Structure-function relationships and crystal structures of the vitamin D receptor bound 2α-methyl-(20S,23S)- and 2α-methyl-(20S,23R)- epoxymethano-1α,25-dihydroxyvitamin D3

Article abstract of DOI:10.1021/jm9014636

The vitamin D nuclear receptor is a ligand-dependent transcription factor that controls multiple biological responses such as cell proliferation, immune responses, and bone mineralization. Numerous 1α,25(OH)₂D ₃ analogues, which exhibit low calcemic side effects and/or antitumoral properties, have been synthesized. We recently showed that the synthetic analogue (20S,23S)-epoxymethano-1α,25-dihydroxyvitamin D ₃ (2a) acts as a 1α,25(OH)₂D₃ superagonist and exhibits both antiproliferative and prodifferentiating properties in vitro. Using this information and on the basis of the crystal structures of humanVDRligand binding domain (hVDRLBD) bound to 1α,25(OH)₂D₃, 2α-methyl-1α,25(OH) ₂D₃, or 2a, we designed a novel analogue, 2α-methyl-(20S,23S)-epoxymethano-1α,25-dihydroxyvitamin D ₃ (4a), in order to increase its transactivation potency. Here, we solved the crystal structures of the hVDR LBD in complex with the 4a (C23S) and its epimer 4b (C23R) and determined their correlation with specific biological outcomes.

Full text of DOI:10.1021/jm9014636

J. Med. Chem. 2010, 53, 1159–1171 1159
DOI: 10.1021/jm9014636
Structure-Function Relationships and Crystal Structures of the Vitamin D Receptor Bound
2r-Methyl-(20S,23S)- and 2r-Methyl-(20S,23R)-epoxymethano-1r,25-dihydroxyvitamin D3
†
‡
§
‡
‡
‡
Pierre Antony, Rita Sigueiro, Tiphaine Huet, Yoshiteru Sato, Nick Ramalanjaona, Luis Cezar Rodrigues,
§
€
Antonio Mourino, Dino Moras,*,‡ and Natacha Rochel*,‡
§
~
‡Deꢀpartement de Biologie et de Geꢀnomique Structurales, Centre National de la Recherche Scientifique, Institut National de la Santeꢀ de la
Recherche Meꢀdicale, Universiteꢀde Strasbourg, CEBGS-IGBMC (Centre Europeꢀen de Biologie et Geꢀnomique Structurale;Institut de Geꢀneꢀtique
§
et de Biologie Moleꢀculaire et Cellulaire), 1 Rue Laurent Fries, 67404 Illkirch, France and Departamento de Quımica Organica and Unidad
Asociada al CSIC, Universidad de Santiago de Compostela, 15782, Spain
´
ꢀ
Received October 3, 2009
The vitamin D nuclear receptor is a ligand-dependent transcription factor that controls multiple
biological responses such as cell proliferation, immune responses, and bone mineralization. Numerous
1R,25(OH)2D3 analogues, which exhibit low calcemic side effects and/or antitumoral properties, have
been synthesized. We recently showed that the synthetic analogue (20S,23S)-epoxymethano-1R,25-
dihydroxyvitamin D3 (2a) acts as a 1R,25(OH)2D3 superagonist and exhibits both antiproliferative and
prodifferentiating properties in vitro. Using this information and on the basis of the crystal structures of
human VDR ligand binding domain (hVDR LBD) bound to 1R,25(OH)2D3, 2R-methyl-1R,25(OH)2D3,
or 2a, we designed a novel analogue, 2R-methyl-(20S,23S)-epoxymethano-1R,25-dihydroxyvitamin D3
(4a), in order to increase its transactivation potency. Here, we solved the crystal structures of the hVDR
LBD in complex with the 4a (C23S) and its epimer 4b (C23R) and determined their correlation with
specific biological outcomes.
Introduction
cancer, autoimmune diseases, and prevention of graft rejection
are limited because of the hormone’s intrinsic hypercalcemic
effect.11 Therefore, the design of 1R,25(OH)2D3 analogues
exhibiting antiproliferativeand/orimmunoregulatory proper-
ties with concomitant low calcemic side effect is the topic
of intensive investigations at both mechanistic and clinical
levels.12-16 The development of such analogues has led to
more than 3000 compounds with structural modifications on
the A and/or CD rings or the aliphatic side chain. However,
only a few of these are used to treat human diseases.17,18
In order to optimize the aliphatic side chain conformation
with a subsequent entropy benefit, we recently designed and
synthesized the 1R,25(OH)2D3 analogue, (20S,23S)-epoxy-
methano-1R,25-dihydroxyvitamin D3 (AMCR277A, 2a, C23S,
Figure 1), which incorporates an oxolane ring in the side chain.19
We showed that it acts as a 1R,25(OH)2D3 superagonist because
it is able to mediate transcriptional activity with a magnitude at
least 10-fold higher than that of the natural ligand. In addition, it
has the capacity to inhibit the proliferation of the human
promyelocytic HL60 cells and induce their differentiation
into monocyte-like phenotype at concentrations much lower
than 1R,25(OH)2D3. In contrast, we demonstrated that its
epimer, (20S,23R)-epoxymethano-1R,25-dihydroxyvitamin D3
(AMCR277B, 2b,C23R, Figure 1), behaves like 1R,25(OH)2D3.
Previous studies have shown that incorporation of an R-methyl
at C2 of the A-ring of 1R,25(OH)2D3 does not result in super-
agonistic activity, although this compound (3, Figure 1) exhibits
a 3-fold higher affinity to the VDR LBD relative to the natural
ligand.20,21 In order to improve the capacity of 1R,25(OH)2D3
agonists in stimulating transcription and cellular growth arrest,
we undertook the structure-based design of novel 1R,25(OH)2D3
The pleiotropic actions of the hormonally active metabolite
of vitamin D3, the seco-steroid 1R,25-dihydroxyvitamin D3
(1, 1R,25(OH)2D3, Figure 1), are mediated through itsbinding
to the cognate nuclear vitamin D receptor (VDRa), a ligand-
dependent transcriptional modulator that belongs to the
superfamily of steroid/thyroid hormone/retinoid nuclear
receptors (NR).1-3 Among the structural multidomains of
NR, the ligand binding domain (LBD), located at the carboxy
terminus, is a key transcriptional regulator because it harbors
the ligand induced transactivation function 2 (AF-2).4 Upon
ligand complexation, the LBD undergoes a major conforma-
tional change involving the folding back of H12 onto the core
of LBD, termed the “mouse-trap” mechanism.5,6
Activated VDR controls multiple long-term biological
responses including cell growth, apoptosis, angiogenesis, anti-
proliferation, differentiation, bone mineralization, and cal-
cium/phosphate homeostasis.7-10 Therapeutic applications
of pharmacological doses of 1R,25(OH)2D3, which encompass
treatments for renal osteodystrophy, osteoporosis, psoriasis,
†The atomic coordinates and structure factors (PDB codes 3A3Z and
3A40) have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ (http://www.rcsb.org/).
*To whom correspondence should be addressed. For D.M.: phone,
33.3.88.65.32.20; fax, 33.3.88.65.32.76; e-mail, moras@igbmc.fr.
For N.R.: phone, 33.3.88.65.57.81; fax, 33.3.88.65.32.76; e-mail:
rochel@igbmc.fr.
a Abbreviations: NR, nuclear receptor; 1R,25(OH)2D3, 1R,25-dihy-
droxyvitamin D3; LBD, ligand binding domain; VDR, vitamin D
receptor; rmsd, root-mean-square deviation; (S)-p-tolBINAP, (S)-(-)-
2,20-bis(di-p-tolylphosphino)-1,10-binaphthyl.
r
2010 American Chemical Society
Published on Web 01/13/2010
pubs.acs.org/jmc

1160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
Figure 1. Chemical structures of the 1R,25(OH)2D3 and the analogues 2a, 2b, 4a, and 4b.
Scheme 1. Synthesis of Vitamin D3 Analogues 2a and 2b by the
Wittig-Horner Approach
Scheme 2. Asymmetric Reduction of Unsaturated Esters 9a
and 9ba
a Reagents and conditions:(a) PMHS, CuCl, KOt-Bu, (S)-tolBINAP,
i-PrOH, hexanes, -25 °C, ultrasound, 1 h; then MeLi, THF, -78 °C f
room temp; (b) same as in step a but with (R)-tolBINAP instead of
(S)-tolBINAP.
analogues, the (20S,23S)-epoxymethano-1R,25-dihydroxyvita-
min D3 (2R-methyl-AMCR277A, 4a, C23S, Figure 1) and its
epimer, the (20S,23R)-epoxymethano-1R,25-dihydroxyvitamin
D3 (2R-methyl-AMCR277B, 4b, C23R, Figure 1). These new
1R,25(OH)2D3 analogues were synthesized to increase their
stability in the ligand binding pocket (LBP) to enhance 1R,25-
(OH)2D3 superagonistic activity.
The present study aims at gaining insight into the struc-
ture-activity relationships of 4a,b. To achieve this goal, we
solved the crystal structures of the hVDR LBD bound to4a or
to its epimer 4b and highlight a sharp difference between the in
vitro and the in vivo biological effects of the two 1R,25-
(OH)2D3 analogues.
methylation, separation of the resulting tertiary alcohols, desi-
lylation, and oxidation (Scheme 1).19 We now report the
synthesis of 1R,25(OH)2D3 analogues 4a and 4b from (R)-
carvone (A-ring precursor) and involving a stereoselective route
to the CD-building block. The key features of the new synthetic
strategy involve (1) palladium-catalyzed coupling between en-
oltriflate 8 and alkenylzinc intermediates derived from 7, (2)
stereoselective synthesis of bromides 7 from 6, and (3) synthesis
of enoltriflate 8 from (R)-carvone.
Synthesis of Alkenyl Bromides 7. Separation of a 1:1
mixture of (E,Z)-unsaturated esters 619 by HPLC provided
pure (E)-ester (6a, less polar compound) and (Z)-ester (6b,
more polar compound) (Scheme 2). The stereochemistry of
both isomers was established by NOE experiments
(dCHCO2Et). Asymmetric conjugate reduction of unsatu-
rated ester 6a under Buchwald conditions22 employing catalytic
Chemistry
We have recently described the synthesis of active 1R,25-
(OH)2D3 analogues 2a and 2b (Figure 1) by Lythgoe’s Wittig-
Horner approach using ketones 5as the CD side chain building
block. These ketones were prepared by catalytic hydrogenation
of unsaturated esters 6 followed by the following sequence:

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1161
Scheme 3. Synthesis of the Upper Alkenyl Bromides 7a and 7ba
Scheme 5. Formation of the Triene System by Pd(0)-Catalyzed
Tandem Cyclization Negishi Couplinga
a Reagents and conditions: (a) TBAF, THF, reflux, 4 days; PDC,
CH2Cl2, room temp, 12 h; (b) (Ph3PCH2Br)Br, NaHMDS, THF; (c)
Et3SiCl (TESCl), Im, DMAP, DMF, 0 °C f room temp, 12 h.
Scheme 4. Synthesis of the A-Ring Fragmenta
a Reagents and conditions: (a) t-BuLi, THF, -78 °C, 1 h; ZnBr2,
THF, -78 °C f 0 °C, 1 h; (b) 8, (Ph3P)4Pd, Et3N , THF, 12 h; (c) n-
Bu4NF, THF, room temp, 24 h; (d) HF Py, CH2Cl2, CH3CN, Et3N,
room temp, 2 h.
3
with the corresponding dialkylated compound (29%). The
stereochemistry of the methylation product 12 can be
explained by protonation of the corresponding enolate.
Degradation of the isopropenyl side chain25b of 12 by
ozonolysis and subsequent reaction of the resulting inter-
mediates with acetic anhydride followed by treatment with
sodium acetate provided the desired alcohol 13a in 80%
yield. Silylation of 13a with tert-butyldimethylsilyl chlor-
ide followed by reduction of the resulting silylated com-
pound 13b with lithium selectride followed by protection
afforded the disilylated epoxide 14b (77% yield for the
three steps). The sterochemical outcome of the reduction
product 14a can be explained by L-Selectride attack to
ketone 13b from the less hindered face. The structure of
compound 14a was established by NOE experiments (see
Supporting Information). Periodic acid-oxidative clea-
vage of 14b generated the aldehyde 15 which was trans-
formed into the vinylic dibromide 16 by olefination with
Ph3PdCBr2 (75% yield, 2 steps). Conversion of 16 to the
desired enoltriflate 8 was carried out in 75% yield using
conditions previously reported for the corresponding un-
alkylated compound:24i base treatment (LDA and n-BuLi)
to form the triple bond and the enolate and trapping of the
latter with N-(5-chloro-2-pyridyl)bis(trifluoromethanesul-
fonimide).
Synthesis of the Target 1r,25(OH)2D3 Analogues 4a and
4b. For the construction of the vitamin D triene system, we
chose the convergent Pd(0)-catalyzed tandem cyclization
Negishi coupling approach recently developed in our labor-
atories.24k The alkenylzinc intermediate 17a (CD side chain
building block, Scheme 5) was prepared by metalation of
alkenyl bromide 7a with t-BuLi followed by transmetalation
with ZnBr2. Addition of the enoltriflate 8, triethylamine, and
catalytic tetrakis(triphenylphosphine)palladium(0) gave,
after desilylation, the desired analogue 4a in 55% yield.
The other analogue 4b was prepared in a similar way (53%
yield) from alkenyl bromide 7b.
a Reagents and conditions: (a) H2O2, LiOH H2O, MeOH, 0 °C, 30
3
min; (b) NaH, THF, room temp, 1 h; MeI, 0 °C, 5 h (47%); (c) O3,
MeOH-CH2Cl2, -78 °C; Ac2O, Et3N, DMAP, -35 °C f 78 °C, 2 h;
NaOAc, MeOH, 37 °C, 12 h; (d) TBSCl (t-BuMe2SiCl), imidazole,
DMF, room temp, 12 h; (e) L-Selectride, THF, -78 °C, 30 min; (f )
H5IO6, Et2O, room temp, 12 h; (g) Ph3P, Zn, CBr4, CH2Cl2, 45 min; (h)
LDA, THF, -78 °C, 1 h; n-BuLi, 15 min,; 5-Cl-py-2-NTf2, -78 °C f
room temp, 12 h.
(R)-p-tolBINAP/CuCl/KOt-Bu and poly(methylhydrosilox-
ane) (PMHS) as the reducing agent and i-PrOH as the proton
source provided (C23S)-alcohol 9a19 in 92% yield. The use of
the enantiomeric ligand (S)-p-tolBINAP gave (C23R)-alcohol
9b19 in 86% yield. This strategy allows the preparation of 9a
(precursor of superagonist 2a) or 9b as the only products from
either unsaturated esters 6a or 6b. The above synthetic route
allows the preparation of alcohol 9a or 9b in high yield from
either ester 6a or 6b. The previous synthetic route19 provides a
mixture of alcohols 9a and 9b.
Alcohols 9a and 9b were converted to the corresponding
ketones 5a and 5b as previously described.19 Wittig olefina-
tion of 5a and 5b by the method of Trost23 followed by
silylation provided the required alkenyl bromides 7a and 7b
in 46% and 48% yield, respectively (two steps) (Scheme 3).
Synthesis of A-Ring Fragment 8. The synthesis of enoltri-
flate 8, precursor of the A-ring, started with (R)-carvone24
(Scheme 4). Stereoselective epoxidation of (R)-carvone pro-
vided the known epoxide 1124g,25 in 84% yield. Treatment of
11 with sodium hydride and alkylation with methyl iodide
gave the monoalkykated product 12 in 47% yield, together

1162 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
Figure 3. Detailed structural representation of both analogues
bound to hVDR LBD around the aliphatic chain (A) and the
2R-methyl group (B) showing the characteristic residues involved
in interactions. 4a and 4b are shown in green and blue, respectively.
Oxygen and nitrogen atoms in VDR/4a and VDR/4b structures are
shown in red and blue, respectively. Hydrogen and van der Waals
bonds are shown in red and black dotted lines, respectively.
Secondary structure of VDR is shown in cartoon. H2, H3, H6,
H7, and H11 indicate helices.
Figure 2. Conformation of the bound ligand. (A) The 4a (left) and
4b (right) are shown in the Fo - Fc electron density omit map
contoured at 3σ. The ligands are shown in stick representation with
carbon atoms in green for 4a and blue for 4b, and oxygen atoms are
in red. (B) A stereoview of the ligand conformations of 4a (green)
and 4b (blue) in the VDR ligand binding pocket after superimposi-
tion showing the different side chain conformation of the two
epimers. The chiral atoms at C23 are marked by asterisks.
hydrogen bonds forming the anchoring points. Compared
to the structure of hVDR LBD in complex 1R,25(OH)2D3,
the atomic coordinates of all CR atoms of hVDR bound to 4a
˚
and to 4b show an rmsd of 0.18 and 0.15 A, respectively. The
ligand is buried in the predominantly hydrophobic pocket.
3
The volumes of the ligands are 418, 416, and 396 A for 4a,
Results
˚
Overall Structures of the hVDR LBD Bound to 4a or 4b.
The hVDR LBD mutant lacking 50 residues in the loop
connecting helices H2 and H3 was used for the X-ray
analyses of the hVDR LBD in complex with 4a or 4b. The
same mutant was used to solve the structure of the hVDR
LBD bound to 1R,25(OH)2D3 and to several 1R,25(OH)2D3
analogues, since the biological properties of this mutant such
as ligand binding and transactivation in distinct cell lines are
the same as those of the wild type.6,26-28 Isomorphous
crystals were obtained in similar conditions, and the crystal
structures of hVDR LBD bound to 4a or 4b were determined
4b, and 1R,25(OH)2D3, respectively. The volume of the
ligand binding cavity is 685, 682, and 673 A and the ligands
occupy 61%, 61%, and 59% of the pocket for 4a, 4b, and
3
˚
1R,25(OH)2D3, respectively.
Ligand-Protein Interactions. The A, seco-B, C, and D
rings present conformations that are similar to those ob-
served in the presence of the natural ligand (Figure 2). The
distances between the 1-OH and the 25-OH groups are 12.9,
˚
13.1, and 12.8 A for hVDR LBD bound to 4a, 4b, and
1R,25(OH)2D3, respectively. The hydroxyl groups make the
same hydrogen bonds as hVDR LBD bound to 1R,25-
(OH)2D3 complex, 1-OH with Ser237 and Arg274, 3-OH
with Tyr143 and Ser278, and 25-OH with His305 and
His397.
A comparison of the aliphatic chain of 4a to those of the
previous nonmethylated 2a and 2b19 reveals that the specific
van der Waals interactions of the side chain with His-305,
His-397, Val-418 and of the O21 atom of the oxolane ring
with Val300 are conserved (Figure 3A). Similar to 2b, the
additional van der Waals contact of O21 with Val300 is
weaker in the VDR-4b complex. This interaction with
Val300 has been observed in some hVDR LBD crystal
˚
at a resolution of 1.7 and 1.45 A, respectively (Supporting
Information Table 1).
Complexes of hVDR LBD with 4a or 4b adopt the
canonical conformation of all previously reported agonist-
bound nuclear receptor LBDs with 12 or 13 R-helices orga-
nized in a three-layered sandwich. In all the structures of
hVDR LBD bound to agonist ligands, a unique conforma-
tion of the complex is observed. The position and conforma-
tion of the activation helix H12 are strictly maintained. The
ligand adopts the same orientation in the pocket. An adapta-
tion of their conformation is observed to maintain the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1163
2
˚
structures with superagonist ligands 1R,25-dihydroxy-20-
epi-22-oxa-24a,26a,27a-trihomovitamin D3 (KH1060) or
19-nor-14-epi-23-yne-1,25-(OH)2D3 (TX522).28,29 The stereo-
isomer 4b presents an energetically unfavorable oxolane ring
and adopts a different side chain conformation because of
the inverse configuration at C23 which affects the positions
of C25, C26, C27, and 25-OH and destabilizes the activation
helix-12 (Val418) compared to 4a.
32.3 A for the hVDR LBD/4a complex and 4b complex,
respectively).
Binding Affinity of the SRC-1 Peptide to hVDR LBD
Bound to 2a or 4a Is Similar. To check the ligand effect on
the recruitment of coactivator peptide to VDR, binding of
fluorescently labeled SRC-1 peptide to hVDR LBD bound
to 1R,25(OH)2D3, 2a, 2b, 4a, and 4b was monitored by
fluorescence anisotropy. In its apo form, hVDR LBD does
not bind the SRC-1 coactivator peptide. The magnitude of
the SRC-1 binding when complexed to 2a or 4a was similar to
that obtained with the natural ligand. In contrast, the 4b
ligand induces a 3-fold weaker binding between hVDR LBD
and the SRC-1, whereas its parental analogue 2b lowers it by
1.7-fold (Table 1). Therefore, stereochemistry of the C23 is a
significant parameter for the recruitment of the SRC-1
peptide.
4a Is a More Potent 1R,25(OH)2D3 Superagonist Than 2a.
Using gene reporter assays, we recently reported that 2a
shows superagonistic biological activities because it stimu-
lates transcription 12-fold more than the natural ligand at
0.1 nM.19 Methylation at the C2R position of the 1R,25-
(OH)2D3 A ring induces a 3-fold increase of the binding
affinity of hVDR LBD. Hence, we postulate that C2R
methylation of 2a may further increase its superagonist
character.
The introduction of a methyl group at the 2R position does
not modify the A-ring chair conformation of each ligand.
The 2R-methyl group fills a small cavity of the pocket and
makes additional van der Waals interactions with Phe150,
Leu233, and Ser237 (Figure 3B), contacts also observed in
the hVDR LBD complexed to 2R-methyl-1R,25(OH)2D3.20
The previously reported crystal structures of hVDR LBD in
complex with 1R,25(OH)2D3 and several synthetic ligands
revealed the presence of tightly bound water molecules
forming a channel near the C2 position of the ligand, which
may play important roles in protein stability.20 This water
channel is also conserved in the two present 4a and 4b
complexes, and the B-factors of the three waters (7.0, 6.4,
2
˚
and 3.3 A for the hVDR LBD/4a complex and 12.4, 11.9,
2
and 10.3 A for the 4b complex, respectively) are significantly
˚
lower than the average value of all water molecules (28.1 and
Here, we demonstrate that at 0.1 nM, the transcription
activity upon 4a stimulation is 13- and 3.8-fold higher than
those induced by the natural ligand and 2a, respectively.
Furthermore, at 5 nM 4a achieves optimal VDR-directed
transcription, whereas 4b is 4 times less potent under similar
stimulatory conditions in transactivation assays and behaves
like the natural ligand (Figure 4). The dose-dependent
comparison between 2a and its new to 2R-methyl derivative
reveals that at 0.1, 1, and 5 nM, the transcription activities
induced by 2a are only 26%, 42%, and 73% of that obtained
Table 1. Dissociation Constants of the SRC-1 Peptide to hVDR LBD in
Response to 1R,25(OH)2D3 Analogues
analogues
relative dissociation constants
no ligand
1R,25(OH)2D3
no binding
1
2a
2b
4a
4b
0.9 ( 0.2
1.7 ( 0.2
1.3 ( 0.2
3.2 ( 0.2
Figure 4. 4a acts as an 1R,25(OH)2D3 superagonist in vitro. Transient transfections and luciferase reporter gene assays using MCF-7 human
breast cancer cells were performed as reported in Experimental Procedures. Relative luciferase activities were calculated by dividing the
luciferase activity by the respective β-galactosidase activity to correct for differences in transfection efficiency. For every triplicate the mean and
the standard deviation were calculated. A two-tailed, paired Student’s t test was performed when appropriate, and p values were calculated with
reference to stimulation of hVDR with the solvent.

1164 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
Figure 5. 4a displays higher HL60 cell antiproliferation and prodifferentiating potency relative to its parental analogue 2a. (A) 4a mediated
HL60 cell growth arrest is achieved for a dose of 0.1 nM. HL60 cells were incubated for 96 h with various concentrations of 1R,25(OH)2D3, 2a,
2b, 4a, or 4b versus control (ethanol 0.7%) and are counted. Data are presented as the mean ( SEM. (B) 4a mediated HL60 differentiation into
monocyte-like cells is achieved for a dose of 0.1 nM. Cells were labeled with PE-labeled antihuman CD11c and FITC-labeled antihuman CD14,
and HL60 cell differentiation was estimated by the double-positive CD11c/CD14 subpopulation. Data are representative of three distinct
experiments.
in the presence of 4a, respectively. In summary, 4a is a more
potent superagonist compared to 2a while its epimer 4b is as
active as the natural ligand (1).
differentiation was obtained with 0.1 nM 4a, as a new double
positive CD11c/CD14 subpopulation was detected com-
pared to the other 1R,25(OH)2D3 analogues (Figure 5B).
Moreover, single positive CD11c and CD14 cell populations
were more abundant relative to 2a and the natural ligand (at
least 1.5- and 3-fold increase, respectively). A similar profile
was observed with 10 nM 1R,25(OH)2D3 analogues treat-
ment, and a lower or equal dose of 2R-methyl-1R,25(OH)2D3
achieves neither antiproliferation nor differentiation of
HL60 cells (data not shown). Therefore, C2R methylation
of 2a increases its ability to inhibit HL60 cell proliferation
with concomitant differentiation into monocyte-like pheno-
type.
At low dose, 4a is more calcemic than 1r,25(OH)2D3.
Whereas 4b Displayed in Vivo Low Calcemic Action Com-
pared to the Natural Ligand. Using C57BL/6J mice, we
previously showed that 2a exhibits higher calcemic action
than 1R,25(OH)2D3, whereas 2b has an in vivo low calcemic
effect than the natural ligand.19 We monitored the calcium
serum level of male mice that were subjected to 0.1 μg/kg
intraperitoneal injections (ip) of 1R,25(OH)2D3 analogues
versus untreated (control) ones. Mice survive during the
Only 4a at 0.1 nM Induces HL60 Cell Proliferation Arrest
with Concomitant Differentiation. Our previous data showed
that the superagonistic property of 2a is associated with a
higher ability, compared to the natural ligand, to inhibit the
proliferation of HL60 promyelocytic cell and to trigger their
subsequent differentiation into monocyte-like phenotype as
evidenced by the up-regulation of cell surface markers
CD11c and CD14.19 We further examined the effects of 4a
and 4b in directing HL60 cell fate. Here, we found that the
dose-dependent profile of 4b in mediating HL60 cell arrest
and differentiation is similar to that induced by 2b. In sharp
contrast, a dose of only 0.1 nM of 4a is able to significantly
reduce HL60 cell proliferation, whereas no effect at this
concentration was observed for the other 1R,25(OH)2D3
analogues, including its parental analogue 2a, compared to
control incubations (Figure 5A). A similar proliferation
pattern was detected when HL60 cells were incubated with
1 nM 1R,25(OH)2D3 agonists. Consistent with cell prolifera-
tion data, a 10-fold gain in the induction of HL60 cells

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1165
adopts the canonical orientation, sealing the ligand binding
cavity.22,30,31 Comparison of complexes formed by hVDR
LBD with 2a, 1R,25(OH)2D3 or 2R-methyl-1R,25(OH)2D3,
indicates that the new ligand 4a displays an increased number
of van der Waals contacts with the LBP than the other ones.
These additional contacts may explain in part the higher
potency of 4a in mediating VDR-dependent transcription.
Although 2R-methyl-1R,25(OH)2D3 displays a higher binding
affinity for hVDR LBD relative to its natural ligand, it does
not exhibit superagonist properties. On the other hand, we
have shown that 2a displays superagonistic biological activ-
ities.19 Therefore, methylation at the C2R of the 2a analogue
synergistically increases its superagonistic character
(Figure 4). In contrast, C2R methylation of 2b does not
improve its ability to further induce VDR-directed transcrip-
tion compared to its parental 2b analogue or the natural ligand
(Figures 2 and 3). Because of the inverse configuration at C23,
the stereoisomers 4b and 2b adopt a different side chain
conformation compared to 4a and 2a affecting their respective
contacts with activation helix 12 and resulting in a weaker
activation. This property is conserved in the 4b relative to 4a,
demonstrating that the active form of the bound ligand favored
by the ring pucker is an essential parameter that directs the
magnitude of VDR-induced transcription and HL60 cell anti-
proliferation/prodifferentiation (Figures 4 and 5).
Previous studies have shown that stronger VDR-coacti-
vator interactions underlie the superagonistic activity of
1R,25(OH)2D3 analogues, such as 14-epi analogues.28 To gain
insights into the potential molecular mechanisms that may
explain the superagonistic strength of 4a relative to 2a, we
determined the binding affinity of the coactivator SRC-1
peptide to hVDR LBD incubated with each analogue. A
higher recruitment of SRC-1 to hVDR LBD upon binding
to 4a does not account for its increased superagonist char-
acter, as 4a induces weaker binding between hVDR LBD and
the SRC-1 peptide compared to either 1R,25(OH)2D3 or 2a
which induces a binding efficiency fully comparable to that
obtained with the natural ligand (Table 1). Therefore, larger
domains of SRC-1 or other coactivators may play a key rolein
VDR-dependent transcription in response to 4a.
Since the 2a superagonist character is associated with a
more potent ability to inhibit HL60 cell proliferation with
concomitant differentiation into monocyte-like cells, we pos-
tulate that increased stabilization of the complex hVDR LBD
bound to 4a would result in a higher capacity to reduce HL60
cell growth arrest at low concentrations. We recapitulated the
profile of each previously described 1R,25(OH)2D3 analogue
in the modulating HL60 cell fate, namely, 2a at 1 nM induces
HL60 cell antiproliferation, whereas 2b behaves like the
natural ligand.19 We now show that 4a induces a 10-fold gain
in lowering HL60 cell proliferation, as 0.1 nM 4a leads to a
significant decrease of HL60 cells number, whereas no effect
was observed at this concentration with the other 1R,25-
(OH)2D3 analogues, including 4b (Figure 5A). A similar
profile is observed at a dose of 1 nM, 4a being more efficient
compared to its parent analogue or 2R-methyl-1R,25(OH)2D3
(Figure 5A and not shown). Consistent with our previous
findings, the antiproliferative effect of 4a correlates with its
capacity to better differentiate HL60 cells into monocyte-like
cells comparedto2a (Figure5B). It shouldbeemphasizedthat
the 2R-methyl-1R,25(OH)2D3 has been reported to be be-
tween 2- and 4-fold more potent relative to 1R,25(OH)2D3 in
inducing differentiation of HL60 cells, depending on the
quantified parameter (nitroblue tetrazolium reduction assay
Figure 6. 4a is more calcemic than 2a or 1R,25(OH)2D3, whereas 4b
is less calcemic. Mice (6 weeks old; 6-7 mice/group) were injected
intraperitoneally with 1R,25(OH)2D3, 4a, 4b, or sesame oil (vehicle)
for 3 weeks. A single dose of 0.1 μg/kg was administrated every
second day, and calcium serum level was weekly monitored: (open
diamond) control, (black diamond) 1R,25(OH)2D3, (open triangle)
4a, and (black triangle) 4b incubations. Data are presented as the
mean ( SEM.
3 weeks of the experiments, and their physiological state was
satisfactory in response to the different analogues (data not
shown). We observed that mice treated with 4b leads to
calcemic values that are similar to those induced by sesame
oil as vehicle incubations. In contrast, ip injections of 4a
promote a robust calcium increase in the serum of mice,
which is even higher than that detected with the natural
ligand and similar to 2a19 (Figure 6). Thus, C2R methylation
of 2a improves the superagonist character of its parental
analogue but not its calcemic property.
Discussion
The present study reports the crystal structures of the
human VDR LBD bound to novel 1R,25(OH)2D3 analogues,
the 4a, and its epimer4b anddescribestheir in vitro and in vivo
biological effects. The rationale to undertake this study was
based on three distinct observations: (i) compared to the
crystal structure of hVDR LBD in complex with its natural
ligand, those with 2R-methyl-1R,25(OH)2D3 and 2a show
different van der Waals interactions that may explain in part
their biological action; (ii) 2R-methyl-1R,25(OH)2D3 displays
higher binding affinity for hVDR LBD compared to 1R,25-
(OH)2D3; (iii) 2a is a 1R,25(OH)2D3 superagonist or has the
capacity to better induce the differentiation of HL60 cells into
monocyte-like phenotype than the natural ligand, while its
epimer behaves like the natural ligand.19,20 Thus, a methyla-
tion at the C2R of the A ring combined with an incorporation
of an oxolane ring in the side chain leads to a new 1R,25-
(OH)2D3 analogue that may behave as a more potent super-
agonist associated with stronger antitumoral properties in
vitro. Therefore, we synthesized the 4a and its epimer 4b
(Figure 1) and investigated their structure-function relation-
ships.
Atomic resolution of the crystal structure of the hVDR
LBD bound to 4a reveals that this complex inherits structural
features of both 2R-methyl-1R,25(OH)2D3 and 2a, as evi-
denced by the specific van der Waals contacts of the 2R-
methyl group with Phe150, Leu233, Ser237 and of the O21
atom of theoxolane ring withVal300(Figure3). Aspreviously
reported for hVDR LBD bound to agonist ligands, helix 12

1166 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
and CD11b surface cell marker expression).21,32,33 However,
no information of the concentration of the analogues used in
these studies was provided. Our present data indicate that
1 nM 2R-methyl-1R,25(OH)2D3 was not sufficient to induce
HL60 cells antiproliferation or differentiation (not shown).
However, when used at the same low concentration, the C2R-
methylated derivative of 2a does achieve HL60 cell differen-
tiation. This property is associated with a better binding
affinity of 4a for VDR compared to 2a and 1R,25(OH)2D3,
as evidenced by electrospray ionization mass spectrometry
experiments (data not shown).
The in vitro biological data were not accompanied with a
better capacity to reduce the serum calcium level in 4a-treated
mice. Nevertheless, we report that ip administration for
3 weeks of 4a or 4b every 2 days results in an optimal survival
of mice, as no mice death was observed. Further dissocia-
tion between antiproliferative and calcemic effects has to be
improved.
In summary, we have synthesized a novel 1R,25(OH)2D3
analogue, the 2R-methyl-20S,23S-epoxymethano-1R,25-di-
hydroxyvitamin D3 (4a), which exhibits superagonistic and
cellular antiproliferation/prodifferentiation properties while
showing similar calcemic effects. In addition, its epimer 4b
(23R), which behaves like the natural ligand in vitro, is a
noncalcemic vitamin D in vivo. To validate the potential
application of these two analogues, further in vitro studies
in distinct cell types are required to determine the specific gene
activity and metabolic degradation.
concentrated in vacuo. The residue was purified by flash chro-
matography (SiO2, 1.5 cm ꢀ 9 cm, 10% EtOAc/hexanes) to give
9a19 [45 mg, 92% (two steps), Rf = 0.2 (20% EtOAc/hexanes)].
Following a similar procedure, 9a was also prepared from 6b in
76% yield. Spectral data for the compound 9a were identical to
those reported.19
8E-Bromomethylene-(20S,23S)-epoxymethano-25-hydroxy-des-
A,B-cholesta-8-one (10a). Sodium hexametildisilazide (0.75 mL,
1.5 mmol, 2 M, 3 equiv) was added to (bromomethylene)-
triphenylphosphonium bromide (0.680 g, 1.56 mmol, 3.1 equiv)
in 10 mL of THF at -60 °C. After 1 h, a solution of ketone 5a
(0.155 g, 0.502 mmol, 1 equiv) in THF (5 mL) was added via
cannula. The solution was stirred at -60 °C for 5 min and then at
room temperature for 3 h. Hexanes were added, and the
suspension was filtered over a pad of silica gel (elution with
20% Et2O/hexanes). After concentration in vacuo, the residue
was purified by flash chromatography (SiO2, 2 cm ꢀ 12 cm, 10%
EtOAc/hexanes) to give the bromide 10a [94 mg, 49%, colorless
oil, Rf = 0.6 (50% EtOAc/ hexanes)]. 1H NMR (250 MHz,
CDCl3): δ 5.61 (broad s, 1H, H-7), 4.09 (t, 1H, J = 7.9 Hz,
H-28), 3.32 (t, 1H, J = 9.2 Hz, H-28), 2.83 (m, 1H, H-9), 2.45
(m, 1H, H-23), 1.22 (s, 3H, CH3-21), 1.20 (s, 6H, CH3-26 and
CH3-27), 0.66 (s, 3H, CH3-18). 13C NMR (62.9 MHz, CDCl3): δ
144.8 (dC, C-8), 97.5 (dCH, C-7), 84.8 (C, C-20), 75.0 (CH2, C-
28), 70.7 (C, C-25), 59.0 (CH, C-17), 56.0 (CH, C-14), 46.3
(CH2), 46.0 (CH2), 45.5 (C, C-13), 40.0 (CH2), 34.1 (CH, C-23),
30.9 (CH2), 30.0 (CH3, C-27), 29.7 (CH3, C-26), 27.2 (CH3,
C-21), 22.5 (CH2), 22.3 (CH2), 21.7 (CH2), 13.3 (CH3, C-18).
8E-Bromomethylene-(20S,23S)-epoxymethano-25-[triethylsi-
lyl]oxy]-des-A,B-cholestan-8-one (7a). Triethylsilyl chloride
(0.140 mL, 0.84 mmol, 3 equiv) was added dropwise to a
solution of alcohol 10a (0.108, 0.280 mmol, 1 equiv), imidazol
(0.06 g, 0.84 mmol, 3 equiv), and DMAP (0.007 g, 0.06 mmol,
0.2 equiv) in dry DMF (2 mL) at 0 °C. The cooling bath was
removed, and the mixture was stirred for 12 h and poured onto a
mixture of ice and saturated NH4Cl (10 mL). The aqueous phase
was extracted with hexanes (3 ꢀ 10 mL). The combined organic
phase was dried, filtered, and concentrated in vacuo. The residue
was purified by flash chromatography (SiO2, 2 cm ꢀ 10 cm, 5%
EtOAc/hexanes) to give 7a [0.132 g, 94%, solid, Rf = 0.66 (10%
EtOAc/hexanes). IR (dissolved in CHCl3, cm-1): 1630 (νCdC).
1H NMR (250 MHz, CDCl3): δ 5.63 (broad s, 1H, H-7), 4.09 (t,
1H, J = 7.9 Hz, H-28), 3.32 (dd, 1H, J1 = 9.7 Hz, J2 = 8.6 Hz,
H-28), 2.84 (m, 1H, H-9), 2.47 (m, 1H, H-23), 2.05 (d, 1H, J =
12.5 Hz, H-12), 1.97 (dd, 1H, J1 = 11.9 Hz, J2 = 7.3 Hz, H-14),
1.22 (s, 3H, CH3-21), 1.20 (s, 6H, CH3-26 and CH3-27), 0.93
(t, 9H, J = 7.8 Hz, 3 ꢀ CH3CH2-Si), 0.68 (s, 3H, CH3-18), 0.55
(c, 6H, J = 7.8 Hz, 3 ꢀ CH3CH2-Si). 13C NMR (62.9 MHz,
CDCl3): δ 144.9 (dC, C-8), 95.6 (dCH, C-7), 84.4 (C, C-20),
75.2 (CH2, C-28), 73.1 (C, C-25), 59.1 (CH, C-17), 56.0 (CH, C-
14), 47.9 (CH2), 46.2 (CH2), 45.5 (C, C-13), 40.0 (CH2), 34.2
(CH, C-23), 30.9 (CH2), 30.7 (CH3, C-27), 30.0 (CH3, C-26),
27.2 (CH3, C-21), 22.6 (CH2), 22.4 (CH2), 21.7 (CH2), 13.4
(CH3, C-18), 7.1 (3 ꢀ CH3, 3 ꢀ CH3CH2-Si), 6.7 (3 ꢀ CH2, 3 ꢀ
CH3CH2-Si). HMRS ([CI]þ): calculated for [C26H48O2Si79Br]þ
([M þ H]þ), 499.2607; found, 499.2609.
Experimental Procedures
Chemistry. General. For general experimental procedures,
see ref 19. CD-ring intermediates were named following the
steroidal nomenclature.34 Vitamin D analogues are named as
vitamin D derivatives. IUPAC rules were used for the other
compounds. In addition to NMR, HPLC analysis was used to
determine the purity (>95%) of the vitamin D analogues.
Ethyl 8β-[(tert-Butyldimethylsilyl)oxy]-(20R,23)-epoxymeth-
ano-des-A,B-cholest-23E-en-25-oate (6a) and Ethyl 8β-[(tert-
Butyldimethylsilyl)oxy]-(20R,23)-epoxymethano-des-A,B-cholest-
23Z-en-25-oate (6b). A mixture of 6a and 6b (1.01 g), prepared
by phosphonate chemistry,19 was separated by HPLC
(Phenomenex Silica(2), 250 mm ꢀ 21.2 mm, isocratic 3% EtOAc/
hexanes) to give 6a (E, 0.52 g, 46%, colorless oil) and 6b (Z, 0.48 g,
42%, white solid, mp = 101 °C). Spectral data for the compounds
6a and 6b were identical to those reported.19
8β-[(tert-Butyldimethylsilyl)oxy]-(20S,23S)-epoxymethano-25-
hydroxy-des-A,B-cholestane (9a) from 6a and 6b. PMHS
[(poly(methylhydrosiloxane)] (0.11 mL, 1.82 mmol, 16 equiv)
was added to a suspension of CuCl (8 mg, 0.08 mmol, 0.7 equiv),
KOt-Bu (10 mg, 0.086 mmol, 0.75 equiv), and (R)-p-tolBINAP
(9.3 mg, 0.014 mmol, 0.12 equiv) in hexanes (2 mL). The mixture
was sonicated for 2 h. The color changed from yellow to deep-
red. After the mixture was cooled to -25 °C, a solution of 6a
(50 mg, 0.114 mmol, 1 equiv) in hexanes (2 mL) and i-PrOH
(0.035 mL, 0.456 mmol, 4 equiv) was added. After 1 h, the
reaction was quenched by the addition of saturated NH4Cl
(5 mL) and EtOAc (5 mL). The mixture was extracted with
EtOAc (2 ꢀ 10 mL), and the combined organic phase was dried,
filtered, and concentrated in vacuo. The residue was dissolved in
dry THF and cooled to -78 °C. After 15 min, a solution of MeLi
in Et2O (0.18 mL, 0.285 mmol, 1.6 M, 2.5 equiv) was added via
syringe. The resulting solution was stirred for 5 min at -78 °C
and then at room temp for 1 h. After the mixture was cooled to
-78 °C, the reaction was quenched by the slow addition of
saturated NH4Cl (3 mL). The mixture was extracted with Et2O
(3 ꢀ 5 mL). The combined organic phase was dried, filtered, and
(1R,3S,4R,6R)-1,3-Dimethyl-4-(prop-1-en-2-yl)-7-oxa-bicyclo-
[4.1.0]heptan-2-one (12). A solution of ketone 11 (10 g, 59.4 mmol,
1 equiv) in dry THF (50 mL) was added via syringe to a suspension
of NaH (2.16 g, 89.1 mmol, 1.5 equiv) in THF (65 mL). After being
stirred for 1 h, the mixture was cooled to 0 °C and then stirred for
10 min. MeI (4.5 mL, 72.3 mmol, 1.2 equiv) was added dropwise
via syringe. The reaction mixture was stirred for 4 h. The reaction
was quenched with saturated NH4Cl (100 mL). The mixture was
extracted with Et2O (3 ꢀ 50 mL). The combined organic phase was
dried, filtered, and concentrated. The residue was purified by
MPLC (SiO2, 5 cm ꢀ 45 cm, 3% EtOAc/hexanes) to give the
monoalkylated compound 12 [5.03 g, 47%, colorless liquid, Rf =
0.37 (10% EtOAc/hexanes)]. Starting material recovered: 2.49 g
(63% conversion). [R]D25 147 (c 2.4, CHCl3). IR (neat, cm-1): 1732

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1167
(νCdO), 1659 (νCdC). 1H NMR (400 MHz, CDCl3): δ 4.81 (s, 1H,
H-20), 4.79 (s, 1H, H-20), 3.40 (d, 1H, J = 3.1 Hz, H-6), 2.49 (td,
1H, J1 = 11.9 Hz, J2 = 4.2 Hz, H-4), 2.21 (dt, 1H, J1 = 14.8 Hz,
J2 = 3.4 Hz, H-5), 1.97 (2H, m, H-3 and H-5), 1.63 (3H, s, CH3-30),
1.41 (3H, s, CH3C-1), 1.04 (3H, d, J =6.9 Hz, CH3C-3). 13C NMR
(100.6 MHz, CDCl3): 207.6 (CdO, C-2), 144.9 (=C, C-10), 113.2
(dCH2, C-20), 60.6 (CH, C-6), 58.5 (C, C-1), 44.7 (CH, C-3), 42.2
(CH, C-4), 29.2 (CH2, C-5), 18.4 (CH3, C-30), 15.8 (CH3, CH3C-1),
13.8 (CH3, CH3C-3). HMRS ([CI]þ): calculated for [C11H17O2]þ
([M þ H]þ), 181.1228; found, 181.1222.
HMRS ([CI]þ): calculated for [C14H27O3Si]þ ([M þ H]þ),
271.1729; found, 271.1724.
(1S,2S,3R,4R,6R)-4-[(tert-Butyldimethylsilyl)oxy]-1,3-dimet-
hyl-7-oxabicyclo[4.1.0]heptan-2-ol (14a). A solution of L-Selec-
tride (22 mL, 22.2 mmol, 1 M, 1.5 equiv) in dry THF was added
dropwise to a solution of ketone 13b (4 g, 14.8 mmol, 1 equiv) in
dry THF (40 mL) at -78 °C. After 30 min, the reaction was
quenched by the dropwise addition of MeOH (10 mL) and H2O
(10 mL). A solution of NaOH (10 mL, 10%) and H2O2 (10 mL,
30% v/v) was successively and slowly added. The mixture was
allowed to reach room temperature over 12 h. A saturated
solution of NH4Cl (30 mL) was added. The aqueous phase
was extracted with a solution of EtOAc/hexanes (20%, 3 ꢀ 50 mL).
The combined organic phase was dried, filtered, and concen-
trated. The residue was purified by flash chromatography (SiO2,
4 cm ꢀ 10 cm, 20% EtOAc/hexanes) to give 14a [3.51 g, 87%,
colorless, Rf = 0.4 (20% EtOAc/hexanes)]. [R]2D5 -79.7 (c 3.7,
(1R,3S,4R,6R)-4-Hydroxy-1,3-dimethyl-7-oxabicyclo[4.1.0]-
heptan-one (13a). A stream of O3/O2 (0.7 bar, 0.1 NL/h, 50 w)
was bubbled through a -78 °C cooled solution of ketone 12 (5 g,
27.74 mmol, 1 equiv) in dry MeOH (5.1 mL, 125 mmol,
4.5 equiv) and dry CH2Cl2 (50 mL) until the solution turned
blue (30 min). The excess of O3 was removed with a flow of argon
for 30 min. The reaction mixture was allowed to reach room
temperature (40 min) under a slow flow of argon and then
cooled to -35 °C. After 10 min, Et3N (30 mL, 222 mmol,
8 equiv) and DMAP (0.7 g, 5.6 mmol, 0.2 equiv) were succes-
sively and slowly added. Once DMAP has dissolved, Ac2O
(21 mL, 222 mmol, 8 equiv, freshly distilled from P2O5 under
argon) was added. The reaction mixture was allowed to reach
-8 °C and stirred for 2 h. The reaction was quenched by the slow
addition of MeOH (20 mL). The mixture was stirred at room
temperature for 5 min, diluted with EtOAc (100 mL), and
successively washed with an aqueous solution of citric acid
(2 ꢀ 50 mL, 10%) and saturated NaHCO3 (2 ꢀ 50 mL). The
combined organic phase was dried, filtered, and concentrated.
The residue was dissolved in MeOH (60 mL). NaOAc (0.460 g,
5.55 mmol, 0.2 equiv) was added. The mixture was heated at
37 °C for 12 h and then concentrated to half volume. The residue
was dissolved in EtOAc (30 mL) and washed with saturated
NH4Cl (25 mL). The aqueous phase was extracted with EtOAc
(2 ꢀ 20 mL). The combined organic phase was dried, filtered,
and concentrated. The residue was purified by flash chroma-
tography (SiO2, 5 cm ꢀ 10 cm, 20% EtOAc/hexanes) to give
alcohol 13a [3.47 g, 80%, colorless oil, Rf = 0.30 (50% EtOAc/
hexanes)]. [R]2D5 79.8 (c 1.3, CHCl3). IR (neat, cm-1): 3445
(νO-H), 1707 (νCdO). 1H NMR (400 MHz, CDCl3): δ 3.79 (td,
1H, J1 = 13.6 Hz, J2 = 9.1 Hz, H-4), 3.38 (s, 1H, H-6), 2.62 (dt,
1H, J1 = 14.4 Hz, J2 = 3.6 Hz, H-5), 2.41 (s, 1H, OH), 2.01 (m,
1H, H-3), 1.92 (dd, 1H, J1 = 14.4 Hz, J2 = 10.0 Hz, H-5), 1.38
(s, 3H, CH3C-1), 1.21 (d, 3H, J = 7.0 Hz, CH3C-3). 13C NMR
(100.6 MHz, CDCl3): δ 206.1 (CdO, C-2), 67.9 (CH, C-4), 59.9
(CH, C-6), 58.5 (C, C-1), 50.8 (CH, C-3), 32.1 (CH2, C-5), 15.2
(CH3, CH3C-1), 12.5 (CH3, CH3C-3). Anal. Calcd for
[C8H12O3]: C, 62.06; H, 8.46. Found: C, 61.52; H, 7.94.
1
CHCl3). IR (neat, cm-1): 3471 (νO-H). H NMR (400 MHz,
CDCl3): δ 3.82 (dd, 1H, J1 = 10.3 Hz, J2 = 4.5 Hz, H-2), 3.51
(td, 1H, J1 = 9.0 Hz, J2 = 4.6 Hz, H-4), 3.24 (sa, 1H, H-6), 2.37
(dd, 1H, J1 = 14.8 Hz, J2 = 4.5 Hz, H-5), 1.81 (d, 1H, J = 10.3
Hz, OH), 1.66 (dd, 1H, J1 = 14.8 Hz, J2 = 9.0 Hz, H-5), 1.49 (m,
1H, H-3), 1.42 (s, 3H, CH3C-1), 0.98 (d, 3H, J = 7.0 Hz, CH3C-3),
0.87 (s, 9H, Me3C-Si), 0.04 (s, 3H, CH3-Si), 0.03 (s, 3H, Me-Si).
13C NMR (100.6 MHz, CDCl3): δ 72.8 (CH, C-2), 66.3 (CH,
C-4), 63.7 (CH, C-6), 60.9 (C, C-1), 42.2 (CH, C-3), 34.9 (CH2,
C-5), 25.8 (3 ꢀ CH3, Me3C-Si), 21.6 (CH3, CH3C-1), 18.0 (C,
C-Si), 12.4 (CH3, CH3C-3), -4.4 (CH3, Me-Si), -4.8 (CH3,
Me-Si). Anal. Calcd for [C8H12O3]: C, 61.72; H, 10.36. Found:
C, 61.93; H, 10.49.
(1R,2S,3S,4R,6R)-2,4-Bis[(tert-butyldimethylsilyl)oxy)-1,3-
dimethyl-7-oxabicyclo[4.1.0]heptane (14b). Imidazole (0.63 g,
9.2 mmol, 2.5 equiv) and TBSCl (1.1 g, 7.34 mmol, 2 equiv)
were successively added to a solution of alcohol 14a (1 g, 3.67 mmol,
1 equiv) in dry DMF (10 mL). After 12 h, the reaction was quenched
by the addition of ice pieces and saturated NH4Cl (10 mL). The
aqueous phase was extracted with hexanes (3 ꢀ 15 mL). The
combined organic phase was dried, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography (SiO2,
3 cm ꢀ 10 cm, hexanes) to give 14b [1.37 g, colorless oil, Rf = 0.78
(20% EtOAc/hexanes)]. [R]2D5 -42.3 (c 4.2, CHCl3). 1H NMR
(400 MHz, CDCl3): δ 4.22 (d, 1H, J = 5.7 Hz, H-2), 3.79 (td, 1H,
J1 = 9.1 Hz, J2 = 4.4 Hz, H-4), 2.97 (d, 1H, J = 4.5 Hz, H-6), 2.19
(dd, 1H, J1 = 15.5 Hz, J2 = 4.1 Hz, H-5), 1.84 (m, 1H, H-3), 1.71
(dt, 1H,J1 =15.5Hz, J2 = 4.2 Hz, H-5), 1.32 (s, 3H, CH3C-1), 0.94
(s, 9H, Me3C-Si), 0.93 (d, 3H, J = 6.8 Hz, CH3C-3), 0.88 (s, 9H,
Me3C-Si), 0.11 (s, 3H, Me-Si), 0.07 (s, 3H, Me-Si), 0.03 (s, 6H, 2 ꢀ
Me-Si). 13C NMR (100.6 MHz, CDCl3): δ 71.2 (CH, C-2), 70.5
(CH, C-4), 59.8 (C, C-1), 59.5 (CH, C-6), 41.5 (CH, C-3), 31.1
(CH2, C-5), 25.9 (3 ꢀ CH3, Me3C-Si), 25.7 (3 ꢀ CH3, Me3C-Si),
21.4 (CH3, CH3C-1), 18.3 (C, C-Si), 17.9 (C, C-Si), 11.7 (CH3,
CH3C-3), -4.5 (CH3, Me-Si), -4.6 (CH3, Me-Si), -4.8 (CH3,
Me-Si), -4.9 (CH3, Me-Si). Anal. Calcd for [C20H43O3Si2]:
C, 62.12; H, 10.95. Found: C, 62.36; H, 11.26.
(3R,4S,5S)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methyl-6-
oxoheptanal (15). H5IO6 (2.65 g, 11.64 mmol, 3 equiv) was added
to a solution of epoxide 14b (1.5 g, 3.73 mmol, 1 equiv) in dry
Et2O (20 mL). After 12 h, the reaction mixture was poured into
an aqueous saturated solution of Na2S2O3 (20 mL). The aqu-
eous phase was extracted with Et2O (3 ꢀ 15 mL). The combined
organic phase was dried, filtered, and concentrated in vacuo to
give aldehyde 11 (1.56 g), which was immediately used in the
next step. 1H NMR (250 MHz, CDCl3): δ 9.71 (t, 1H, J = 2.4 Hz,
H-1), 4.17 (c, 1H, J = 5.4 Hz, H-3), 3.88 (d, 1H, J = 5.1 Hz, H-
5), 2.65 (m, 2H, CH2-4), 2.10 (s, 3H, CH3-7), 1.93 (m, 1H, H-4),
0.86 (s, 9H, Me3C-Si), 0.80 (d, 3H, J = 7.0 Hz, CH3C-4), 0.79
(s, 9H, Me3C-Si), -0.01 (s, 3H, Me-Si), -0.02 (s, 3H, Me-Si),
-0.03 (s, 3H, Me-Si), -0.04 (s, 3H, Me-Si). 13C NMR(62.9MHz,
CDCl3): δ 211.4 (CdO, C-6), 201.9 (CdO, C-1), 79.9 (CH, C-5),
69.0 (CH, C-3), 48.9 (CH2, C-2), 44.0 (CH, C-4), 26.6 (CH3, C-7),
(1R,3S,4R,6R)-[(tert-Butyldimethylsilyl)oxy]-1,3-dimethyl-7-
oxabicyclo[4.1.0]heptan-2-one (13b). Imidazole (2.6 g, 38.4 mmol,
2 equiv) and TBSCl (4.34 g, 28.8 mmol, 1.5 equiv) were succes-
sively added to a solution of alcohol 13a (3 g, 19.2 mmol, 1 equiv)
in dry DMF (30 mL). After 12 h, the reaction was quenched by
the addition of a few pieces of ice and hexanes. The aqueous phase
was extracted with hexanes (2 ꢀ 50 mL). The combined organic
phase was dried, filtered, and concentrated. The residue was
purified by flash chromatography (SiO2, 4 cm ꢀ 10 cm, hexanes)
to give 13b [4.62 g, 89%, colorless oil, Rf = 0.75 (20% EtOAc/
hexanes)]. [R]2D5 24.2 (c 1.34, CHCl3). IR (neat, cm-1): 1712
1
(νCdO). H NMR (400 MHz, CDCl3): δ 3.80 (td, 1H, J1 = 9.0
Hz, J2 = 4.8 Hz, H-4), 3.33 (dd, 1H, J1 = 3.1 Hz, J2 = 1.3 Hz, H-
6), 2.49 (ddd, 1H, J1 =14.6 Hz, J2 = 4.6 Hz, J3= 3.1 Hz, H-5),
2.06 (m, 1H, H-3), 1.9 (ddd, 1H, J1 = 14.6 Hz, J2 = 9.0 Hz, J3 =
1.3Hz, H-5), 1.38(s, 3H, CH3C-1), 1.14 (d, 3H, J=7Hz, CH3C-3),
0.86 (s, 9H, Me3C-Si), 0.06 (s, 3H, Me-Si), 0.04 (s, 3H, Me-Si). 13
C
NMR (100.6 MHz, CDCl3): δ 206.2 (CdO, C-2), 68.8 (CH, C-4),
60.1 (CH, C-6), 58.2 (C, C-1), 51.4 (CH, C-3), 32.8 (CH2, C-5),
25.6 (3xCH3, Me3C-Si), 17.8 [C, (C, C-Si], 15.3 (CH3, CH3C-1),
12.8 (CH3, CH3C-3), -4.4 (CH3, CH3-Si), -4.8 (CH3, CH3-Si).

1168 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
25.7 (3 ꢀ CH3, Me3C-Si), 25.6 (3 ꢀ CH3, Me3C-Si), 17.9 (C, C-
Si), 17.8 (C, C-Si), 12.5 (CH3, CH3C-4), -3.7 (CH3, Me-Si),
-4.6 (CH3, Me-Si), -5.1, (CH3, Me-Si), -5.2 (CH3, Me-Si).
(3S,4S,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-8,8-dibromo-
4-methyloct-7-en-2-one (16). CBr4 (3.86 g, 11.64 mmol, 3 equiv)
was added to a suspension of Ph3P (3.05 g, 11.64 mmol, 3 equiv)
and Zn (0.761 g, 11.64 mmol, 3 equiv) in CH2Cl2 (15 mL) at 0 °C.
After 5 min, the cooling bath was removed and the reaction
mixture was stirred at room temperature for 2 h. The color
changed from green to deep-red. A solution of aldehyde 15 (1.56 g,
4.88 mmol, 1 equiv) in CH2Cl2 (5 mL) was added via cannula.
After 45 min, the reaction mixture was filtered through a path of
silica gel (elution with hexanes and 20% EtOAc/hexanes). After
concentration in vacuo, the residue was purified by flash chroma-
tography (SiO2, 3 cm ꢀ 12 cm, 2% EtOAc/hexanes) to give the
dibromide 16 [1.63g, 75%(twosteps), colorless oil,Rf =0.50(5%
EtOAc/hexanes). [R]D25 -25.4 (c 2.0, CHCl3). IR (neat, cm-1):
1708 (νCdO), 1633 (νCdC). 1H NMR (400 MHz, CDCl3): δ 6.46
(dd, 1H, J1 = 8.1 Hz, J2 = 5.9 Hz, H-7), 3.88 (d, 1H, J = 5.6 Hz,
H-3), 3.83 (c, 1H, J = 5.4 Hz, H-5), 2.59 (ddd, 1H, J1 = 15.5 Hz,
J2 =8.2Hz,J3 =5.1Hz,H-6),2.3(dt,1H,J1= 15.5 Hz, J2=5.7Hz,
H-6), 2.17 (s, 3H, CH3-1), 1.81 (m, 1H, H-4), 0.94 (s, 9H, Me3C-
Si), 0.89 (d, 3H, J = 5.5 Hz, CH3C-4), 0.88 (s, 9H, Me3C-Si), 0.08
(s, 3H, Me-Si), 0.06 (s, 3H, Me-Si), 0.04 (s, 6H, 2 ꢀ Me-Si). 13C
NMR (100.6MHz, CDCl3):δ212.3(CdO,C-2), 135.5 (CH, C-7),
90.0 (C, C-8), 79.6 (CH, C-3), 71.2(CH, C-5), 43.6(CH, C-4), 38.8
(CH2, C-6), 27.0 (CH3, C-1), 25.9 (3 ꢀ CH3, Me3C-Si), 25.7 (3 ꢀ
CH3, Me3C-Si), 18.1 (C, C-Si), 18.0 (C, C-Si), 11.8 (CH3, CH3-
C-4), -4.2 (CH3, Me-Si), -4.4 (CH3, Me-Si), -4.8 (CH3, Me-Si),
-4.9 (CH3, Me-Si). HMRS ([CI]þ): calculated for [C21H43Br2-
O3Si]þ, 557.1117; found, 557.1115.
(3S,4S,5R)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-4-methyloct-
1-en-7-yne-2-yl Trifluoromethanesulfonate (8). A freshly pre-
pared solution of LiN(i-Pr)2 in THF (6 mL, 0.9 M, 3 equiv)
was added dropwise to a solution of the dibromide 16 (1 g, 1.79
mmol, 1 equiv) in THF (10 mL) at -78 °C. After being stirred for
1 h, a solution of n-BuLi in hexanes (0.25 mL, 0.54 mmol, 2.5 M,
0.3 equiv) was added. After 15 min, N-(5-chloro-2-pyridyl)bis-
(trifluoromethanesulfonimide) (2.1 g, 5.37 mmol, 3 equiv) was
added at once. The reaction mixture was allowed to reach room
temperature over 12 h. The reaction was quenched by the
addition of saturated NaCl (20 mL). The aqueous phase was
extracted with Et2O (2 ꢀ 20 mL). The combined organic phase
was dried, filtered, and concentrated in vacuo. The residue was
purified by flash chromatography (SiO2, 3 cm ꢀ 12 cm, hexanes)
to give enoltriflate 8 [0.712 g, 75%, colorless oil, Rf = 0.75 (5%
Et2O/hexanes)]. [R]2D5 -1.52 (c 2.1 mg/mL, CHCl3). IR (neat,
cm-1): 3312 (νtC-H), 1663 (νCdC). 1H NMR (500 MHz, CDCl3):
δ 5.24 (d, 1H, J = 3.7 Hz, H-1), 5.15 (d, 1H, J = 3.7 Hz, H-1), 4.10
(d, 1H, J = 7.3 Hz, H-3), 4.07 (ddd, 1H, J1 = 7.4 Hz, J2 =
5.3 Hz, J3 = 3.2 Hz, H-5), 2.43 (ddd, 1H, J1 = 16.8 Hz, J2 =5.3Hz,
J3 = 2.7 Hz, H-6), 2.38 (ddd, 1H, J1 = 1.7 Hz, J2 = 7.5 Hz, J3 =
2.7 Hz, H-6), 2.12 (cd, 1H, J1 = 7.1 Hz, J2 = 3.2 Hz, H-4), 1.99
(t, 1H, J = 2.7 Hz, H-8), 0.92 (s, 9H, Me3C-Si), 0.89 (s, 9H,
Me3C-Si), 0.87 (d, 3H, J = 7.0 Hz, CH3C-4), 0.11 (CH3, Me-Si),
0.10 (CH3, Me-Si), 0.09 (CH3, Me-Si), 0.08 (CH3, Me-Si). 13C
NMR (125.7 MHz, CDCl3): δ 156.2 (dC, C-2), 118.4 (C, c, J =
320 Hz, CF3), 104.4 (dCH2, C-1), 80.8 (tCH, C-8), 74.1 (CH,
C-3), 70.7 (tC, C-7), 70.1 (CH, C-3), 42.1 (CH, C-4), 25.8 (3 ꢀ
CH3, Me3C-Si), 25.7 (3 ꢀ CH3, Me3C-Si), 25.7 (CH2, C-6), 18.2
(C, C-Si), 18.1 (C, C-Si), 9.8 (CH3, CH3C-4), -3.8 (CH3, Me-Si),
-4.4 (CH3, Me-Si), -5.0 (2 ꢀ CH3, 2 ꢀ Me-Si). HMRS ([CI]þ):
calculated for [C22H42F3O5SSi2]þ, 531.2244; found, 531.2239.
2r-Methyl-20S,23S-epoxymethano-1r,25-dihydroxyvitamin
D3 (4a). A solution of t-BuLi in pentane (0.280 mL, 0.427 mmol,
1.55 M, 2.2 equiv) was added dropwise to a solution of bromide
7a (0.097 g, 0.194 mmol, 1 equiv) in dry THF (2 mL) at -78 °C.
After the mixture was stirred for 1 h, a solution of dry ZnBr2 in
THF (0.46 mL, 0.232 mmol, 0.5 M, 1.2 equiv) was added
dropwise. After 5 min, the -78 °C cooling bath was replaced
by an ice-water cooling bath and the yellow solution was stirred
for 1 h. A solution of enoltriflate 8 (0.070 g, 0.132 mmol,
0.68 equiv), (Ph3P)4Pd (15 mg, 0.012 mmol, 6 mol %), and dry
Et3N (0.1 mL, 0.75 mmol, 4 equiv) in dry THF (2 mL) was
added. The yellow color changed to orange. The reaction
mixture was stirred in the dark for 5 min at 0 °C and then at
room temperature for 12 h. The reaction was quenched by the
addition of H2O (1 mL). The aqueous phase was extracted with
Et2O (3 ꢀ 3 mL). The combined organic phase was dried,
filtered, and concentrated in vacuo. The residue (17a) was
dissolved in THF (2 mL) under argon. A solution of n-Bu4NF
in THF (1 mL, 0.97 mmol, 1 M, 5 equiv) was added. The mixture
was stirred for 24 h. The reaction was quenched by the addition
of saturated NH4Cl (5 mL). The aqueous phase was extracted
with EtOAc (3 ꢀ 10 mL). The combined organic phase was
dried, filtered, and concentrated in vacuo. The residue was
purified by flash chromatography (SiO2, 1.5 cm ꢀ 10 cm, 50%
EtOAc/hexanes) to give 4a [0.033 g, 55%, white solid, Rf = 0.48
1
(60% EtOAc/hexanes)]. H NMR (500 MHz, CDCl3): δ 6.37
(d, 1H, J = 11.3 Hz, H-6), 6.01 (d, 1H, J = 11.3 Hz, H-7), 5.27
(s, 1H, H-19), 5.00 (d, 1H, J = 1.6 Hz, H-19), 4.31 (d, 1H, J =
3.1 Hz, H-1), 4.10 (t, 1H, J = 7.9 Hz, H-28), 3.84 (td, 1H, J1 =
7.5 Hz, J2 = 4.3 Hz, H-3), 3.34 (dd, 1H, J1 = 9.7 Hz, J2 = 8.5 Hz,
H-28), 2.81 (d, 1H, J1 = 12.5 Hz, H-9), 2.66 (dd, 1H, J1 = 13.5 Hz,
J2 = 3.8 Hz, H-4), 2.46 (m, 1H, H-23), 2.22 (dd, 1H, J1 = 13.5 Hz,
J2 = 7.6 Hz, H-4), 2.05 (d, 1H, J = 11.7 Hz, H-12), 1.99 (dd, 1H,
J1 = 11.2 Hz, J2 = 7.9 Hz, H-14), 1.92 (cd, 1H, J1 = 7.0 Hz,
J2 = 3.7 Hz, H-2), 1.23 (s, 3H, CH3-21), 1.22 (s, 6H, CH3-26 and
CH3-27), 1.06 (d, 3H, J = 6.9 Hz, CH3C-2R), 0.66 (s, 3H, CH3-
18). 13C NMR (125.7 MHz, CDCl3): δ 146.5 (dC, C-10), 142.7
(dC, C-8), 133.1 (dC, C-5), 124.7 (dCH, C-6), 117.3 (dCH, C-
7), 113.1 (dCH2, C-19), 84.9 (C,C-20), 75.2 (CH, C-1), 73.2
(CH2, C-28), 71.7 (CH, C-3), 70.9 (CH, C-25), 59.8 (CH, C-14),
56.5 (CH, C-17), 47.1 (CH2, C-22), 46.9 (CH2, C-24), 46.1 (C, C-
13), 44.1 (CH2, C-2), 43.2 (CH2, C-4), 40.7 (CH2, C-12), 35.9
(CH, C-23), 30.2 (CH3, C-27), 29.7 (CH3, C-26), 29.0 (CH2,
C-9), 27.6 (CH3, CH3-21), 23.4 (CH2), 22.7 (CH2), 21.9 (CH2),
13.2 (CH3, C-18), 12.4 (CH3, CH3C-2R). HMRS (ESI-TOF):
calculated for [C29H47O4]þ ([M þ H]þ), 459.3475; found,
459.3469.
2r-Methyl-20S,23R-epoxymethano-1r,25-dihydroxyvitamin
D3 (4b). A solution of t-BuLi in pentane (0.370 mL, 0.630 mmol,
1.7 M, 2.1 equiv) was added dropwise to a solution of bromide
7b (0.150 g, 0.3 mmol, 1 equiv) in dry THF (2 mL) at -78 °C.
After the mixture was stirred for 1 h, a solution of dry ZnBr2 in
THF (0.5 mL, 0.41 mmol, 0.82 M, 1.37 equiv) was added
dropwise. After 5 min, the -78 °C cooling bath was replaced
by an ice-water cooling bath and the yellow solution was stirred
for 1 h. A solution of enoltriflate 8 (0.080 g, 0.150 mmol,
0.5 equiv), (Ph3P)4Pd (20 mg, 0.017 mmol, 6 mol %), and dry
Et3N (0.2 mL, 1.5 mmol, 5 equiv) in dry THF (2 mL) was added.
The yellow color changed to orange. The reaction mixture was
stirred in the dark for 5 min at 0 °C and then at room
temperature for 6 h. The reaction was quenched by the addition
of H2O (1 mL). The aqueous phase was extracted with Et2O (3 ꢀ
3 mL). The combined organic phase was dried, filtered, and
concentrated in vacuo. The residue (17b) was dissolved in a
mixture of deoxygenated CH2Cl2 (2 mL), CH3CN (2 mL), and
Et3N (1 mL). HF-Py complex (0.2 mL) was added. The mixture
was stirred at room temperature for 2 h. The reaction was
quenched by the addition of saturated NaHCO3 (5 mL). The
aqueous phase was extracted with EtOAc (3 ꢀ 10 mL). The
combined organic phase was dried, filtered, and concentrated in
vacuo. The residue was purified by flash chromatography (SiO2,
1.5 cm ꢀ 10 cm, 50% EtOAc/hexanes) and then by HPLC
(Phenomenex Silica(2), 250 mm ꢀ 1.2 mm, 10% i-PrOH/
hexanes) to give 4b [0.036 g, 53%, white solid, Rf = 0.48
(60% EtOAc/hexanes)].1H NMR (500 MHz, CDCl3): δ 6.37
(d, 1H, J = 11.3 Hz, H-6), 6.00 (d, 1H, J = 11.3 Hz, H-7), 5.27
(s, 1H, H-19), 5.00 (d, 1H, J= 1.9 Hz, H-19), 4.31 (d, 1H, J=3.6Hz,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1169
H-1), 4.07 (t, 1H, J = 7.7 Hz, H-28), 3.84 (td, 1H, J1 = 7.5 Hz,
J2 = 4.2 Hz, H-3), 3.37 (dd, 1H, J1 = 9.8 Hz, J2 = 8.9 Hz, H-
28), 2.81 (dd, 1H, J1 = 11.9 Hz, J2 = 3.7 Hz, H-9), 2.66 (dd, 1H,
J1 = 13.6 Hz, J2 = 3.9 Hz, H-4), 2.32 (m, 1H, H-23), 1.27 (s, 3H,
CH3-21), 1.21 (s, 6H, CH3-26 and CH3-27), 1.06 (d, 3H, J = 6.9
Hz, CH3C-2), 0.66 (s, 3H, CH3-18). 13C NMR (125.7 MHz,
CDCl3): δ 146.5 (dC, C-10), 142.7 (dC, C-8), 133.1 (dC, C-5),
124.7 (dCH, C-6), 117.3 (dCH, C-7), 113.1 (dCH2, C-19), 84.9
(C,C-20), 75.2 (CH, C-1), 73.2 (CH2, C-28), 71.7 (CH, C-3), 70.9
(CH, C-25), 59.8 (CH, C-14), 56.5 (CH, C-17), 47.1 (CH2, C-22),
46.9 (CH2, C-24), 46.1 (C, C-13), 44.1 (CH2, C-2), 43.2 (CH2, C-
4), 40.7 (CH2, C-12), 35.9 (CH, C-23), 30.2 (CH3, C-27), 29.7
(CH3, C-26), 29.0 (CH2, C-9), 27.6 (CH3, CH3-21), 23.4 (CH2),
22.7 (CH2), 21.9 (CH2), 13.2 (CH3, C-18), 12.4 (CH3, CH3C-2R).
HMRS (ESI-TOF): calculated for [C29H47O4]þ ([M þ H]þ),
459.3475; found, 459.3478.
Purification and Crystallization. Purification and crystalliza-
tion of the human VDR LBD complexes with 4a or 4b were
performed as previously described.6 The LBD of the human
VDR (residues 118-427, Δ166-216) was cloned in pET28b
expression vector to obtain an N-terminal hexahistidine-tagged
fusion protein and was overproduced in E. coli BL21 (DE3)
strain. Cells were grown in Luria-Bertani medium and subse-
quently incubated for 3 h at 25 °C with 1 mM isopropyl thio-β-D-
galactoside. The protein purification included a metal affinity
chromatography step on a cobalt-chelating resin (TALON,
Clontech). The tag was removed by thrombin digestion over-
night at 4 °C, and the protein was further purified by gel
filtration on a Superdex S200 16/60 column (Amersham). The
protein buffer prior to concentration of the protein contains
20 mM Tris, pH 7.5, 200 mM NaCl, and 2 nM TCEP. The
protein was concentrated to 3.5 mg/mL and incubated in the
presence of a 1.5-fold excess of ligands. The purity and homo-
geneity of the protein were assessed by SDS-PAGE. Crystals of
complexes were obtained at 4 °C by vapor diffusion method
using crystals of VDR LBD/1R,25(OH)2D3 as microseeds. The
reservoir solution contained 0.1 M Mes and 1.4 M ammonium
sulfate at pH 6.0.
Supporting Information Table 1. The volumes of each ligand
binding pocket and each ligand were calculated as previously
reported.
Steady-State Fluorescence Anisotropy Measurements. Steady-
state anisotropy measurements were performed with a T-format
SLM 8000 spectrofluorometer. Anisotropy titrations were car-
ried out by adding increasing hVDR LBD concentrations to
1 μM fluorescent labeled TAMRA (tetramethylrhodamine)-
SRC-1 (RHKILHRLLQEGSPS) peptide in 20 mM Tris-HCl
(pH 7.5), 200 mM NaCl. The binding affinity constants of the
SRC-1 peptide to hVDR LBD was determined in response to
1R,25(OH)2D3, 2a, 4a, or 4b. The excitation wavelength was
550 nm, and the emitted light was monitored through high-pass
filters (550 nm) (Kodak). A home-built device ensured the
automatic rotation of the excitation polarizer. Assuming that
hVDR binds with the TAMRA-SRC-1 peptide in a 1:1 stoichio-
metry, the model used to describe the binding experiment was
the following:
Kd
R þ L T P
L and R represent the hVDR LBD and the TAMRA-SRC-1
peptide, respectively, and P designates the hVDR LBD/TAM-
RA-SRC-1 complex.
The Scatchard equation was rewritten to fit the anisotropy, r,
as follows:
0
1
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2
ðRt þ Lt þ KdÞ - ðRt þ Lt þ KdÞ -4RtLt
2Rt
B
C
r ¼ r þ ðr -r Þ
@
A
0
f
0
Lt and Rt are the total concentrations of hVDR LBD and
TAMRA-SRC-1 peptides, respectively; rf represents the aniso-
tropy at the plateau when all the complex is formed, whereas r0
and r correspond to the anisotropy values of the TAMRA-SRC-
1 peptide in the absence and in the presence of a given concen-
tration of hVDR, respectively. Kd corresponds to the dissocia-
tion constant of the complex. All experiments were performed at
20 °C, and results are representative of three distinct experi-
ments.
X-ray Data Collection and Structure Determination. The
crystals were mounted in fiber loop and flash-cooled in liquid
nitrogen after cryoprotection with a solution containing the
reservoir solution plus 30% glycerol and 2% polyethylene glycol
400. Data collection from a single frozen crystal was performed
at 100 K on beamlines ID23-2 and ID29 of the ESRF (Grenoble,
France) for 4a and 4b complexes, respectively. The crystals
belong to the orthorhombic space group P212121 with one
monomer per asymmetric unit. Data were integrated and scaled
using HKL2000.35 A rigid body refinement was used with the
structure of the hVDR LBD/1R,25(OH)2D3 complex as a start-
ing model. Refinement involved iterative cycles of manual
building and refinement calculations. The programs Refmac36
and COOT37 were used throughout structure determination and
refinement. The ligand molecule was included only at the last
stage of the refinement. The omit map from the refined atomic
model of VDR LBD was used to fit the ligand to its electron
density, shown in Figure 2A. Anisotropic scaling and a bulk
solvent correction were used. Individual B atomic factors were
refined isotropically for the 4a complex and anisotropically for
the 4b complex. Solvent molecules were then placed according to
unassigned peaks in the difference Fourier map. In the VDR/4a
Transient Transfections and Luciferase Reporter Gene Assays.
The chimera Gal4-VDR LBD (90-427) was constructed by
PCR using the appropriate oligonucleotides with restriction
sites (XhoI/BamHI) and the pSG5 VDR plasmid (1-427) as
template and cloned into the vector PXJ440 encoding the DBD
of the yeast activator Gal4 (1-147). MCF-7 human breast
cancer cells were seeded into 24-well plates (105 cells per well)
and grown overnight in phenol red-free Dulbecco’s modified
Eagle’s medium (DMEM) supplemented with 10% charcoal-
treated fetal bovine serum (FCS), 5% gentamycin, and 0.6 μg/
mL insulin. Liposomes were formed using the transfection
reagent jetPEI (Polyplus transfection) and used according to
the manufacturer’s instructions. Cells were transfected with
25 ng of the expression plasmid pXJ440-Gal4DBD-hVDR
(90-427), 250 ng of the reporter plasmid ptk-LUC, 50 ng of
the pCH110-β-galactosidase vector (used as an internal control
to normalize variation in transfection efficiency), and 675 ng of
the carrier plasmid pBluescript (Stratagene). Ten hours later,
cells were washed with freshly prepared phosphate buffered
saline (PBS) and various concentrations of 1R,25(OH)2D3, 4a,
4b, 2a, and 2b versus control (solvent) were added to the cells in
phenol red-free DMEM supplemented with 10% FCS. Twenty-
four hours after the onset of stimulation, cells were rinsed in PBS
and lysed in 100 μL of reporter gene lysis buffer (Roche
Diagnostics). Cell extracts were assayed for luciferase and
β-galactosidase activities. Luciferase reporter activity was de-
termined by using luciferin as the substrate, and β-galactosidase
activity was measured using o-nitrophenyl-β-galactoside as the
substrate. The chemiluminescence from activated luciferin was
measured on a luminometer plate reader LB96P (Berthold
˚
complex, refined at 1.7 A with no σ cutoff, the final model
consists of residues 118-423 (Δ166-216), the ligand, two
sulfate ions, and 447 water molecules. In the VDR/4b complex
˚
refined at 1.45 A with no σ cutoff, the final model consists of
residues 118-423 (Δ166-216), the ligand, two sulfate ions, and
458 water molecules. According to PROCHECK,38 93.0% of
peptide lies in the most favored regions and 7.0% in additional
allowed regions for the VDR/4a complex, and 93.4% of peptide
lies in most favored regions and 6.6% in additional allowed
regions for the VDR/4b complex. Data are summarized in

1170 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Antony et al.
Technologies). Luciferase values were normalized to the β-
galactosidase activity. Luciferase activities are expressed as
arbitrary units of light intensity. Data points represent the mean
of assays performed in triplicate for at least three independent
experiments. For every triplicate, the mean and the standard
deviation of the mean were calculated.
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HL60 Cell Culture and Differentiation. HL60 cells were
cultured in complete RPMI with 10% fetal calf serum (Sigma)
and 5% gentamycine (Kalys) at 37 °C without CO2 as previously
described.19 Cells were plated at a density of 4 ꢀ 105 cells/mL
and cultured for 96 h in the presence of 1R,25(OH)2D3, 2a, or 4a
and 4b at various concentrations. Control incubations were
performed with 0.7% ethanol. HL60 cell differentiation was
determined by flow cytometry using PE-conjugated antihuman
CD11c and FITC-conjugated antihuman CD14 antibodies
(Pharmingen/BD Biosciences). Topro-3 (Molecular probes)
was added immediately before laser excitation to exclude dead
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cells. Cells were analyzed on
a FACSCalibur (Becton
Dickinson) and using the FlowJo (TreeStar Inc.) software.
Mice and Serum Calcium Quantitation. Animal protocols
were approved by the Alsace Regional Ethics Committee. Male
C57BL/6J mice were obtained from Charles River Laboratories,
France (L’Arbresle, France). Mice (6-7 weeks old) were main-
tained in a temperature-controlled (23 °C) facility with a 12 h
light/dark cycle and were given free access to food and water.
Mice were fed with the standard mouse chow (D03 SAFE;
Augy, France) and tap water. The different 1R,25(OH)2D3
agonists were dissolved in sesame oil and administered intraper-
itoneally every 2 days at a dose of 0.1 μg/kg. Mice were fasted 3 h
prior to blood harvest, and subsequent calcium measurements
were determined with a kit supplied by Olympus according to
the manufacturer’s procedure on an Olympus AU400 analyzer
(Olympus SA, Ringis, France).39
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Deluca, H. F. New 1R,25-dihydroxy-19-norvitamin D3 com-
pounds constrained in a single A-ring conformation: synthesis of
the analogs by ring-closing methathesis route and their biological
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vitamin D analogs. Expert Opin. Invest. Drugs 2003, 12, 825–840.
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P; Van Haver, D.; Mathieu, C.; Bouillon, R.; Verstuyf, A. Mechan-
ism and potentialof the growth-inhibitory actions of vitamin D and
analogs. Curr. Med. Chem. 2007, 14, 1893–1910.
Acknowledgment. We thank the beamline staff at the
ESRF (Grenoble, France) for help during data collection.
The study described here was supported by CNRS, INSERM,
ULP, the European Commission as SPINE2 complexes
(Contract No. LSHG-CT-2006-031220) under the RDT pro-
gramme “Quality of Life and Management of Living Re-
sources”, the Spanish MEC (Grants SAF2004-01885 and
SAF2007-67205), and Xunta de Galicia (Grants INCITE08P-
XIB-209130PR and ACEUIC-2006/XA050). The authors are
grateful to Jean-Marc Bornert, Cindy Vincent, and Magali
Huc for mouse experiments, to the clinical biochemistry team
of the Mouse Clinics Institute for serum calcium measure-
ments, and to Dr. Daniel Metzger for helpful discussions. We
(19) Hourai, S.; Rodrigues, L. C.; Antony, P.; Reina-San Martin, B.;
~
Ciesielski, F.; Magnier, B. C.; Schoonjans, K.; Mourino, A.;
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ligand for the vitamin D nuclear receptors. Chem. Biol. 2008, 15,
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(20) Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.; Takayama, H.;
Rochel, N.; Moras, D. Probing a water channel near the A-ring of
receptor-bound 1a,25-dihydroxyvitamin D3 with selected 2 alpha-
substituted analogs. J. Med. Chem. 2006, 49, 5199–5205.
(21) Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura, D.; Chokki,
M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.; Kurihara, M.; Miyata,
N.; Smith, C.; DeLuca, H. F.; Takayama, H. Synthesis, biological
evaluation, and conformational analysis of A-ring diastereomers of
2-methyl-1,25-dihydroxyvitamin D3 and their 20-epimers: unique
activity profiles depending on the stereochemistry of the A-ring and
at C-20. J. Med. Chem. 2000, 43, 4247–4265.
ꢀ
thank Yves Mely for advice with fluorescence experiments. We
thank Claudine Ebel and Sara Milosevic for advice with flow
cytometry experiments and the cytometry common service. R.
S. thanks the Spanish MEC for a FPI grant and T.H. for a
fellowship from the Regional Council of Alsace. The authors
declare that they have no competing financial interests.
(22) Hughes, G.; Kimura, M.; Buchwald, S. L. Catalytic enantioselec-
tive conjugate reduction of lactones and lactams. J. Am. Chem. Soc.
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of vitamin D metabolites via Pd-catalyzed reactions. J. Am. Chem.
Soc. 1992, 114, 9836–9845.
Supporting Information Available: Table 1 listing crystal-
lographic data and refinement statistics, experimental proce-
dures for the chemical synthesis, and NMR spectra of
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
(24) For the use of carvones in the synthesis of vitamin D3 A-ring
fragments, see the following: (a) Baggiolini, E. G.; Iacobelli, J. A.;
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