%, Rf = 0.62 (60% EtOAc−hexanes), colorless oil]. 1H NMR
(CDCl3, 250 MHz): 3.61−3.48 (2H, m, H-24), 1.17 (6H, s, Me2C-
OH), 0.91 (3H, d, J = 6.8, H-21), 0.84 (9H, s, Me3C-Si), 0.58 (3H, s,
H-18), 0.00 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz): 212.5 (C,
C-8), 70.8 (C, C-OH), 63.3 (CH2, C-24), 62.2 (CH), 57.1 (CH), 52.4
(C, C-13), 48.9 (CH), 43.9 (CH2), 40.4 (CH2), 33.0 (CH), 31.8
(CH2), 30.7 (CH2), 30.1 (CH2), 29.8 (CH2), 29.6 (CH2), 29.1 (2 ×
CH3, Me2C-OH), 29.0 (CH2), 28.2 (CH2), 25.8 (3 × CH3, Me3C-Si),
24.2 (CH2), 21.9 (CH3), 21.1 (CH2), 19.3 (CH2), 18.2 (C, C-Si), 10.1
(CH3), −5.3 (2 × CH3, Me2Si). IR (film, cm−1): 3460 (νO−H), 2955
(νC−H), 2930 (νC−H), 2858 (νC−H), 1717 (νCO). MS ([CI]+, m/z,
%): 477 ([M − OH]+, 25), 419 ([M − OH − t-Bu]+, 33), 327 ([M −
2H2O − TBSO]+, 100). HRMS: [CI]+, calcd for [C30H57O2Si]+:
477.4128; found: 477.4131.
8β-Hydroxy-12β-(7-hydroxy-7-methyloctyl)-de-A,B-24-norcho-
lane-23-carbonitrile (26b). See 26a for reaction procedure. Reagents:
potassium cyanide (159 mg, 2.44 mmol), 25b (255 mg, 0.488 mmol),
and DMSO (10 mL). Product: 26b [167 mg, 0.442 mmol, 91%, Rf =
(E)-8-(Bromomethylene)-24-(tert-butyldimethylsilyloxy)-12β-(7-
hydroxy-7-methyloctyl)-de-A,B-cholane (29c). See 29a for reaction
procedure. Reagents: (Ph3PCH2Br)Br (782 mg, 1.79 mmol) in
toluene (12 mL), KOt-Bu in THF (1.76 mL, 1 M, 1.76 mmol), ketone
28c (111 mg, 0.22 mmol) in toluene (6 mL). Product: 29c [90 mg,
1
0.50 (70% EtOAc−hexanes), colorless oil]. H NMR (CDCl3, 250
MHz): 3.96 (1H, broad s, H-8), 2.39−2.16 (2H, m, H-23), 1.14 (6H,
s, Me2C−OH), 0.90 (3H, d, J = 6.8, H-21), 0.82 (3H, s, H-18). 13C
NMR (CDCl3, 63 MHz): 119.9 (C, CN), 70.9 (C, C-OH), 68.7
(CH, C-8), 57.0 (CH), 53.6 (CH), 49.7 (CH), 45.6 (C, C-13), 43.9
(CH2), 34.0 (CH2), 32.3 (CH), 31.4 (CH2), 30.2 (CH2), 29.9 (CH2),
29.2 (CH2), 29.1 (2 × CH3, Me2C-OH), 28.2 (CH2), 24.3 (CH2),
23.5 (CH2), 22.4 (CH2), 21.4 (CH3), 20.8 (CH2), 15.9 (CH2), 11.4
(CH3). IR (film, cm−1): 3445 (νO−H), 2931 (νC−H), 2873 (νC−H),
2247 (νC≡N). MS ([CI]+, m/z, %): 377 ([M]+, 7), 360 ([M − OH]+,
48), 342 ([M − OH − H2O]+, 100). HRMS: [CI]+, calcd for
[C24H42NO]+, 360.3266; found, 360.3269.
1
0.16 mmol, 70%, Rf = 0.52 (20% EtOAc−hexanes), colorless oil]. H
NMR (CDCl3, 250 MHz): 5.61 (1H, s, H-7), 3.61−3.48 (2H, m, H-
24), 1.20 (6H, s, Me2C-OH), 0.92 (3H, d, J = 6.9, H-21), 0.88 (9H, s,
Me3C-Si), 0.49 (3H, s, H-18), 0.03 (6H, s, Me2Si). 13C NMR (CDCl3,
63 MHz): 144.9 (C, C-8), 97.0 (CH, C-7), 71.0 (C, C-OH), 63.5
(CH2, C-24), 56.7 (CH), 56.3 (CH), 49.5 (CH), 48.7 (C, C-13), 44.0
(CH2), 33.6 (CH), 32.0 (CH2), 31.2 (CH2), 31.1 (CH2), 30.2 (CH2),
30.0 (CH2), 29.2 (2 × CH3, Me2C-OH), 29.0 (CH2), 28.3 (CH2),
28.2 (CH2), 25.9 (3 × CH3, Me3C-Si), 24.3 (CH2), 22.3 (CH2), 22.0
(CH3), 21.2 (CH2), 18.3 (C, C−Si), 9.6 (CH3), −5.3 (2 × CH3,
Me2Si). IR (film, cm−1): 3369 (νO−H), 3084 (ν=C−H), 2953 (νC−H),
2930 (νC−H), 2858 (νC−H), 1632 (νCC). MS ([CI]+, m/z, %): 553
([M − OH]+, 36), 421 ([M − OH − TBSOH]+, 42), 341 ([M −
C6H18BrO2Si]+, 100). HRMS: [CI]+, calcd for [C31H58BrOSi]+,
553.3440; found, 553.3444.
12β-(7-Hydroxy-7-methyloctyl)-de-A,B-cholan-8β,24-diol (8c).
See 8b for reaction procedure. Reagents: diisobutylaluminium hydride
in CH2Cl2 (2.01 mL, 1 M, 2.01 mmol) and CH2Cl2 (5 mL), 26b (152
mg, 0.402 mmol) in CH2Cl2 (5 mL) and aqueous HCl (10 mL, 3 M)
in Et2O (10 mL), then THF (6 mL) and diisobutylaluminium hydride
in CH2Cl2 (2.01 mL, 1 M, 2.01 mmol). Product: 8c [95 mg, 0.248
(E)-24-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloc-
tyl)-8-[(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)methylene]-de-
A,B-cholane (6c). See 6a for reaction procedure. Reagents: PCy3 (2
mg, 0.008 mmol) and PdCl2(dppf)·CH2Cl2 (4 mg, 0.004 mmol) in
DMSO (2 mL), 29c (82 mg, 0.14 mmol) in DMSO (2 mL), KOAc
(41 mg, 0.42 mmol), and Pin2B2 (71 mg, 0.28 mmol). Product: 6c [61
mg, 0.10 mmol, 69%, Rf = 0.28 (15% EtOAc−hexanes), colorless oil].
1H NMR (CDCl3, 250 MHz): 4.89 (1H, s, H-7), 3.62−3.52 (2H, m,
H-24), 1.25 (12H, s, 2 × Me2C-BO), 1.20 (6H, s, Me2C-OH), 0.94−
0.85 (12H, m, H-21 and Me3C-Si), 0.47 (3H, s, H-18), 0.03 (6H, s,
Me2Si). 13C NMR (CDCl3, 63 MHz): 166.1 (C, C-8), 82.5 (2 × C, 2
× C-OB), 71.0 (C, C-OH), 63.6 (CH2, C-24), 57.4 (CH), 56.3 (CH),
50.0 (CH), 49.4 (C, C-13), 44.0 (CH2), 33.7 (CH), 33.2 (CH2), 32.0
(CH2), 31.3 (CH2), 30.2 (2 × CH2), 30.0 (CH2), 29.2 (2 × CH3,
Me2C-OH), 29.0 (CH2), 28.2 (CH2), 25.9 (3 × CH3, Me3C-Si), 24.9
(2 × CH3, Me2C-OB), 24.8 (2 × CH3, Me2C-OB), 24.3 (CH2), 22.5
(CH2), 22.0 (CH3), 21.0 (CH2), 18.3 (C, C-Si), 9.9 (CH3), −5.3 (2 ×
CH3, Me2Si). IR (film, cm−1): 3369 (νO−H), 2953 (νC−H), 2930
(νC−H), 2858 (νC−H), 1640 (νCC). MS ([CI]+, m/z, %): 601 ([M −
OH]+, 71), 469 ([M − OH− TBSOH]+, 100), 343 ([M −
C12H28BO4Si]+, 29). HRMS: [CI]+, calcd for [C37H70BO3Si]+,
601.5187; found, 601.5197.
mmol, 61%, Rf = 0.38 (70% EtOAc−hexanes), white foam, [α]D
=
1
+38.4 (c = 0.2, CHCl3)]. H NMR (CDCl3, 250 MHz): 4.01 (1H,
broad d, J = 1.4, H-8), 3.71−3.51 (2H, m, H-24), 1.19 (6H, s, Me2C-
OH), 0.91 (3H, d, J = 6.8, H-21), 0.83 (3H, s, H-18). 13C NMR
(CDCl3, 63 MHz): 71.0 (C, C-OH), 69.0 (CH, C-8), 63.1 (CH2, C-
24), 57.5 (CH), 53.6 (CH), 49.8 (CH), 45.4 (C, C-13), 43.9 (CH2),
33.9 (CH2), 32.8 (CH), 31.7 (CH2), 31.3 (CH2), 30.1 (CH2), 29.9
(CH2), 29.6 (CH2), 29.1 (2 × CH3, Me2C-OH), 28.1 (CH2), 24.3
(CH2), 23.5 (CH2), 22.5 (CH2), 22.3 (CH3), 20.8 (CH2), 11.3 (CH3).
IR (film, cm−1): 3335 (νO−H), 2932 (νC−H), 2864 (νC−H). MS
([FAB]+, m/z, %): 365 ([M − OH]+, 53), 342 ([M − OH − H2O]+,
100). HRMS: [FAB]+, calcd for [C24H45O2]+, 365.3420; found,
365.3422.
24-(tert-Butyldimethylsilyloxy)-12β-(7-hydroxy-7-methyloctyl)-
de-A,B-cholan-8β-ol (27c). See 27a for reaction procedure. Reagents:
imidazole (30 mg, 0.441 mmol), TBSCl (30 mg, 0.199 mmol), 8c (35
mg, 0.091 mmol), and DMF (10 mL). Product: 27c [35 mg, 0.07
mmol, 78%, Rf = 0.78 (70% EtOAc−hexanes), colorless oil]. 1H NMR
(CDCl3, 250 MHz): 4.01 (1H, broad d, J = 1.2, H-8), 3.64−3.52 (2H,
m, H-24), 1.20 (6H, s, Me2C-OH), 0.93−0.82 (15H, m, H-18, H-21
and Me3C-Si), 0.04 (6H, s, Me2Si). 13C NMR (CDCl3, 63 MHz): 71.0
(C, C-OH), 69.1 (CH, C-8), 63.5 (CH2, C-24), 57.6 (CH), 53.8
(CH), 49.9 (CH), 45.4 (C, C-13), 44.0 (CH2), 34.0 (CH2), 32.8
(CH), 32.0 (CH2), 31.3 (CH2), 30.2 (CH2), 30.0 (CH2), 29.6 (CH2),
29.1 (2 × CH3, Me2C-OH), 28.2 (CH2), 25.9 (3 × CH3, Me3C-Si),
24.3 (CH2), 23.5 (CH2), 22.6 (CH2), 22.3 (CH3), 20.8 (CH2), 18.3
(C, C-Si), 11.3 (CH3), −5.3 (2 × CH3, Me2Si). IR (film, cm−1): 3400
(νO−H), 2930 (νC−H), 2858 (νC−H). MS ([CI]+, m/z, %): 479 ([M −
OH]+, 53), 461 ([M − OH − H2O]+, 18), 329 ([M − 2H2O −
1α,24-Dihydroxy-12β-(7-hydroxy-7-methyloctyl)-25,26,27-trinor-
vitamin D3 (5c). See 5a for reaction procedure. Reagents: aqueous
solution of K3PO4 (1.2 mL, 2 M), PdCl2(PPh3)2 (3 mg, 0.004 mmol),
6c (54 mg, 0.087 mmol), 7 (61 mg, 0.102 mmol), and THF (3 mL).
Then THF (6 mL) and TBAF in THF (560 μL, 1 M, 0.560 mmol).
Product: 5c [37 mg, 0.072 mmol, 83%, Rf = 0.18 (90% EtOAc−
hexanes), white solid, mp 88−92 °C (Et2O−hexanes), [α]D = −20.4 (c
1
= 0.5, EtOH 96%)]. H NMR (CDCl3, 400 MHz): 6.34 (1H, d, J =
L
dx.doi.org/10.1021/jm3008272 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
11.2, H-6), 6.01 (1H, d, J = 11.2, H-7), 5.32 (1H, s, H-19), 4.98 (1H, s,
H-19), 4.42 (1H, dd, J1 = 4.2, J2 = 7.7, H-1), 4.24−4.18 (1H, m, H-3),
3.61 (2H, td, J1 = 2.2, J2 = 6.5, H-24), 2.80 (1H, dd, J1 = 3.8, J2 = 13.3,
H-9), 2.58 (1H, dd, J1 = 2.9, J2 = 13.2, H-4), 2.30 (1H, dd, J1 = 6.5, J2 =
13.2, H-4), 1.21 (6H, s, Me2C-OH), 0.94 (3H, d, J = 6.8, H-21), 0.48
(3H, s, H-18). 13C NMR (CDCl3, 100 MHz): 147.6 (C, C-10), 142.7
(C, C-8), 133.0 (C, C-5), 124.9 (CH, C-6), 116.9 (CH, C-7), 111.7
(CH2, C-19), 71.1 (C, C-OH), 70.7 (CH, C-1), 66.8 (CH, C-3), 63.3
(CH2, C-24), 57.2 (CH), 57.0 (CH), 50.1 (CH), 49.3 (C, C-13), 45.2
(CH2, C-4), 44.0 (CH2), 42.8 (CH2, C-2), 33.8 (CH), 31.8 (CH2),
31.4 (CH2), 30.2 (CH2), 30.0 (CH2), 29.7 (CH2), 29.4 (CH2), 29.2 (2
× CH3, Me2C-OH), 29.0 (CH2, C-9), 28.2 (CH2), 24.3 (CH2), 22.5
(CH2), 22.0 (CH3, C-21), 21.3 (CH2), 9.9 (CH3, C-18). IR (KBr,
cm−1): 3388 (νO−H), 2930 (νC−H), 2861 (νC−H), 1632 (νCC). MS
([ESI-TOF]+, m/z, %): 539 ([M + Na]+, 100), 481 ([M − OH −
H2O]+, 24). HRMS: [ESI-TOF]+, calcd for [C33H56O4Na]+, 539.4071;
found, 539.4069. UV (96% EtOH): λmax = 264 nm, λmin = 230 nm.
Functional Studies. Cell Culture. Human MCF-7 breast
adenocarcinoma and SW480-ADH colon cancer cells were grown in
DMEM and RPMI media, respectively, supplemented with 10% FBS,
100 U/mL penicillin, 100 U/mL streptomycin, and 2 mM L-glutamine
(all from Invitrogen, Paisley, UK), in air-CO2 (95:5) atmosphere a 37
°C. Confluent cells were washed twice with phosphate-buffered saline
(PBS) and harvested by a brief incubation with trypsin−EDTA
solution (Sigma Aldrich St. Louis, USA) in PBS. Treatments with
1α,25-(OH)2D3 (1), or compounds 5a−c were carried out using
medium supplemented with charcoal-treated FCS to remove lip-
osoluble hormones. Control cells were treated with the corresponding
vehicle.
MTT Metabolization. Cell proliferation experiments were carried
out using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assays, where MTT is reduced to purple formazan by
the mitochondria of living cells. Increase in cell number is detected by
augmented MTT metabolization. MCF-7 cells were plated at a 5 × 104
cells per well in 24-well plates. Twenty-four hours klater, the cells were
treated with compounds 5a−c at concentrations of 10, 100, or 500 nM
during 48 h. MTT (0.5 μg/μL) was added to each well, and the
mixture was incubated for 1 h. The medium was then removed, and
DMSO (500 μL) was added to each well. Absorbance of samples was
measured at 570 nm in a Mithras LB 940 from Berthold Technologies
(Bad Wildbad, Germany).
competition assay that provides a sensitive and robust method for
high-throughput screening of potential vitamin D receptor ligands.
VDR is added to a fluorescent VDR ligand to form a receptor/tracer
complex resulting in a high polarization value. This complex is then
added to individual test compounds in microwell plates. Competitors
will displace the tracer from the complex, causing the fluorescent
ligand to tumble more rapidly during its fluorescence lifetime and
resulting in a low polarization value. The polarized fluorescence was
measured in a 384-well black plate during 200 ms/well using a Mithras
LB 940 (Berthold Technologies). All compounds (1, 5a−c) were
evaluated within the range from 10−11 M to 10−5 M. IC50 values were
calculated using average of measured values. The activity of each
compound is also shown as percentage, in which the activity of the
natural hormone 1α,25-(OH)2D3 was normalized to 100%.
Western Blotting. Western blotting was performed as previously
described.27 Protein concentration was measured using the Bio-Rad
DC protein assay kit. Analysis of cell lysates was performed by
electrophoresis in SDS gels and protein transfer to Immobilon P
membranes (Millipore Corp., Billerica, MA, USA). The membranes
were incubated with the appropriate primary and secondary
horseradish peroxidase-conjugated antibodies, and the antibody
binding was visualized using the ECL detection system (Amer-
sham−GE Healthcare, Barcelona, Spain). We used rabbit polyclonal
antibodies generated against Cystatin D,29 mouse monoclonal
antibodies against E-cadherin (BD Transduction Laboratories, San
Diego, CA, USA), c-MYC (Santa Cruz Biotechnology, Heidelberg,
Germany), and goat polyclonal antibody against β-actin (Santa Cruz
Biotechnology).
Quantitative RT-PCR. Total RNA was extracted from cultured cells
using NucleoSpin miRNA extraction kit (Macherey-Nagel) and
retrotranscribed using the iScript cDNA Synthesis Kit (Bio-Rad).
The quantitative PCR reaction was performed in a CFX384 real-time
PCR detection system (Bio-Rad) using the TaqMan Universal Master
Mix (Applied Biosystems) and TaqMan probes for PIP4K2B
(Hs01552176_m1) and RPLP0 (Hs99999902_m1) (both from
Applied Biosystems). Thermal cycling consisted of an initial
denaturing step at 95 °C for 10 min and 40 cycles of denaturing at
95 °C for 15 s and annealing and elongation at 60 °C for 30 s. RNA
expression values were normalized versus the housekeeping gene
RPLP0 (ribosomal protein, large, P0) using the comparative CT
method. All measurements were performed in triplicate.
Serum Calcium Quantitation and Weight Measure. All animal
studies were approved by the University of Santiago de Compostela
Ethics Committee for Animal Experiments. Female CD-1 mice (age
matched, between 6 and 8 weeks) were obtained from Charles River
Laboratories (L’Arbresle, France). The compounds (1, 5a−c) were
dissolved in sesame oil and administered intraperitoneally (0.3 μg/kg)
every other day for three weeks. Calcium measurement was
determined a day after the last dose using the QuantiChom Calcium
Assay Kit (BioAssay Systems, Hayward, CA, USA) following the
manufacturer’s guidelines. Weight of mice was carried out every other
day for a week.
Luciferase Reporter Assays. Cells were cultured as described above.
Then 12−24 h before transfection, 2 × 105 cells per well were seeded
in 24-well plates and allowed to attach overnight. Cells were then
transfected using JetPEI transfection reagent (PolyPlus Transfection,
Illrich, France) following the manufacturer’s guidelines. Transfections
were performed in triplicate using 1 μg of pCYP24A1-Luc plasmid
(kindly provided by Dr. Aranda, Instituto de Investigaciones
́
Biomedicas Alberto Sols, Madrid). This vector encoding the luciferase
gene under control of a consensus vitamin D response element (24-
hydroxylase promoter, CYP24A1), and it is very responsive to the
1α,25-(OH)2D3 treatment. After incubation for 24 h in DMEM
supplemented with 10% of charcoal-stripped FCS, culture medium was
replaced to phenol red free DMEM containing 10% FCS for 24 h with
each compound (1, 5a−c) at several concentrations (1 × 10−11 to 1 ×
10−6 M). Cells were then treated during 10 min with luciferin
potassiumsalt (100 mg/L) (Regis Technologies, Morton Grove, IL),
and bioluminescence images acquired with the In Vivo Imaging
System (IVIS, Caliper Life Sciences, Alameda, CA, USA), quantified as
total photon counts, and processed by Living Image software (Caliper
Life Sciences). The EC50 values are derived from dose−response
curves and represent the analogue concentration capable of increasing
the luciferase activity by 50%. The luciferase activity ratio is the
average ratio of the EC50 for the analogue to the EC50 for 1α,25-
(OH)2D3. All experiments were carried out in duplicate on at least two
different occasions.
Statistical Analysis. Each experiment was performed at least three
times. Values are expressed as means SD. Means were compared by
unpaired t-tests or one-way ANOVA with the Tukey−Kramer multiple
comparison for post hoc comparisons. Statistical significance is taken
to be indicated by * P < 0.05, *** P < 0.001. Dose−response curves
for the luciferase reporter assay and competitive VDR binding, as well
as calculation of EC50 and IC50 values, were performed using
GraphPad Prism 5 software (San Diego, CA, USA).
ASSOCIATED CONTENT
■
S
* Supporting Information
Data from docking calculations, RT-PCR data, NMR spectra
for the new compounds, X-ray structure and data of triol 8a,
and HPLC traces of vitamins 5a−c. This material is available
free of charge via the Internet at http://pubs.acs.org.
Human VDR Binding Assay. Binding affinity to VDR was evaluated
using a 1α,25-(OH)2D3 assay kit under manufacturer conditions
(Polarscreen Vitamin D receptor competitor assay, Red, catalogue no.
PV4569, Invitrogen). This kit is a fluorescence polarization (FP)-based
M
dx.doi.org/10.1021/jm3008272 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Vitamin D Analogues: A Challenge for Process Research. Curr. Opin.
AUTHOR INFORMATION
Corresponding Author
*Phone: +34 881 814 224. E-mail: mercedes.torneiro@usc.es.
■
Drug Discov. Dev. 2001, 4, 808−833. (c) Carlberg, C.; Mourino, A.
̃
New Vitamin D Receptor Ligands. Expert Opin. Ther. Pat. 2003, 13,
761−772. (d) Nagpal, S.; Na, S.; Rathnachalam, R. Noncalcemic
Actions of Vitamin D Receptor Ligands. Endocr. Rev. 2005, 26, 662−
Present Addresses
#
́
́
Departamento de Quımica Organica, Pontificia Universidad
́
687. (e) Carlberg, C.; Molnar, F. Current Status of Vitamin D
Signaling and its Therapeutic Applications. Curr. Top. Med. Chem.
2012, 12, 528−547.
́
Catolica de Chile, Macul, 7020436 Santiago de Chile, Chile.
▽The Walter and Eliza Hall Institute of Medical Research,
Parkville, VIC 3052, Australia.
(5) (a) Vitamin D: Molecular Biology, Physiology, and Clinical
Applications; Holick, M. F., Ed.; Humana: Totowa, NJ, 1999.
Notes
(b) Prufer, K.; Racz, A.; Lin, G. C.; Barsony, J. Dimerization with
̈
The authors declare no competing financial interest.
Retinoid X Receptors Promotes Nuclear Localization and Subnuclear
Targeting of Vitamin D Receptors. J. Biol. Chem. 2000, 275, 41114−
41123. (c) Jurutka, P. W.; Whitfield, G. K.; Hsieh, J.-C.; Thompson, P.
D.; Haussler, C. A.; Haussler, M. R. Molecular Nature of the Vitamin
D Receptor and its Role in Regulation of Gene Expression. Rev.
Endocr. Metab. Disord. 2001, 2, 203−216. (d) Christakos, S.; Dhawan,
P.; Benn, B.; Porta, A.; Hediger, M.; Oh, G. T.; Jeung, E.-B.; Zhong, Y.;
Ajibade, D.; Dhawan, K.; Joshi, S. Vitamin DMolecular Mechanism
of Action. Ann. N. Y. Acad. Sci. 2007, 1116, 340−348.
ACKNOWLEDGMENTS
We thank the Spanish Ministry of Education and Innovation
(SAF2007-67205 and SAF2010-15291 to A. Mourino,
■
̃
SAF2010-18302 to A. Munoz, and SAF2009-07631 to RPF)
̃
for financial support and CESGA for computer time. S.S.
́
́
Cientıfica de la Asociacion Espanola
̃
thanks the Fundacion
Contra el Cancer for a postdoctoral fellowship. F.Z. thanks the
́
́
(6) (a) Moras, D.; Gronemeyer, H. The Nuclear Receptor Ligand-
Binding Domain: Structure and Function. Curr. Opin. Cell Biol. 1998,
10, 384−391. (b) Huang, P.; Chandra, V.; Rastinejad, F. Structural
Overview of the Nuclear Receptor Superfamily: Insights into
Physiology and Therapeutics. Annu. Rev. Physiol. 2010, 72, 247−272.
(7) (a) Robyr, D.; Wolffe, A.; Wahli, W. Nuclear Hormone Receptor
Coregulators in Action: Diversity for Shared Tasks. Mol. Endocrinol.
2000, 14, 329−347. (b) Kim, S.; Shevde, N. K.; Pike, J. W. 1,25-
Dihydroxyvitamin D3 Stimulates Cyclic Vitamin D Receptor/Retinoid
X Receptor DNA-Binding, Co-activator Recruitment, and Histone
Acetylation in Intact Osteoblasts. J. Bone Miner. Res. 2005, 20, 305−
Universidad Nacional del Sur (Argentina) for financial support.
We thank DishmanNetherlands for the generous gift of
vitamin D2 and Dr. Fredy Sussman for advice on docking
studies.
DEDICATION
†Dedicated to Prof. Milan Uskokovic.
■
ABBREVIATIONS USED
■
CI, chemical ionization; 1,25D, 1α,25-dihydroxyvitamin D3;
DEPT, distortionless enhancement by polarization transfer;
DIBAL-H, diisobutylaluminium hydride; dppf, 1,1′-bis-
(diphenylphosphino)ferrocene; ESI-TOF, electrospray ioniza-
tion-time-of-flight; FAB, fast atom bombardment; HMPA,
hexamethylphosphoramide; Im, imidazole; LBD, ligand binding
domain; LDA, lithium diisopropylamide; Ms, methylsulfonyl;
1α,25-(OH)2D3, 1α,25-dihydroxyvitamin D3; PDC, pyridinium
dichromate; Pin, pinacolato; RXR, retinoic X receptor; TBS,
tert-butyldimethylsilyl; Tf, trifluoromethanesulfonyl; VDR,
vitamin D receptor; MCF-7, human adenocarcinoma breast
cancer cell line; SW480-ADH, human colon cancer cell line
́
317. (c) Carlberg, C.; Molnar, F. Current Status of Vitamin D
Signaling and its Therapeutic Applications. Curr. Top. Med. Chem.
2012, 12, 528−547.
́ ́
(8) Ordonez-Moran, P.; Munoz, A. Nuclear Receptors: Genomic and
̃ ̃
Non-Genomic Effects Converge. Cell Cycle 2009, 8, 1675−1680.
(9) Haussler, M. R.; Jurutka, P. W.; Mizwicki, M.; Norman, A. W.
Vitamin D Receptor (VDR)-Mediated Actions of 1α,25(OH)2Vitamin
D3: Genomic and Non-Genomic Mechanisms. Best Pract. Res., Clin.
Endocrinol. Metab. 2011, 25, 543−559.
(10) PDB ID: 1DB1. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klaholz,
B.; Moras, D. The Crystal Structure of the Nuclear Receptor for
Vitamin D Bound to its Natural Ligand. Mol. Cell 2000, 5, 173−179.
́
(11) (a) Carlberg, C.; Molnar, F.; Mourino, A. Vitamin D Receptor
̃
Ligands: The Impact of Crystal Structures. Expert Opin. Ther. Pat.
2012, 22, 417−435. (b) Berman, H. M.; Henrick, K.; Nakamura, H.
Announcing the Worldwide Protein Data Bank. Nature Struct. Biol.
2003, 10, 980−980, www.wwpdb.org.
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