Butyl pocket formation in the vitamin D receptor strongly affects the agonistic or antagonistic behavior of ligands

Article abstract of DOI:10.1021/jm300230a

Previously, we reported that 22S-butyl-25,26,27-trinor-1α24- dihydroxyvitamin D₃2 represents a new class of antagonist for the vitamin D receptor (VDR). The crystal structure of the ligand-binding domain (LBD) of VDR complexed with 2 showed the formation of a butyl pocket to accommodate the 22-butyl group and insufficient interactions between ligand 2 and the C-terminus of VDR. Here, we designed and synthesized new analogues 5a-c and evaluated their biological activities to probe whether agonistic activity is recovered when the analogue restores interactions with the C-terminus of VDR. Analogues 5a-c exhibited full agonistic activity in transactivation. Interestingly, 5c, which bears a 24-diethyl group, completely recovered agonistic activity, although 3c and 4c act as an antagonist and a weak agonist, respectively. The crystal structures of VDR-LBD complexed with 3a, 4a, 5a, and 5c were solved, and the results confirmed that butyl pocket formation in VDR strongly affects the agonistic or antagonistic behaviors of ligands.

Full text of DOI:10.1021/jm300230a

Article
pubs.acs.org/jmc
Butyl Pocket Formation in the Vitamin D Receptor Strongly Affects
the Agonistic or Antagonistic Behavior of Ligands
Nobuko Yoshimoto,†,‡,⊥ Yuta Sakamaki,†,§,⊥ Minoru Haeta,† Akira Kato,† Yuka Inaba,† Toshimasa Itoh,†
Makoto Nakabayashi,∥ Nobutoshi Ito,∥ and Keiko Yamamoto*,†,‡
‡
†Laboratory of Drug Design and Medicinal Chemistry and High Technology Research Center, Showa Pharmaceutical University,
3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan
§Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo
101-0062, Japan
∥Graduate School of Biomedical Science, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japan
ABSTRACT: Previously, we reported that 22S-butyl-25,26,27-
trinor-1α,24-dihydroxyvitamin D3 2 represents a new class of
antagonist for the vitamin D receptor (VDR). The crystal
structure of the ligand-binding domain (LBD) of VDR
complexed with 2 showed the formation of a butyl pocket to
accommodate the 22-butyl group and insufficient interactions
between ligand 2 and the C-terminus of VDR. Here, we
designed and synthesized new analogues 5a−c and evaluated
their biological activities to probe whether agonistic activity is
recovered when the analogue restores interactions with the C-
terminus of VDR. Analogues 5a−c exhibited full agonistic
activity in transactivation. Interestingly, 5c, which bears a 24-
diethyl group, completely recovered agonistic activity, although 3c and 4c act as an antagonist and a weak agonist, respectively.
The crystal structures of VDR-LBD complexed with 3a, 4a, 5a, and 5c were solved, and the results confirmed that butyl pocket
formation in VDR strongly affects the agonistic or antagonistic behaviors of ligands.
tives.8−11 VDR antagonism is believed to be based on an
unstable conformation of VDR generated upon ligand binding.
INTRODUCTION
Active vitamin D3, 1α,25-dihydroxyvitamin D3 1a (Chart 1), is
■
A VDR that accommodates an antagonist would prevent the
a hormone that plays a major role in calcium homeostasis. This
heterodimerization with RXR and/or the recruitment of
hormone is also involved in cell differentiation and proliferation
and immunomodulation.1 The actions of 1a and its active
coactivators. However, the molecular basis for VDR antagonism
is not clearly understood.
analogues are mediated by the vitamin D receptor (VDR),
which is a member of the nuclear receptor superfamily that
Recently, we reported that 22S-butyl-25,26,27-trinor-1α,24-
dihydroxyvitamin D3 2 acts as a third type of VDR antagonist.12
includes the receptors for steroid, retinoid, and thyroid
hormones.2 These nuclear receptors function through a
The crystal structure of the ligand binding domain (LBD) of
VDR complexed with 2 shows the formation of an extra cavity
common mechanism by which the receptors regulate the
transcription of their target genes. VDR forms a heterodimer
with retinoid X receptor (RXR), and upon ligand binding, VDR
to accommodate the butyl group.12 We have termed this extra
cavity the “butyl pocket”. Several research groups have solved
crystal structures of VDR-LBD complexed with a variety of
changes to an active conformation that provides the activation
ligands.13−25 Most of the crystal structures show the canonical
Moras’ active conformation13 of VDR-LBD and similar
architectures of the ligand binding pocket (LBP). Exceptions
are the complex of zebrafish VDR-LBD with “GEMINI”,16,20
which has two identical side chains,26,27 and the complex of
VDR-LBD with 22-butyl analogue 2.12 The crystal structure of
the zebrafish VDR-LBD/GEMINI complex revealed the
formation of a new cavity, which is an extension of the original
LBP, in order to accommodate the second side chain. The
crystal structure of the VDR-LBD/2 complex also revealed the
function 2 (AF2) surface to allow binding of a coactivator.3
Since the discovery of 1a, thousands of vitamin D analogues
have been synthesized,3 of which more than 10 have been
clinically used to treat metabolic bone diseases and skin
diseases such as psoriasis, immune disorders, and malignant
tumors.4,5 All these clinically used analogues are VDR agonists.
VDR antagonists have been synthesized by several groups and
are classified structurally into two types. The first group is
composed of analogues containing a bulky side chain, such as
carboxylic ester (ZK series) compounds6 and adamantane
compounds,7 while the second group is composed of analogues
lacking a bulky side chain, such as (23S)-25-dehydro-1α-
hydroxyvitamin D3-26,23-lactone (TEI9647)8 and its deriva-
Received: February 21, 2012
Published: April 18, 2012
© 2012 American Chemical Society
4373
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
Chart 1. Structures of 1a and Vitamin D Analogues
formation of a cavity similar to that of the VDR-LBD/GEMINI
complex. These observations suggest that the region around
helix 6, loop 6−7, and the N-terminus of helix 7 is somewhat
flexible. Interestingly, although both GEMINI 1b and 22-butyl
analogue 2 induce the formation of an extra cavity, 1b acts as a
VDR agonist whereas 22-butyl analogue 2 acts as a VDR
antagonist. We hypothesized that the contrasting behaviors of
1b and 2 against VDR were derived from differences in
hydrophobic interactions between the side chain terminus of
each ligand and the C-terminus of VDR.
To investigate the effects on antagonism of the induction of
the butyl pocket and of insufficient interactions of the ligand
with the C-terminus of VDR, we synthesized a series of vitamin
D3 analogues with and without a 22-alkyl substituent and
evaluated their biological activities.28 We found that these
synthetic ligands show strong antagonist, partial agonist, or full
agonist activities, depending on the primary or tertiary alcohol
at the side chain terminus and on the length of the 22-alkyl
group. The results suggested that both the induction of a butyl
pocket and insufficient hydrophobic interactions with the VDR
C-terminus are necessary for VDR antagonism.
The present study was conducted to further confirm the
molecular basis of agonistic and antagonistic activity of a new
class of ligands that induce the formation of a butyl pocket.
Here, we report the design, synthesis, and biological evaluation
of new analogues in order to probe whether agonistic activity
increases when the analogue exhibits increased interactions
with the C-terminus of VDR. Furthermore, X-ray crystallo-
graphic analyses of VDR-LBD complexed with those analogues,
which contain or lack a 22-butyl substituent, are also reported.
of a more hydrophobic substituent at the 24-position would
enhance VDR activation. Therefore, we designed 22-butyl
analogue 5c with a diethyl substituent instead of a dimethyl at
C(24). 22-Ethyl analogue 5b and 22-H analogue 5a, both of
which have a diethyl substituent at C(24), were also designed
as counterparts of 5c. As described in earlier paper, we selected
the 2-methylene-19-nor structure because of its superior
chemical stability, convenient synthesis, and its increased
biological activity compared to the original A-ring structure.28,29
Synthesis of the 1,24-Dihydroxyvitamin D3 Ana-
logues. As shown in Scheme 1, 1,24-dihydroxyvitamin D3
derivative 5a was synthesized from ester 6. Ester 6, prepared by
the procedure reported previously,28 was treated with ethyl-
lithium in THF to afford 24,24-diethylated compound 7.
Diethyl compound 7 was deprotected with CSA in methanol to
provide desired compound 5a. 22-Ethyl analogue 5b was
derived from 22-ethyl ester 8.28,30 Ester 8 was treated with
EtMgBr to afford 24,24-diethylated compound 9. Compound 9
was deprotected with CSA in methanol to provide desired
compound 5b. 22-Butyl analogue 5c was derived from 22-butyl
ester 10.28,30 Ester 10 was treated with EtMgI to afford 24,24-
diethylated compound 11. Compound 11 was deprotected with
CSA in methanol to provide desired compound 5c.
BIOLOGICAL ACTIVITIES
■
VDR Binding. Binding affinity to VDR was evaluated by a
competitive binding assay using recombinant human VDR-LBD
expressed as a C-terminus GST-tagged protein using the
pGEX-VDR vector28,31 in Escherichia coli BL21. The results are
summarized in Table 1 including previously reported results.28
Three synthetic compounds 5a−c showed specific binding to
VDR, indicating they are ligands for VDR. In particular, 5a
showed stronger affinity than 1a.
DESIGN AND SYNTHESIS
■
Design. Previously we showed that a 22-butyl analogue with
a 24-primary alcohol, 3c, is a VDR ligand but does not activate
VDR whereas the corresponding 24-tertiary alcohol 4c activates
VDR, albeit weakly.28 The results implied that the introduction
Transactivation. The ability of synthetic compounds 5a−c
to induce transcription of a vitamin D-responsive gene was
tested using the mouse osteopontin luciferase reporter gene
4374
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
Scheme 1. Synthesis of Compounds 5a−c
assay system in Cos7 cells.12 The results are shown in Figure 1,
together with the results for 3a−c and 4a−c.28 New analogues
5a−c showed concentration-dependent transcriptional activity
and acted as potent agonists. Of the 22-butyl analogues, new
analogue 5c completely restored full agonistic activity. In the
22-H analogues, the EC50 of transcriptional activity decreased
in the order 24OH 3a > 24OHDM 4a > 24OHDE 5a. In the
22-Et analogues, it is interesting that 5b acted as a full agonist
while 3b and 4b are partial agonists.
Table 1. VDR Binding Affinity of Synthetic Analogues 3−6,
X-RAY CRYSTAL STRUCTURE
a
■
IC50 (nM)
We attempted to crystallize the VDR-LBD complex with the
22-H analogues (3a, 4a, and 5a) and 22-butyl analogues (3c,
4c, and 5c). We were able to obtain crystals in the presence of,
but not in the absence of, a coactivator peptide, which is the
same result as in our previous report.12 Good quality crystals of
VDR complexed with 3a, 4a, 5a, or 5c were obtained, but good
crystals accommodating antagonists 3c or 4c were not
obtained. As a result, we could solve the crystal structure of
the complex with 3a, 4a, 5a (Figure 2), or 5c (Figure 3) but not
the complex with 3c or 4c. The crystallographic analysis data
are summarized in Table 2. The overall protein structure of
VDR-LBD complexed with 3a, 4a, 5a, or 5c adopted the
canonical Moras’ active conformation observed in the complex
with 1a.13 Coactivator peptide is also closely bound to the AF2
a
Competitive binding of 1a and synthetic compounds 5a−c to the
human vitamin D receptor. Affinity of related compounds (3a−c and
4a−c) was cited from previous report28 to compare with 5a−c. The
experiments were carried out in duplicate. The IC50 values are derived
from dose−response curves and represent the compound concen-
tration required for 50% displacement of radiolabeled 1α,25-
dihydroxyvitamin D3 from the receptor protein.
4375
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
surface. These results indicate that the complexes with 3c or 4c,
for which good quality crystals were not obtained, are difficult
to adopt the canonical Moras’ active conformation.
The entire main-chain structure of the VDR-LBD/5c
complex is similar to the complex with 1a or antagonist 2.
The 1α-, 3β-, and 25-hydroxyl groups of 5c form pincer-type
hydrogen bonds, as is the case with most vitamin D analogues
(Figure 3a). Formation of a butyl pocket to accommodate the
22-butyl group was clearly observed (Figure 3b). The residues
within 4.5 Å from each ligand are summarized in Table 3; these
residues in 5c are identical with those, except Leu400, in 1a.
In the complexes with 3a, 4a, or 5a, the side chain
conformations of the amino acid residues lining the LBP are
superimposed onto those of the VDR-LBD/1a complex. In the
crystallographic analysis, ligands 4a and 5a docked in the VDR
are well-defined, whereas the electron density map of the side
chain of ligand 3a in the VDR is unclear, probably because of its
flexibility. Therefore, the side chain conformation of 3a was
tentatively determined based on the electron density map and
the conformation that allowed hydrogen bonding with His393
and/or His301. Ligands 3a, 4a, and 5a were accommodated
into the VDR-LBP in a manner similar to 1a. The hydroxyl
groups at the 1α and 3β positions form pincer-type hydrogen
bonds, evident in the VDR-LBD/1a complex (Figure 2a).
Furthermore, the 24-hydroxyl group of 4a and 5a forms
hydrogen bonds with both His393 and His301, and that of 3a
forms a hydrogen bond with His393 (Figure 2a). As shown in
Table 3, compared with the complex with 1a, complexes with
3a and 4a showed reduced interactions.
Figure 1. Transactivation of compounds 5a−c in Cos7 cells, together
with the results of 3a−c and 4a−c previously reported.28 Transcrip-
tional activity was evaluated by the dual luciferase assay using a full-
length human VDR expression plasmid (pCMX-hVDR), a reporter
plasmid containing three copies of mouse osteopontin VDRE (SPPx3-
TK-Luc), and an internal control plasmid containing sea pansy
luciferase expression constructs (pRL-CMV) in Cos7 cells as described
previously.12 Luciferase activity of 10−8 M 1a was defined as 1.
DISCUSSION
■
All synthetic analogues with a 24-hydroxyl group at the side
chain showed specific binding for hVDR (Table 1). Therefore,
compounds 5a−c synthesized here are true ligands for hVDR.
Interestingly, compound 5a showed stronger affinity for hVDR
Figure 2. Crystal structures of VDR-LBD complexed with 1a, 3a, 4a, and 5a: (a) hydrogen bonds between VDR-LBD and 1a (green), 3a (gray), 4a
(purple), or 5a (yellow); (b) interactions between the VDR C-terminus (helix 11, loop 11−12, and helix 12) and the ligands. The C-terminus (helix
11, loop 11−12, and helix 12) is presented as a green ribbon, and the ligands are presented in the same colors as in (a). Residues within 4.5 Å from
each ligand are presented.
4376
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
Figure 3. Crystal structures of VDR-LBD complexed with 5c: (a) hydrogen bonds between VDR-LBD and 5c (pink); (b) Conolly channel surface
of the ligand-binding pocket of the VDR-LBD/1a complex (left, green), VDR-LBD/2 complex (middle, cyan), and VDR-LBD/5c complex (right,
pink); (c, d) superposition of VDR-LBD complexed with 1a (green), 2 (cyan), or 5c (pink). (a) The 1α-, 3β-, and 25-hydroxyl groups of 5c form
pincer-type hydrogen bonds with Ser233 and Arg270, Tyr143 and Ser274, and His301 and His393, respectively. (b) Formation of the butyl pocket
to accommodate the 22-butyl group is clearly observed in the complex with 2 and 5c. (c) The terminal S-methyl group of Met268 in the VDR/2
complex (cyan) rotated in about 120° compared with that in VDR/1a complex (green), but that in VDR/5c (pink) did not rotate. Leu305 in VDR/
5c (pink) adopted intermediate conformation between conformations of the complexes with 1a and 2. (d) 5c (pink) and Phe418 at helix 12 make
C−H···π interactions. The distance between the terminal carbon of one of 24-ethyl groups in 5c and the center of the π-system (dC−X) is 3.9 Å.
Table 2. Summary of Data Collection Statistics and Refinement
ligand
parameter
X-ray source
3a
KEK-PF BL-6A
4a
5a
5c
KEK-PFAR NW-12A
KEK-PFAR NW-12A
KEK-PFAR NW-12A
wavelength (Å)
space group
0.97800
1.00000
1.00000
1.00000
C2
C2
C2
C2
unit cell dimensions
bond (Å)
a = 152.99, b = 43.87, c = 42.43 a = 154.34, b = 42.29, c = 42.21 a = 154.14, b = 42.77, c = 42.12 a = 154.21, b = 43.21, c = 42.38
α = 90.00, β = 95.94, γ = 90.00 α = 90.00, β = 95.89, γ = 90.00 α = 90.00, β = 95.42, γ = 90.00 α = 90.00, β = 95.49, γ = 90.00
angle (deg)
a
resolution range (Å)
38.04−2.40 (2.49−2.40)
76.76−2.00 (2.11−2.00)
56092
76.73−1.90 (2.00−1.90)
75735
76.75−2.40 (2.53−2.40)
34783
total no. of reflections
41156
no. of unique reflections 11168
17728
20511
10065
a
% completeness
ab
99.5 (99.6)
96.4 (96.3)
0.074 (0.308)
94.6 (95.1)
0.047 (0.343)
91.9 (91.4)
0.081 (0.336)
,
Rmerge
Refinement Statistics
0.045 (0.299)
a
resolution range (Å)
38.04−2.40 (2.49−2.40)
26.98−2.00 (2.11−2.00)
0.279/0.219
29.74−1.90 (2.00−1.90)
0.248/0.209
33.01−2.40 (2.53−2.40)
0.256/0.218
ac
,
R factor (Rfree/Rwork
)
0.273/0.225
a
b
Values in parentheses are for the highest-resolution shell. Rmerge = ∑|(Ihkl − ⟨Ihkl⟩)|/(∑Ihkl), where ⟨Ihkl⟩ is the mean intensity of all reflections
c
equivalent to reflection hkl. Rwork (Rfree) = ∑||Fobs| − |Fcalc||/∑|Fobs|, where 5% of randomly selected data were used for Rfree
.
than natural hormone 1a. All three analogues 5a−c showed
potent transcriptional activity (Figure 1). 22-Ethyl analogue 5b
acts as a full agonist, whereas 3b and 4b are partial agonists.
More surprisingly, 22-butyl analogue 5c completely restored
agonistic activity, although 2 and 3c are antagonists and 4c is a
quite weak agonist.
This drastic change in transcriptional activity was explained
by comparison of the crystal structures. As expected, 5c
induced the formation of a butyl pocket similar to 2 (Figure
3b), but in contrast to 2, 5c interacts intimately with helix 12 to
form a stable complex with VDR. As shown in Table 3, the
number of amino acid residues within 4.5 Å of 5c is 27, whereas
for 2 there are only 22 residues within that distance.
Compound 5c therefore forms sufficient interactions with
VDR, and especially with helix 12, to form a stable complex, but
2 does not. This is a primary reason why 2 is an antagonist
whereas 5c is an agonist.
4377
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
Table 3. Residues within 4.5 Å from ligand
compd
residues within 4.5 Å from ligand
a
1a (28 residues)
Tyr143, Tyr147, Phe150, Leu223, Leu226, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284, Tyr291,
Val296, Ala299, His301, Leu305, Leu309, His393, Tyr397, Leu400, Leu410, Val414, Phe418
a
2 (22 residues)
Tyr143, Tyr147, Phe150, Leu226, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284, Tyr291, Val296,
His301, Leu305, Leu309, His393, Leu398
a
a
a
3a (20 residues)
4a (24 residues)
5a (27 residues)
Tyr143, Tyr147, Phe150, Leu226, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284, Tyr291, Val296,
His301, Leu309, His393
Tyr143, Tyr147, Phe150, Leu223, Leu226, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284, Tyr291,
Val296, Ala299, His301, Leu305, His393, Tyr397, Phe418
Tyr143, Tyr147, Phe150, Leu223, Leu226, Ala227, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284,
Tyr291, Val296, Ala299, His301, Leu305, Leu309, His393, Tyr397, Val414, Phe418
a
5c (27 residues)
Tyr143, Tyr147, Phe150, Leu223, Leu226, Leu229, Val230, Ser233, Ile264, Ile267, Met268, Arg270, Ser271, Ser274, Trp282, Cys284, Tyr291,
Val296, Ala299, His301, Leu305, Leu309, His393, Tyr397, Leu410, Val414, Phe418
a
Total number of residues within 4.5 Å from ligand.
Interestingly, the butyl pocket of the VDR-LBD/5c complex
Second, among ligands that induce butyl pocket formation (2,
3c, and 5c), a ligand acts as an antagonist when it does not
interact with the C-terminus of VDR, whereas a ligand acts as
an agonist when it interacts intimately with the C-terminus of
VDR. Third, ligands that do not induce an extra cavity like a
butyl pocket (3a, 4a, and 5a) increase agonistic activity when
the ligand increases interactions with the C-terminus of VDR.
Thus, butyl pocket formation in VDR strongly affects the
agonistic or antagonistic behavior of ligands. These results
indicate that structural modification of a target protein at a
flexible region such as loop region may be an important strategy
for the discovery of new drugs for the treatment of various
diseases.
is slightly smaller than that of VDR-LBD/2; this is mainly due
to the conformation of Met268 and Leu305 (Figure 3c). The
terminal S-methyl group of Met268 in the VDR-LBD/2
complex rotated in about 120° compared with that in the
VDR-LBD/1a complex, but that in VDR-LBD/5c did not
rotate. In addition, Leu305 adopted a conformation inter-
mediate between conformations of the complexes with 1a and
2. Furthermore, we found that 5c and Phe418 in helix 12 make
C−H···π interactions (Figure 3d). The distance between the
terminal carbon of one of the 24-ethyl groups in 5c and the
center of the π-system (dC−X) is 3.9 Å, indicating that this C−
H···π interaction would contribute significantly to the overall
stability of the protein.32 The results agree well with our
hypothesis that increased hydrophobic interactions overcome
the strain caused by the formation of an extra cavity such as a
butyl pocket.
EXPERIMENTAL SECTION
■
All reagents were purchased from commercial sources. Unless
otherwise stated, NMR spectra were recorded at 300 MHz for H
1
In 22-H analogues, the EC50 of transcriptional activity
decreased in the order 3a > 4a > 5a. These results are explained
by the crystal structures. Specific differences between the three
complexes are observed in the interactions between the ligand
side chain and the C-terminus of VDR-LBD. As shown in
Figure 2b, among the C-terminal residues within 4.5 Å of
hormone 1a, five residues (Tyr397, Leu400, Leu410, Val414,
and Phe418), three residues (Leu400, Leu410, and Val414),
and two residues (Leu400 and Leu410) are not within 4.5 Å of
3a, 4a, and 5a, respectively, indicating that interactions with
helices 11 and 12 increase in the order 3a < 4a < 5a. These
results indicate that intimate interactions with VDR reinforce
transcriptional activity.
Structural modification of the secondary structure of a
protein by the binding of a small molecule such as a ligand, a
substrate, or an inhibitor is difficult, but modification of a loop
region would be possible. Our synthetic compounds with a 22-
butyl substituent, 2 and 5c, push out the loop region, as
observed in the crystal structures. This study suggests that
modification of the three-dimensional structure of a protein at a
flexible loop region by a ligand binding is a novel strategy for
the discovery of new drugs that have the desired selectivity.
NMR and 75 MHz for 13C NMR in CDCl3 solution with TMS as an
internal standard, and the chemical shifts are given in δ values. High
and low resolution mass spectra were obtained with JEOL JMS D-300,
JEOL AccuTOF LC-plus JMS-T100LP, and JEOL JMS-HX110A
spectrometers. Relative intensities are given in parentheses in low
mass. IR spectra were recorded on a Shimadzu FTIR-8400S
spectrophotometer, and data are given in cm−1. UV spectra were
recorded on a Beckman DU7500 spectrophotometer. All air and
moisture sensitive reactions were carried out under argon or nitrogen
atmosphere. Purity was determined by HPLC [PEGASIL silica SP100,
4.6 mm × 150 mm, hexane/CHCl3/MeOH (100:25:8), flow rate 1.0
mL/min] and was >95% for all compounds tested.
6-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-
methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylheptan-3-
ol (7). To a solution of ester 6 (54.6 mg, 0.0867 mmol) in THF (1
mL) at −78 °C was added EtLi (870 μL of 0.5 M benzene/
cyclohexane (90/10) solution, 0.435 mmol), and the mixture was
stirred for 1 h. The reaction was quenched with saturated NH4Cl, and
the mixture was extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (AcOEt/hexane = 5/95) to afford 7
(30.4 mg, 0.0462 mmol, 53%). 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each
3 H, s, SiMe), 0.55 (3 H, s, H-18), 0.80−0.96 (6H, m, CH2CH3 × 2),
0.86, 0.89 (each 9 H, s, t-Bu), 0.94 (3 H, d, J = 6.1 Hz, H-21), 2.80 (1
H, m, H-9), 4.42 (2 H, m, H-1, 3), 4.92, 4.96 (each 1 H, s, −C
CH2), 5.83 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz, H-6).
13C NMR δ −5.1, −4.9, −4.8 (2 C), 7.7, 7.9, 12.0, 18.1, 18.2, 18.9,
CONCLUSIONS
■
We designed and synthesized analogues 5a−c bearing a diethyl
substituent at C(24) and evaluated their biological activities.
The crystal structures of VDR-LBD complexed with 3a, 4a, 5a,
or 5c were solved and the results confirmed the following. First,
analogues with a 22S-butyl group, such as 2 and 5c, induce
butyl pocket formation to accommodate the butyl group.
22.2, 23.4, 25.7 (3 C), 25.8 (3 C), 27.7, 28.7, 29.2, 30.9, 31.1, 34.2,
36.5, 38.6, 40.6, 45.6, 47.6, 56.2, 56.3, 71.7, 72.5, 74.7, 106.2, 116.1,
122.4, 132.7, 141.1, 152.9. MS (EI) m/z (%): 658 (M+, 2), 526 (26),
508 (8), 366 (20), 234 (14), 147 (15), 73 (100). HRMS (EI) calcd for
C40H74O3Si2 658.5177, found 658.5196. IR (neat) 2954, 2856, 1461,
1251, 1101, 1072, 835 cm−1. UV (hexane) λmax 246, 254, 264 nm.
4378
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
2-Methylidene-26,27-dimethyl-19,24-dinor-1α,25-dihydrox-
yvitamin D3 (5a). A solution of 7 (29.4 mg, 0.0447 mmol) and
camphor sulfonic acid (39.0 mg, 0.168 mmol) in MeOH (1 mL) was
stirred at room temperature for 0.5 h. Aqueous NaHCO3 was added,
and the mixture was extracted with AcOEt. The organic layer was
washed with brine, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (AcOEt/hexane = 1/1) to afford 5a
(17.0 mg, 89%). 1H NMR δ 0.55 (3 H, s, H-18), 0.86 (6H, td, J = 2.0,
7.4 Hz, CH2CH3 × 2), 0.95 (3 H, d, J = 6.0 Hz, H-21), 4.47 (2 H, m,
H-1, 3), 5.09, 5.11 (each 1 H, s, −CCH2), 5.88 (1 H, d, J = 11.2 Hz,
H-7), 6.36 (1 H, d, J = 11.2 Hz, H-6). 13C NMR δ 7.7, 7.8, 12.1, 18.9,
22.3, 23.5, 27.6, 29.0, 29.1, 30.9, 31.1, 34.2, 36.4, 38.1, 40.4, 45.8 (2 C),
56.2, 56.3, 70.6, 71.8, 74.7, 107.7, 115.3, 124.2, 130.5, 143.4, 152.0. MS
(EI) m/z (%): 430 (M+, 40), 383 (36), 365 (25), 285 (32), 187 (20),
175 (30), 161 (46), 147 (57), 135 (75), 119 (55), 69 (100). HRMS
(EI) calcd for C28H46O3 430.3447, found 430.3462. IR (neat) 3377,
2941, 2875, 1658, 1612, 1452, 1145, 1074, 1045, 977, 912, 756 cm−1.
UV (EtOH) λmax 245, 254, 263 nm.
(5S,6R)-6-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)-
silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-
3,5-diethylheptan-3-ol (9). To a solution of ester 8 (118.9 mg, 0.18
mmol) in THF (3 mL) at room temperature was added EtMgBr (530
μL of 1.0 M THF solution, 0.53 mmol), and the mixture was stirred
for 3 h. The reaction was quenched with saturated NH4Cl, and the
mixture was extracted with AcOEt. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (AcOEt/hexane = 3/97) to afford 9
(104.0 mg, 0.15 mmol) in 86% yield. 1H NMR δ 0.03, 0.04, 0.06, 0.08
(each 3 H, s, SiMe), 0.53 (3 H, s, H-18), 0.83 (3 H, J = 6.3 Hz, d, H-
21), 0.84−0.94 (9H, m, CH2CH3 × 3), 0.86, 0.89 (each 9 H, s, t-Bu),
2.82 (1 H, m, H-9), 4.42 (2 H, m, H-1,3), 4.92, 4.96 (each 1 H, s,
−CCH2), 5.84 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz,
H-6). 13C NMR δ −4.8, −4.69, −4.66, −4.63, 8.2, 8.3, 12.2, 13.5, 13.8,
18.4, 18.5, 22.3, 22.4, 23.7, 26.0 (3 C), 26.1 (3 C), 27.7, 29.0, 31.0,
32.0, 37.4, 38.9, 39.9, 40.3, 40.9, 45.9, 47.8, 54.8, 56.5, 71.9, 72.6, 75.8,
106.5, 116.3, 122.6, 133.0, 141.5, 153.2. MS (APCI) m/z (%):711 (M
+ Na+, 1), 429 (45), 234 (35), 220 (25), 202 (100), 167 (95). HRMS
(APCI) calcd for C42H79O3Si2Na 710.5465, found 710.5462. IR (neat)
3489, 2955, 2930, 2883, 2856, 1730, 1655, 1618, 1472, 1462, 1256,
1101, 935, 897, 835, 775, 667 cm−1. UV (hexane) λmax 246, 255, 265
nm.
−CCH2), 5.83 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz,
H-6); 13C NMR δ −5.1, −4.9, −4.8 (2 C), 7.9, 8.1, 12.0, 13.5, 14.2,
18.1, 18.2, 22.1, 23.3, 23.5, 25.7 (3 C), 25.8 (3 C), 27.5, 28.8, 29.4,
30.7, 30.9, 31.7, 35.1, 38.6, 39.5, 40.6, 40.7, 45.6, 47.5, 54.5, 56.2, 71.7,
72.4, 75.6, 106.2, 116.1, 122.4, 132.7, 141.2, 152.9. MS (EI) m/z (%):
714 (M+, 2), 582 (36), 564 (10), 366 (35), 351 (10), 257 (11), 234
(12), 197 (10), 147 (15), 73 (100). HRMS (EI) calcd for C44H82O3Si2
714.5802, found 714.5804. IR (neat) 3487, 2954, 2927, 2856, 1726,
1658, 1620, 1461, 1251, 1101, 1072, 835, 775 cm−1. UV (hexane) λmax
246, 255, 264 nm.
22S-Butyl-2-methylidene-26,27-dimethyl-19,24-dinor-
1α,25-dihydroxyvitamin D3 (5c). In a manner similar to that for the
synthesis of 5a from 7, target compound 5c (16.6 mg, 0.0342 mmol)
was obtained from 11 (26.7 mg, 0.0374 mmol) in 91% yield. 1H NMR
δ 0.54 (3 H, s, H-18), 0.82 (3 H, d, J = 6.3 Hz, H-21), 0.86 (6 H, t, J =
7.4 Hz, 2 × CH3 of Et), 0.90 (3 H, t, J = 6.8 Hz, CH3 of Bu), 4.47 (2
H, m, H-1, 3), 5.09, 5.11 (each 1 H, s, −CCH2), 5.88 (1 H, d, J =
11.2 Hz, H-7), 6.35 (1 H, d, J = 11.2 Hz, H-6). 13C NMR δ 7.9, 8.0,
12.1, 13.5, 14.2, 22.1, 23.3, 23.5, 27.4, 29.0, 29.4, 30.8 (2 C), 31.6, 35.1,
38.1, 39.3, 40.5 (2 C), 45.8 (2 C), 54.5, 56.3, 70.6, 71.8, 75.6, 107.7,
115.2, 124.2, 130.4, 143.4, 152.0. MS (EI) m/z (%): 486 (M+, 31), 439
(15), 421 (10), 384 (22), 315 (18), 285 (20), 269 (10), 251 (12), 231
(10), 173 (20), 153 (60), 135 (95), 119 (40), 105 (70), 91 (70), 81
(75), 69 (100). HRMS (EI) calcd for C32H54O3 486.4072, found
486.4073. IR (neat) 3388, 2933, 2871, 1712, 1658, 1612, 1456, 1380,
1076, 910, 754 cm−1. UV (EtOH) λmax 246, 254, 263 nm.
Competitive Binding Assay, Human VDR. The human
recombinant VDR ligand-binding domain (LBD) was expressed as
an N-terminal GST-tagged protein in E. coli BL21 (DE3) pLys S
(Promega).31 The cells were lysed by sonication. The supernatants
were diluted approximately 500 times in 50 mM Tris buffer (100 mM
KCl, 5 mM DTT, 0.5% CHAPS, pH 7.5) containing bovine serum
albumin (100 μg/mL). Binding to GST-hVDR-LBD was evaluated
according to the procedure reported.33 The receptor solution (570
μL) in an assay tube was incubated with [3H]-1α,25-dihydroxyvitamin
D3 (specific activity, 5.85 TBq/mmol, ∼2000 cpm) together with
graded amounts of each vitamin D analogue (0.001−100 nM) or
vehicle for 16 h at 4 °C. The bound and free [3H]-1α,25-
dihydroxyvitamin D3 molecules were separated by treating with
dextran-coated charcoal for 30 min at 4 °C. The assay tubes were
centrifuged at 1000g for 10 min. The radioactivity of the supernatant
was counted. Nonspecific binding was subtracted. These experiments
were done in duplicate.
Transfection and Transactivation Assay. COS-7 cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 5% fetal bovine serum (FBS). Cells were seeded
on 24-well plates at a density of 2 × 104 per well. After 24 h, the cells
were transfected with a reporter plasmid containing three copies of the
mouse osteopontin VDRE (5′-GGTTCAcgaGGTTCA, SPPx3-TK-
Luc), a wild-type or mutant hVDR expression plasmid (pCMX-
hVDR), and the internal control plasmid containing sea pansy
luciferase expression constructs (pRL-CMV) by the lipofection
method as described previously.12 After an 8 h incubation, the cells
were treated with either the ligand or ethanol vehicle and cultured for
16 h. Cells in each well were harvested with a cell lysis buffer, and the
luciferase activity was measured with a luciferase assay kit (Promega,
WI, U.S.). Transactivation measured by the luciferase activity was
normalized with the internal control. All experiments were done in
triplicate.
22S-Ethyl-2-methylidene-26,27-dimethyl-19,24-dinor-
1α,25-dihydroxyvitamin D3 (5b). In a manner similar to that for
the synthesis of 5a from 7, target compound 5b (55.0 mg, 0.120
1
mmol) was obtained from 9 (83.8 mg, 0.122 mmol) in 98% yield. H
NMR δ 0.53 (3 H, s, H-18), 0.81 (3 H, J = 6.3 Hz, d, H-21), 0.81−
0.93 (9H, m, CH2CH3 × 3), 2.81 (1 H, m, H-9), 4.44 (2 H, m, H-1,3),
5.06, 5.08 (each 1 H, s, −CCH2), 5.87 (1 H, d, J = 11.1 Hz, H-7),
6.33 (1 H, d, J = 11.1 Hz, H-6). 13C NMR δ 7.9, 8.0, 12.1, 13.3, 13.6,
22.2 (2 C), 23.6, 27.4, 29.0, 30.8, 31.7, 37.2, 38.1, 39.5, 40.0, 40.6, 45.8
(2 C), 54.6, 56.3, 70.6, 71.8, 75.7, 107.7, 115.3, 124.1, 130.6, 143.3,
152.0. MS (APCI) m/z (%): 481 (M + Na+, 100), 437 (52), 429 (23).
HRMS (APCI) calcd for C30H50O3Na 481.3658 found 481.3664. IR
(neat) 3390, 2961, 2939, 2874, 1715, 1651, 1614, 1462, 1456, 1381,
1074, 1045, 978, 912, 756, 667 cm−1. UV (EtOH) λmax 246, 254, 264
nm.
(5S)-4-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-3-
ethylnonan-3-ol (11). To a solution of EtMgI in Et2O (2 mL)
prepared from EtI (91 μL, 1.13 mmol) and magnesium turnings (27.3
mg, 1.13 mmol) was added ester 10 (37.8 mg, 0.054 mmol) in Et2O (1
mL) at room temperature, and the mixture was stirred for 3.5 h. The
reaction was quenched with 1 N HCl, and the mixture was extracted
with AcOEt. The organic layer was washed with saturated NaHCO3
and brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (AcOEt/hexane = 5/95) to afford 11
(26.7 mg, 0.0374 mmol) in 69% yield. 1H NMR δ 0.03, 0.04, 0.06, 0.08
(each 3 H, s, SiMe), 0.53 (3 H, s, H-18), 0.83 (3 H, d, J = 6.6 Hz, H-
21), 0.80−0.94 (9H, m, CH2CH3 × 3), 0.86, 0.89 (each 9 H, s, t-Bu),
2.82 (1 H, m, H-9), 4.42 (2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s,
Protein Expression. The rat VDR-LBD (residues 116−423,
Δ165−211) was subcloned as an N-terminal His6-tagged fusion
protein into the pET-28a vector. E. coli Rosetta 2 (DE3) was freshly
transformed with the plasmid and grown in four flasks containing 0.75
L of 2xTY medium with kanamycin, 34 μg/mL, and chloramphenicol,
50 μg/mL, at 37 °C until an OD of 0.8 was obtained. The cultures
were then induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside
and further incubated at 23 °C for 18 h. Cells were harvested and
resuspended in 50 mL of lysis buffer (50 mM Na/K phosphate, pH
8.0, 10 mM imdazole, 500 mM NaCl, 5% glycerol, 1% Tween 20, 1
mM TCEP, 1 mM PMSF). Cells were lysed by sonication, and the
4379
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
soluble fraction was isolated by centrifugation (18000g for 20 min).
The supernatant was applied to Ni-NTA agarose (Qiagen, Santa
Clarita, CA), and the resin was thoroughly washed in wash buffer (50
mM Na/K phosphate, pH 8.0, 20 mM imdazole, 500 mM NaCl, 5%
glycerol, 1% Tween 20, 1 mM TCEP, 1 mM PMSF). The rat VDR-
LBD was eluted with elution buffer (150 mM imidazole, 50 mM Na/K
phosphate, pH 8.0, 500 mM NaCl, 5% glycerol, 1% Tween 20, 1 mM
TCEP, 1 mM PMSF). The protein was dialyzed overnight against
dialysis buffer A (20 mM Na/K phosphate, pH 7.0, 5% glycerol, 1 mM
EDTA, 0.5 mM DTT) and then loaded onto a HiTrap SP HP (1 mL)
column (GE Healthcare) equilibrated with buffer A. The elution was
performed by NaCl gradient buffer from 0 to 1.0 M. His-tag of the
protein in elution mixture (17 mL) was cleaved by addition of 70 units
of thrombin and subsequent incubation at 23 °C for 18 h. Then NaCl
(1.96 g) was added to the digested mixture (17 mL), and the resulting
mixture was passed through a HiTrap benzamidine FF (1 mL)
columun (GE healthcare). The flow-through was further purified by
Superdex S75 gel filtration (25 mL) column (GE Healthcare) with a
buffer (100 mM NaCl, Tris-HCl, pH 7.0). Purified rat VDR-LBD was
concentrated in buffer B (10 mM Tris-HCl, pH 7.0, 2 mM NaN3, 10
mM DTT, 0.5 mM PMSF) to 7.5 mg/mL, which was estimated by UV
absorbance at 280 nm.
X-ray Crystallographic Analysis. A ligand (∼10 equiv) was
added to the rVDR-LBD, and then coactivator peptide (H2N-
KNHPMLMNLLKDN-CONH2) derived from DRIP205 in buffer C
(25 mM Tris-HCl, pH 8.0; 50 mM NaCl; 10 mM DTT; 2 mM NaN3)
was added. The mixture of VDR-LBD/ligand/peptide was allowed to
crystallize by the vapor diffusion method using a series of precipitant
solutions containing 0.1 M MOPS-Na (pH 7.0) or 0.1 M MES-Na
(pH 7.0), 0.05−0.4 M sodium formate, 12−22% (w/v) PEG4000, and
5% ethylene glycol. Droplets for crystallization were prepared by
mixing 1 μL of complex solution and 1 μL of precipitant solution, and
droplets were equilibrated against 300 μL of precipitant solution at 20
°C. Prior to diffraction data collection, crystals were soaked in a
cryoprotectant solution containing 0.1 M MOPS-Na, pH 7.0, or 0.1 M
MES-Na, pH 7.0, 0.05−0.3 M sodium formate, 12−22% (w/v)
PEG4000, and 5% ethylene glycol. Diffraction data sets were collected
at 100 K in a stream of nitrogen gas at beamline BL-6A of KEK-PF or
NW-12A of KEK-PFAR (Tsukuba, Japan). Reflections were recorded
with an oscillation range per image of 1.0°. Difflections data were
indexed, integrated, and scaled using the program iMOSFLM34,35 The
structures of ternary complex were solved by molecular replacement
with the software Phaser36 in the CCP4 program37 using a rat VDR-
LBD coordinates (PDB code 2ZLC), and finalized sets of atomic
coordinates were obtained after iterative rounds of model modification
with the program Coot38 and refinement with refmac5.39−43 The
coordinate data for the structures were deposited in Protein Data Bank
with accession numbers 3VRT (VDR-LBD/3a complex), 3VRU
(VDR-LBD/4a complex), 3VRV (VDR-LBD/5a complex), and
3VRW (VDR-LBD/5c complex).
Technology Research Center Project (Grant 19-8) from the
Ministry of Education, Culture, Sports, Science and Technol-
ogy, Japan. The synchrotron-radiation experiment was
performed at the Photon Factory (Proposal Grants
2008G169 and 2009G647). We acknowledge the help provided
by the beamline scientists at the Photon Factory.
ABBREVIATIONS USED
■
VDR, vitamin D receptor; LBP, ligand binding pocket; RXR,
retinoid X receptor; LBD, ligand binding domain
REFERENCES
■
(1) Feldman, D., Pike, J. W., Glorieux, F. H., Eds. Vitamin D, 2nd ed.;
Elsevier Academic Press: Amsterdam, 2005.
(2) 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.
(3) Yamada, S.; Shimizu, M.; Yamamoto, K. Vitamin D Receptor. In
Vitamin D and Rickets; Hochberg, Z., Ed.; Endocrine Develpment, Vol.
6; Karger: Basel, Switzerland, 2003; pp 50−68.
(4) Binderup, L.; Binderup, E.; Godtfredsen, W. O. Development of
New Vitamin D Analogs. In Vitamin D, 2nd ed.; Feldman, D., Pike, J.
W., Glorieux, F. H., Eds.; Elsevier Academic Press: Amsterdam, 2005;
pp 1027−1043.
(5) Kubodera, N. Pharmaceutical studies on vitamin D derivatives
and practical syntheses of six commercially available vitamin D
derivatives that contribute to current clinical practice. Heterocycles
2010, 80, 83−98.
(6) Bury, Y.; Steinmeyer, A.; Carlberg, C. Structure activity
relationship of carboxylic ester antagonists of the vitamin D3 receptor.
Mol. Pharmacol. 2000, 58, 1067−1074.
(7) Igarashi, M.; Yoshimoto, N.; Yamamoto, K.; Shimizu, M.;
Makishima, M.; DeLuca, H. F.; Yamada, S. Identification of a highly
potent vitamin D receptor antagonist: (25S)-26-adamantyl-25-
hydroxy-2-methylene-22,23-didehydro-19,27-dinor-20-epi-vitamin D3
(ADMI3). Arch. Biochem. Biophys. 2007, 460, 240−253.
(8) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.; Norman,
A. W.; Ishizuka, S. Antagonistic action of novel 1α,25-dihydrox-
yvitamin D3-26,23-lactone analogs on differentiation of human
leukemia cells (HL-60) induced by 1α,25-dihydroxyvitamin D3. J.
Biol. Chem. 1999, 274, 16392−16399.
(9) (a) Ishizuka, S.; Kurihara, N.; Miura, D.; Takenouchi, K.;
Cornish, J.; Cundy, T.; Reddy, S. V.; Roodman, G. D. Vitamin D
antagonist, TEI-9647, inhibits osteoclast formation induced by 1α,25-
dihydroxyvitamin D3 from pagetic bone marrow cells. J. Steroid
Biochem. Mol. Biol. 2004, 89−90, 331−334. (b) Roodman, G. D.;
Windle, J. J. Paget disease of bone. J. Clin. Invest. 2005, 115, 200−208.
(10) Saito, N.; Saito, H.; Anzai, M.; Yoshida, A.; Fujishima, T.;
Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama, H.; Kittaka, A.
Dramatic enhancement of antagonistic activity on vitamin D receptor:
a double functionalization of 1α-hydroxyvitamin D3 26,23-lactones.
Org. Lett. 2003, 5, 4859−4862.
(11) Yoshimoto, N.; Inaba, Y.; Yamada, S.; Makishima, M.; Shimizu,
M.; Yamamoto, K. 2-Methylene 19-nor-25-dehydro-1α-hydroxyvita-
min D3 26,23-lactones: synthesis, biological activities and molecular
basis of passive antagonism. Bioorg. Med. Chem. 2008, 16, 457−473.
(12) Inaba, Y.; Yoshimoto, N.; Sakamaki, Y.; Nakabayashi, M.; Ikura,
T.; Tamamura, H.; Ito, N.; Shimizu, M.; Yamamoto, K. A new class of
vitamin D analogues that induce structural rearrangement of the
ligand-binding pocket of the receptor. J. Med. Chem. 2009, 52, 1438−
1449.
Graphical Manipulations and Ligand Docking. Graphical
manipulations were performed using SYBYL 8.0 (Tripos, St. Louis).
The atomic coordinates of the crystal structure of rVDR-LBD
complexed with 1a were retrieved from Protein Data Bank (PDB)
(entry 2ZLC).24
AUTHOR INFORMATION
Corresponding Author
*Phone: +81 42 721 1580. Fax: +81 42 721 1580. E-mail:
yamamoto@ac.shoyaku.ac.jp.
■
Author Contributions
⊥These authors contributed equally.
Notes
The authors declare no competing financial interest.
(13) 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.
(14) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. M.; Mitschler, A.;
Moras, D. Crystal structures of the vitamin D receptor complexed to
ACKNOWLEDGMENTS
Part of this work was supported by a Grant-in-Aid for Scientific
Research (Grant 23590135) and a Grant-in-Aid for High
■
4380
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381

Journal of Medicinal Chemistry
Article
super agonist 20-epi ligands. Proc. Natl. Acad. Sci. U.S.A. 2001, 98,
5491−5496.
(15) Vanhooke, J. L.; Benning, M. M.; Bauer, C. B.; Pike, J. W.;
DeLuca, H. F. Molecular structure of the rat vitamin D receptor ligand
binding domain complexed with 2-carbon-substituted vitamin D3
hormone analogues and a LXXLL-containing coactivator peptide.
Biochemistry 2004, 43, 4101−4110.
(16) Ciesielski, F.; Rochel, N.; Mitschler, A.; Kouzmenko, A.; Moras,
D. Structural investigation of the ligand binding domain of the
zebrafish VDR in complexes with 1α,25(OH)2D3 and Gemini:
purification, crystallization and preliminary X-ray diffraction analysis.
J. Steroid Biochem. Mol. Biol. 2004, 89−90, 55−59.
(17) Tocchini-Valentini, G.; Rochel, N.; Wurtz, J. -M.; Moras, D.
Crystal structures of the vitamin D nuclear receptor liganded with the
vitamin D side chain analogues calcipotriol and seocalcitol, receptor
agonists of clinical importance. Insights into a structural basis for the
switching of calcipotriol to a receptor antagonist by further side chain
modification. J. Med. Chem. 2004, 47, 1956−1961.
(18) Eelen, G.; Verlinden, L.; Rochel, N.; Claessens, F.; De Clercq,
P.; Vandewalle, M.; Tocchini-Valentini, G.; Moras, D.; Bouillon, R.;
Verstuyf, A. Superagonistic action of 14-epi-analogs of 1,25-
dihydroxyvitamin D explained by vitamin D receptor−coactivator
interaction. Mol. Pharmacol. 2005, 67, 1566−1573.
(19) 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 1α,25-dihydroxyvitamin D3 with selected 2α-sub-
stituted analogues. J. Med. Chem. 2006, 49, 5199−5205.
(20) Ciesielski, F.; Rochel, N.; Moras, D. Adaptability of the vitamin
D nuclear receptor to the synthetic ligand Gemini: remodelling the
LBP with one side chain rotation. J. Steroid Biochem. Mol. Biol. 2007,
103, 235−242.
(21) Rochel, N.; Hourai, S.; Perez-Garcia, X.; Rumbo, A.; Mourino,
A.; Moras, D. Crystal structure of the vitamin D nuclear receptor
ligand binding domain in complex with a locked side chain analog of
calcitriol. Arch. Biochem. Biophys. 2007, 460, 172−176.
(22) Vanhooke, J. L.; Tadi, B. P.; Benning, M. M.; Plum, L. A.;
DeLuca, H. F. New analogs of 2-methylene-19-nor-(20S)-1,25-
dihydroxyvitamin D3 with conformationally restricted side chains:
evaluation of biological activity and structural determination of VDR-
bound conformations. Arch. Biochem. Biophys. 2007, 460, 161−165.
(23) Hourai, S.; Rodrigues, L. C.; Antony, P.; Reina-San-Martin, B.;
Ciesielski, F.; Magnier, B. C.; Schoonjans, K.; Mourino, A.; Rochel, N.;
Moras, D. Structure-based design of a superagonist ligand for the
vitamin D nuclear receptor. Chem. Biol. 2008, 15, 383−392.
(24) Shimizu, M.; Miyamoto, Y.; Takaku, H.; Matsuo, M.;
Nakabayashi, M.; Masuno, H.; Udagawa, N.; DeLuca, H. F.; Ikura,
T.; Ito, N. 2-Substituted-16-ene-22-thia-1α,25-dihydroxy-26,27-di-
methyl-19-nor-vitamin D3 analogs: synthesis, biological evaluation,
and crystal structure. Bioorg. Med. Chem. 2008, 16, 6949−6964.
(25) Nakabayashi, M.; Yamada, S.; Yoshimoto, N.; Tanaka, T.;
Igarashi, M.; Ikura, T.; Ito, N.; Makishima, M.; Tokiwa, H.; Deluca, H.
F.; Shimizu, M. Crystal structures of rat vitamin D receptor bound to
adamantyl vitamin D analogs: structural basis for vitamin D receptor
antagonism and partial agonism. J. Med. Chem. 2008, 51, 5320−5329.
(26) Uskokovic, M. R.; Manchand, P. S.; Peleg, S.; Norman, A. W.
Synthesis and Preliminary Evaluation of the Biological Properties of a
1α,25-Dihydroxyvitamin D3 Analogue with Two Side-Chains. In
Vitamin D: Chemistry, Biology and Clinical Applications of the Steroid
Hormone; Norman, A. W., Bouillon, R., Thomasset, M., Eds.;
University of California: Riverside, CA, 1997; pp19−21.
(29) Sicinski, R. R.; Prahl, J. M.; Smith, C. M.; DeLuca, H. F. New
1α,25-dihydroxy-19-norvitamin D3 compounds of high biological
activity: synthesis and biological evaluation of 2-hydroxymethyl, 2-
methyl, and 2-methylene analogues. J. Med. Chem. 1998, 41, 4662−
4674.
(30) Yamamoto, K.; Ogura, H.; Jukuta, J.; Inoue, H.; Hamada, K.;
Sugiyama, Y.; Yamada, S. Stereochemical and mechanistic studies on
conjugate addition of organocuprates to acyclic enones and enoates:
simple rule for diastereofacial selectivity. J. Org. Chem. 1998, 63,
4449−4458.
(31) Inaba, Y.; Yamamoto, K.; Yoshimoto, N.; Matsunawa, M.; Uno,
S.; Yamada, S.; Makishima, M. Vitamin D3 derivatives with
adamantane or lactone ring side chains are cell type-selective vitamin
D receptor modulators. Mol. Pharmacol. 2007, 71, 1298−1311.
(32) Brandl, M.; Weiss, M. S.; Jabs, A.; Suhnel, J.; Hilgenfeld, R. C−
̈
H···π-interactions in proteins. J. Mol. Biol. 2001, 16, 357−377.
(33) Yamamoto, K.; Sun, W.-Y.; Ohta, M.; Hamada, K.; DeLuca, H.
F.; Yamada, S. Conformationally restricted analogs of 1α,25-
dihydroxyvitamin D3 and its 20-epimer: compounds for study of the
three-dimensional structure of vitamin D responsible for binding to
the receptor. J. Med. Chem. 1996, 39, 2727−2737.
(34) Leslie, A. G. W.; Powell, H. R. In Evolving Methods for
Macromolecular Crystallography, ; Read, R. J., Sussman, J. L., Eds.;
NATO Science Series II, Vol. 245; Springer Verlag: Dordrecht, The
Netherlands, 2007; pp 41−51.
(35) Battye, T. G.; Kontogiannis, L.; Johnson, O.; Powell, H. R.;
Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image
processing with MOSFLM. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2011, 67, 271−281.
(36) McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M.
D.; Storoni, L. C.; Read, R. J. Phaser crystallographic software. J. Appl.
Crystallogr. 2007, 40, 658−674.
(37) Collaborative Computational Project, Number 4. The CCP4
Suite: Programs for Protein Crystallography. Acta Crystallogr., Sect. D:
Biol. Crystallogr. 1994, 50, 760−763.
(38) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Features and
development of Coot. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2010,
66, 486−501.
(39) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method. Acta
Crystallogr., Sect. D: Biol. Crystallogr. 1997, 53, 240−255.
(40) Pannu, N. S.; Murshudov, G. N.; Dodson, E. J.; Read, R. J.
Incorporation of prior phase information strengthens maximum-
likelihood structure refinement. Acta Crystallogr., Sect. D: Biol.
Crystallogr. 1998, 54, 1285−1294.
(41) Winn, M. D.; Isupov, M. N.; Murshudov, G. N. Use of TLS
parameters to model anisotropic displacements in macromolecular
refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2001, 57, 122−
133.
(42) Steiner, R. A; Lebedev, A. A.; Murshudov, G. N. Fisher’s
information in maximum-likelihood macromolecular crystallographic
refinement. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2003, 59, 2114−
2124.
́
(43) Murshudov, G. N.; Skubak, P.; Lebedev, A. A.; Pannu, N. S.;
Steiner, R. A.; Nicholls, R. A.; Winn, M. D.; Long, F.; Vagin, A. A.
REFMAC5 for the refinement of macromolecular crystal structures.
Acta Crystallogr., Sect. D: Biol. Crystallogr. 2011, 67, 355−367.
(27) Kurek-Tyrlik, A.; Makaev, F. Z.; Wicha, J.; Zhabinskii, V.;
Calverley, M. J. Pivoting 20-Normal and 20-Epi Calcitriols: Synthesis
and Crystal Structure of a “Double Side Chain” Analogue. In Vitamin
D: Chemistry, Biology and Clinical Applications of the Steroid Hormone;
Norman, A. W., Bouillon, R., Thomasset, M., Eds.; University of
California: Riverside, CA, 1997; pp 30−31.
(28) Sakamaki, Y.; Inaba, Y.; Yoshimoto, N.; Yamamoto, K. Potent
antagonist for the vitamin D receptor: vitamin D analogues with
simple side chain structure. J. Med. Chem. 2010, 53, 5813−5826.
4381
dx.doi.org/10.1021/jm300230a | J. Med. Chem. 2012, 55, 4373−4381