A new class of vitamin D analogues that induce structural rearrangement of the ligand-binding pocket of the receptor

Article abstract of DOI:10.1021/jm8014348

To identify novel vitamin D receptor (VDR) ligands that induce a novel architecture within the ligand-binding pocket (LBP), we have investigated eight 22-butyl-1α,24-dihydroxyvitamin D₃ derivatives (3-10), all having a butyl group as the branch

Full text of DOI:10.1021/jm8014348

1438
J. Med. Chem. 2009, 52, 1438–1449
A New Class of Vitamin D Analogues that Induce Structural Rearrangement of the
Ligand-Binding Pocket of the Receptor
Yuka Inaba,†,‡ Nobuko Yoshimoto,† Yuta Sakamaki,†,§ Makoto Nakabayashi,‡,§ Teikichi Ikura,‡ Hirokazu Tamamura,§
Nobutoshi Ito,‡ Masato Shimizu,‡,§ and Keiko Yamamoto*,†
Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical UniVersity, 3-3165 Higashi-Tamagawagakuen, Machida,
Tokyo 194-8543, Japan, School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental UniVersity,
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
ReceiVed NoVember 13, 2008
To identify novel vitamin D receptor (VDR) ligands that induce a novel architecture within the ligand-
binding pocket (LBP), we have investigated eight 22-butyl-1R,24-dihydroxyvitamin D3 derivatives (3-10),
all having a butyl group as the branched alkyl side chain. We found that the 22S-butyl-20-epi-25,26,27-
trinorvitamin D derivative 5 was a potent VDR agonist, whereas the corresponding compound 4 with the
natural configuration at C(20) was a potent VDR antagonist. Analogues with the full vitamin D3 side chain
were less potent agonist, and whether they were agonists or antagonists depended on the 24-configuration.
X-ray crystal structures demonstrated that the VDR-LBD accommodating the potent agonist 5 has an
architecture wherein the lower side and the helix 11 side of the LBP is simply expanded relative to the
canonical active-VDR situation; in contrast, the potent antagonist 4 induces an extra cavity to accommodate
the branched moiety. This is the first report of a VDR antagonist that generates a new cavity to alter the
canonical pocket structure of the ligand occupied VDR.
Introduction
the subsequent loop 6-7, and the N-terminal of helix 7 has a
certain flexibility without loss of function because, despite the
new pocket structure, GEMINI 1b acts as a VDR agonist.19
We considered that some new ligands having the right side chain
might be able to change the induced pocket structure in such a
way as to modulate the VDR differently. Here, we report a novel
class of vitamin D analogues exhibiting a stereospecific
dichotomy of activities.
The hormonally active metabolite of vitamin D3, 1R,25-
dihydroxyvitamin D3 [1,25-(OH)2D3, 1a], plays an important
role in calcium homeostasis, cell differentiation and proliferation,
and immunomodulation.1 1,25-(OH)2D3 (1a) and its active
analogues exert these effects by binding to the vitamin D
receptor (VDRa), which belongs to the nuclear receptor super-
family.2 The VDR is a ligand-dependent transcription factor that
functions as a heterodimer with another nuclear receptor, retinoid
X receptor (RXR).3 Upon ligand binding, the VDR undergoes
a conformational change that promotes RXR-VDR heterodimer-
ization.3 The RXR-VDR heterodimer binds to the vitamin D
response elements present in the promoter region of responsive
genes, recruits the coactivator, and initiates the transcription.
Since the first report of the X-ray crystal structure of the
ligand binding domain (LBD) of the VDR complexed with the
hormone 1,25-(OH)2D3 (1a) by Moras’ group in 2000,4 several
groups have solved the crystal structures of the VDR-LBD
complexed with a variety of ligands.5-16 All of the crystal
structures showed the canonical Moras’ conformation of the
VDR-LBD and quite similar architectures of the ligand binding
pocket (LBP), except for the complex of zebrafish VDR-LBD
with “GEMINI, 1b”,7,11 an analogue with two identical side
chains.17,18 The zebrafish VDR-LBD/GEMINI complex re-
vealed the formation of a new channel extending the original
LBP in order to accommodate the second side chain. This
particular observation suggests that the region around helix 6,
Design and Synthesis
We have already reported that some 22-alkyl-1R,25-dihy-
droxyvitamin D3, and in particular the 20-epi analogues, act as
superagonists of the VDR.20-22 Docking analysis and alanine
scanning mutational analysis (ASMA) gave a clue that the 22-
alkyl substituent of 22R-butyl-20-epi-1,25-(OH)2D3 2 might
induce a novel cavity in a similar way to GEMINI 1b.22 To
explore further vitamin D compounds possessing a 22-butyl
group, we have now investigated a series of 22-butyl-1R,24-
dihydroxyvitamin D3 derivatives 3-10 as candidate VDR
modulators. The 25-to-24 transposition of the hydroxyl group
is a known agonistic modification, notably seen in several
clinically important vitamin D analogues. In compounds 3-6,
three carbons at the side chain terminus were removed to
decrease the degree of hydrophobic interactions with helix 12,
including the terminal of helix 11, of the VDR. On the basis of
our previous study, we had concluded that insufficient hydro-
phobic interactions between the ligand and the C-terminal of
the VDR cause antagonistic behavior.23 Compounds 7-10 were
designed as the counterparts of side chain truncated analogues
3 and 4 to test the significance of the hydrophobic interactions
(Chart 1).
* To whom correspondence should be addressed. Phone: +81 42 721
1580. Fax: +81 42 721 1580. E-mail: yamamoto@ac.shoyaku.ac.jp.
†
Laboratory of Drug Design and Medicinal Chemistry, Showa Phar-
As shown in Scheme 1, 22-butyl vitamin D derivatives 3 and
4 were synthesized from 22R-butyl compound 11 and 22S-butyl
compound 12, respectively, which are the established products
of a diastereofacial selective conjugate addition reaction of
n-Bu2CuLi with the E- and Z-enoates developed by our group
previously.24 The configuration at C(22) follows from the
maceutical University.
‡
School of Biomedical Science, Tokyo Medical and Dental University.
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental
§
University.
a Abbreviations: VDR, vitamin D receptor; LBP, ligand binding pocket;
1,25-(OH)2D3, 1R,25-dihydroxyvitamin D3; RXR, retinoid X receptor; LBD,
ligand binding domain; ASMA, alanine scanning mutational analysis.
10.1021/jm8014348 CCC: $40.75
2009 American Chemical Society
Published on Web 02/04/2009

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1439
Chart 1. Structures of 1,25-(OH)2D3 (1a) and Its Derivatives
stereochemistry of the starting enoates and the reaction condi-
tions.24 Reduction of the side chain ester together with reductive
cleavage of the carbonates at C(1) and C(3) as protecting groups
of 11 and 12 gave provitamins D, 13 and 14, respectively. These
provitamins were converted to the desired vitamin D analogues
3 and 4 by the biomimetic sequence of photoirradiation with a
high-pressure mercury lamp, followed by thermal isomerization.
The 20-epi-analogues 5 and 6 were synthesized by the analogous
procedure as that described above for the synthesis of the 20-
normal analogues 3 and 4.
in Figure 1a,b. This analysis clearly indicated that the 24-
alcohols 23 and 26 have a 24R-configuration and a 24S-
configuration, respectively. Consequently, the configurations of
24-isomers 24 and 25 were demonstrated to be S and R,
respectively. Methyl carbonates at C(1) and C(3) were saponified
to give provitamins D 27-30. These provitamins were converted
to the desired vitamin D analogues 7-10 by photoirradiation,
followed by thermal isomerization.
Biological Activities
22-Butyl-1R,24-dihydroxyvitamins D3 7-10 were success-
fully synthesized from the same 22-butyl compounds 11 and
12. Controlled chemoselective reduction of the methyl esters
of 11 and 12 by DIBAL afforded 24-alcohols 19 and 20,
respectively. Swern oxidation of the 24-alcohols 19 and 20 gave
the aldehyde 21 and 22, respectively. 22R-aldehyde 21 reacted
with i-PrMgCl to give the 24R- and 24S-isomers 23 (less polar)
and 24 (more polar), while 22S-aldehyde 22 reacted with
i-PrMgCl to give the 24R- and 24S-isomers 25 (less polar) and
26 (more polar). Epimers were separated by column chroma-
tography. The configuration at C24 was determined by the
Kusumi-Mosher method (Scheme 2).25 Thus, less polar
compound 23 was converted to the corresponding S- and
R-MTPA esters of the 24-hydroxyl group (31 and 32), and the
22-isomer 26 was also converted to those of the 24-hydroxyl
group (33 and 34). Chemical shift differences between the
S-MTPA ester and the corresponding R-MTPA ester are shown
Binding affinity for the VDR was evaluated by the standard
competitive binding assay using bovine thymus VDR, which
has a LBD amino acid sequence26 identical to that of the human
VDR.27 The results are summarized in Table 1. Of the four
22-butyl-1R,24-dihydroxy-25,26,27-trinorvitamins D3 3-6, the
22S-compounds 4 and 5 showed significant affinity compared
to the natural hormone 1a, while their 22R-isomers 3 and 6
showed weak affinity for the VDR. Among the four 22-butyl-
1R,24-dihydroxyvitamins D3 7-10, both of the 24S-hydroxyl
compounds 8 and 10 showed significant affinity for the VDR,
while both the 24R-hydroxyl compounds 7 and 9 showed
relatively weak affinity.
The ability of 22-butyl compounds 3-10 to induce transcrip-
tion of a vitamin D-responsive gene was tested using the rat
osteopontin luciferase reporter gene assay system in Cos7 cells,
and the results are shown in Figure 2. In this assay, only

1440 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Inaba et al.
Scheme 1. Synthetic Sequence Leading to Target Compounds 3-10
Scheme 2. MTPA esters from 24-Alcohols 23 and 26
affinity and transactivation potency are not proportional to each
other.22,28 The potency to inhibit VDR activation was evaluated
in the presence of 1 nM 1,25-(OH)2D3 (1a). As shown in Figure
2, compounds 4, 6, 8, and 10 concentration-dependently
inhibited the transactivation induced by 1,25-(OH)2D3 (1a),
indicating that these four analogues work as VDR antagonists.
We performed the same assays in HEK293 cells and obtained
similar results (see Supporting Information Figure S1).
We evaluated the expression of CYP24A1 mRNA in HEK293
cells by quantitative real-time PCR. CYP24A1 is known to be
the most potent target gene of the hormone 1,25-(OH)2D3 (1).29a
In accordance with the transcriptional activities of our synthetic
ligands described above, only compound 5 expressed CYP24A1
mRNA strongly in comparison with the natural hormone 1a,
and compound 9 showed weak expression activity (Figure 3a,b).
All of the others, 3, 4, 6, 7, 8, and 10, showed little activity.
The inhibition potency of synthetic ligands for CYP24A1
mRNA expression was evaluated in the presence of 10 nM 1,25-
(OH)2D3 (1a). As shown in Figure 3a,b, compounds 4, 6, 8,
and 10 significantly inhibited the gene expression induced by
1,25-(OH)2D3 (1a). Figure 3c shows that compound 4 inhibited
the gene expression concentration-dependently.
compound 5 showed full agonistic activity and two analogues
7 and 9 showed weak agonist activity. All of the others, 3, 4,
6, 8, and 10, showed little activity. It is known that receptor

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1441
Figure 1. Determination of stereochemistry at C(24). Chemical shift differences.
Table 1. VDR Binding Affinity of 22-Butyl Compounds 3-10a
1a
3 (22R)
4 (22S)
5 (22S)
6 (22R)
7 (22R,24R)
8 (22R,24S)
9 (22S,24R)
10 (22S,24S)
EC50 (nM)b
0.05
1
11
1/220
1.2
1/24
2.0
1/40
20
1/400
16
1/320
3.4
1/68
20
1/400
3.8
1/76
relative affinityc
a Competitive binding of 1,25-(OH)2D3 (1a) and 22-butyl compounds (3-10) to the bovine thymus vitamin D receptor. The experiments were carried out
in duplicate. b The EC50 values are derived from dose-response curves and represent the compound concentration required for 50% displacement of the
radio-labeled 1,25-(OH)2D3 from the receptor protein. c Affinity relative to 1,25-(OH)2D3 (1a), the activity of 1a being defined as 1.
Cell differentiation and antiproliferation activity were evalu-
ated using human promyelocyte HL-60 cells.29b Compound 5
showed significant activity for differentiation of HL-60 cells
into monocytes/macrophages (Figure 4), being comparable to
that of 1,25-(OH)2D3 (1a), whereas compound 9 showed weak
activity. None of the other compounds induced differentiation
of HL-60 cells. Similar results were obtained in experiments
examining the antiproliferative effect on HL-60 cells (data not
shown). Ligand 4 concentration-dependently inhibited the
differentiation and antiproliferation of HL-60 cells induced by
stimulation with 1,25-(OH)2D3 (1a) (Figure 4c). Morphologic
analysis also indicated that the differentiation of HL-60 cells
was inhibited by ligand 4 (Figure 4d).
the presence of the coactivator peptide at 2.1 Å resolution (Table
2). A ribbon-tube representation of the rVDR-LBD/5/peptide
complex is shown in Figure 5a, and the results of the
crystallographic refinement are summarized in Table 2. The
electron density of the VDR-LBD complex including ligand
5 is well defined. The overall structure of the VDR-LBD is
similar to that observed for VDR-LBD bound to 1,25-(OH)2D3
(1a),15 although the ligand 5 has a branched alkyl side chain.
The coactivator peptide is tightly bound to the activation
function 2 surface formed by helices 3, 4, and 12 of the LBD
and occupies the typical agonist position (Figure 5a). However,
the docking mode of agonist 5 is unusual. In the crystal structure,
the part from the A-ring to the D-ring occupies almost the same
position as that of 1,25-(OH)2D3 (1a), but the mode in which
the side chain is accommodated into the pocket is unique (Figure
5b), i.e., the 22-butyl group, and not the canonical side chain
bearing the 24-hydroxyl group, is oriented toward helix 12, and
the 24-hydroxyl group itself is oriented toward helix 6 to form
a hydrogen bond with the carbonyl oxygen of the main chain
of Val296 (hVal300) on helix 6. Because both of the side chains
of ligand 5 are accommodated tightly in the pocket of the VDR
active conformation, the pocket is slightly expanded in the
bottom (lower) region, where the side chain hydroxyl group of
5 is located, and in the terminal region of helix 11, where the
terminal of the butyl group of 5 is located together with a
molecule. The expansion of the bottom region results from steric
repulsion between the side chains of the residues on helix 6
and the subsequent loop 6-7 of the VDR and the 24-hydroxyl
group of ligand 5. Expansion at the helix 11 side is caused by
twisting of the side chain of Leu400 (hLeu404) on loop 11-12
(Figure 5b).
Thus, in all assays, ligands 4 and 5 were demonstrated to
behave as a potent antagonist and a potent agonist, respectively.
Alanine Scanning Mutational Analysis
We have developed and reported a method for exhaustive
alanine scanning mutational analysis (ASMA) of residues lining
the LBP of the VDR.30 We applied this analysis to all eight
compounds 3-10. Transactivation was examined using 22-butyl
compounds (3-10) at a concentration of 10-6 M. The results
are shown in Figure S2 (see Supporting Information) in
comparison with the natural hormone (1a) and the parent
molecule, 22R-butyl-20-epi-1,25-(OH)2D3 2.
Recruitment of RXR and Coactivator Peptide
We evaluated ligand-dependent recruitment of the RXR and
a coactivator peptide, SRC-1, to the VDR using a mammalian
two-hybrid assay similar to that reported previously.31 As shown
in Figure S3 of the Supporting Information, compound 5
recruited the RXR and SRC-1 strongly, whereas compounds 7
and 9 did so weakly. Inhibition of SRC-1 recruitment induced
by natural hormone 1a was observed for compounds 4, 8, and
10 (data not shown).
To understand the structural basis of the antagonistic effect
of compound 4, we attempted to solve the crystal structure of
the VDR-LBD/4 complex. We were able to obtain crystals of
the complex in the presence of the coactivator peptide, but not
initsabsence.Theresolutionoftheternarycomplex(rVDR-LBD/
4/peptide) was 3.0 Å (Table 2). The electron density of the main
chain of the VDR-LBD and almost all of ligand 4 was clearly
observed. The overall structure of the main chain, including
helices 11 and 12, is quite similar to that of the VDR-LBD/
1a/peptide complex (Figure 6a). The main chains of only two
Structure
To understand the molecular basis of action of our synthetic
ligands, we conducted X-ray crystal structure analysis of the
VDR-LBD/ligand complex and successfully solved the crystal
structure of rat VDR-LBD bound to the potent agonist 5 in

1442 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Inaba et al.
Figure 2. Transactivation of compounds 3-10 in Cos7 cells. Transcriptional activity (a and b) and inhibitory effect on the transactivation induced
by 1,25-(OH)2D3 (1a) (c and d) were evaluated by dual luciferase assay using a full-length human VDR expression plasmid (pCMX-hVDR), a
reporter plasmid containing three copies of the mouse osteopontin VDRE (SPPx3-TK-Luc), and the internal control plasmid containing sea pansy
luciferase expression constructs (pRL-CMV) in Cos7 cells as described previously.32 Luciferase activity in the presence of 10-8 M 1,25-(OH)2D3
(1a) was defined as 1: (a) transcriptional activity of 1a and 3-6, (b) transcriptional activity of 1a and 7-10, (c) transcriptional activity of 3-6 in
the presence of 10-9 M 1,25-(OH)2D3 (1a), (d) transcriptional activity of 7-10 in the presence of 10-9 M 1,25-(OH)2D3 (1a). All experiments were
done in triplicate.
Figure 3. Effect of 1a and 22-butyl compounds (3-10) on expression of the CYP24A1 gene in HEK293 cells. (a,b) Cells were treated with 10-6
M 22-butyl compounds ((a) 3-6, (b) 7-10) in the absence or presence of 10-8 M 1,25-(OH)2D3 (1a) for 24 h. Concentration (10-6 M) of test
compounds was selected to obtain clear results concerning whether they activate CYP24A1 expression or inhibit it. (c) Cells were treated with the
indicated concentrations of compounds for 24 h. All experiments were carried out in triplicate.

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1443
Figure 4. Effect of 1a and 22-butyl compounds (3-10) on differentiation of HL-60 cells. (a-c) Cells were treated with the indicated concentrations
of compounds for four days. Differentiation was determined by nitro blue tetrazolium (NBT) reduction.33 All experiments were carried out in
triplicate. (d) HL-60 cells were stained with Wright-Giemsa and analyzed for morphology by light microscopy.
Table 2. Summary of Data Collection Statistics and Refinement
ligand
X-ray source
compd 4
KEK-PF BL-6A
compd 5
KEK-PF BL-6A
wavelength (Å)
0.97800
0.97800
space group
C2
C2
unit cell dimensions (Å)
(deg)
a ) 153.25, b ) 43.42, c ) 42.37
R ) 90.00, ꢀ ) 95.97, γ ) 90.00
50.00-3.00 (3.11-3.00)
17932
5664
99.1 (99.8)
a ) 153.16, b ) 43.88, c ) 41.89
R ) 90.00, ꢀ ) 95.89, γ ) 90.00
50.00-2.10 (2.18-2.10)
59861
16470
97.5 (92.2)
resolution range (Å)a
total number of reflections
no. of unique reflections
% completenessa
a b
Rmerge
,
0.086 (0.392)
0.045 (0.234)
refinement statistics
resolution range (Å)a
50.00-3.00 (3.11-3.00)
0.295 (0.431)/0.226 (0.321)
50.00-2.10 (2.18-2.10)
0.251 (0.367)/0.209 (0.336)
a,c
R factor (Rfree/Rwork
)
a Values in parentheses are for the highest-resolution shell. b Rmerge ) ∑|(Ihkl - Ihkl )|/(∑Ihkl), where Ihkl is the mean intensity of all reflections equivalent
c
to reflection hkl. Rwork (Rfree) ) ∑||Fobs| - |Fcalc||/∑|Fobs|, where 10% of randomly selected data were used for Rfree
.
amino acid residues, Leu305 (hLeu309) and Ser394 (hSer398),
are slightly twisted outward (Figure 6b). The crystal structure
clearly demonstrated that a novel cavity is produced in the region
surrounded by helix 6, the subsequent loop 6-7, the N-terminal
of helix 7 and helix 11, as in the zVDR/GEMINI complex,
because the terminal of the 22-butyl group of 4 pushes out the
side chain of Leu305 to rearrange the LBP (Figure 6c,d).
Thus, we identified a new class of VDR ligands with a simple
side chain that modulate the structure of the LBP.
Supporting Information Figure S3). These experimental results
corroborate the crystal structure of the VDR-LBD/5/peptide
complex (Figure 5). Thus, the overall surface structure is rather
similar to that of the VDR-LBD/1a/peptide complex. On the other
hand, ligand 5 is accommodated into the LBP by an unusual
docking mode in which two side chains of ligand 5, together with
a molecule, increase the volume of the LBP. The pocket is filled
sufficiently with ligand 5 and a water molecule. These observations
well account for the potent activity of compound 5.
The antagonistic activity of compound 4 was also confirmed
by experiments involving RXR recruitment and SRC-1 peptide
recruitment (see Supporting Information Figure S3). So far, the
X-ray crystal structure of the VDR-LBD/antagonist complex,
which clearly explains the structural basis of the antagonism,
Discussion
Compound 5 was proved to have potent agonistic activity by
additional experiments involving RXR recruitment and SRC-1
peptide recruitment using a mammalian two-hybrid assay (see

1444 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Inaba et al.
Figure 5. X-ray crystal structure of rVDR-LBD/5/peptide complex. (a) Overall view of rVDR-LBD/5/peptide complex. Helices, loops, ꢀ-sheets,
and coactivator peptide are shown in green, yellow, violet, and red, respectively. Ligand 5 is shown in gray with the oxygen atoms in red. (b)
Superposition of rVDR-LBD/1a/peptide (green) and rVDR-LBD/5/peptide (pink) in the ligand binding pocket (LBP) region. Connolly channel
surfaces of the LBP of rVDR-LBD/1a/peptide and rVDR-LBD/5/peptide are shown in green and pink, respectively. Hydrogen bond is shown
with dotted orange line. Water molecule is shown in red sphere.
Figure 6. X-ray crystal structure of rVDR-LBD/4/peptide complex. (a) Overall view of rVDR-LBD/4/peptide complex. Helices, loops, ꢀ-sheets,
and coactivator peptide are shown in green, yellow, violet, and red, respectively. The ligand 4 is shown in gray with the oxygen atoms in red. (b)
Superposition of rVDR-LBD/1a/peptide (green) and rVDR-LBD/4/peptide (gray) in the ligand biding pocket (LBP) region. (c) Connolly channel
surface of the LBP of rVDR-LBD/4/peptide. (d) Connolly channel surface of the LBP of rVDR-LBD/1a/peptide.
has not been reported. Quite recently, the crystal structure of
the VDR-LBD complex accommodating an antagonist with a
coactivator peptide was reported, where the ligand was bound
to the active (agonistic) conformation of the VDR.16 This
situation was also the case in the present study. We were able
to obtain crystals of the VDR-LBD/4 complex in the presence
of, but not in the absence of, the coactivator peptide as a ternary
complex (Figure 6). Considering the action of compound 4 as
a VDR antagonist, the active (agonistic) conformation of the
VDR-LBD/4 complex bound tightly to the peptide seems to
be a temporary and less preferred structure but selected in the
course of crystallization. This idea is consistent with the fact

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1445
that the resolution limit of this crystal (3.0 Å) is much lower
than that of the crystal for compound 5 (2.1 Å). Therefore, the
active conformation observed in our crystal structure may not
exactly correspond to the predominant one in vivo. Meanwhile,
it is true that ligand 4 modifies the pocket structure to induce a
new cavity, as seen in the zVDR-LBD/GEMINI complex.11
Unlike GEMINI, however, compound 4 showed antagonistic
activity. This discrimination can be explained mainly by the
difference in hydrophobic interactions between the side chain
terminals of the ligand and the C-terminal of the VDR. This
explanation agrees with the fact that compound 9, which restored
the three truncated carbons on the side chain, restored the
agonistic activity, albeit weakly.
Compound 6, which is 25,26,27-trinor analogue of the
previously reported 22R-butyl-20-epi-1,25-(OH)2D3 2, having
potent agonistic activity,22 showed weak VDR affinity and little
agonistic or antagonistic activity. ASMA of compound 6 yielded
results similar to those for 22R-butyl-20-epi-1,25-(OH)2D3 2,
indicating that 6 is accommodated into the VDR-LBD by the
similar docking mode to that of the parent compound 2.
However, interaction of a hydrogen bond with His397 is not
important for 6 because H397A did not decrease the transcrip-
tional activity in comparison with the wild type. In addition,
compound 6 lacks the intimate hydrophobic interactions with
the C-terminal residues of the VDR because of truncation of
the three carbons in the side chain terminal. Therefore, the weak
activity of ligand 6 is attributable to poor interaction with the
C-terminal regions (helix 11-helix 12) of the VDR.
Docking analysis of ligand 8 into the crystal structure of the
VDR-LBD/4/peptide complex demonstrated that the two side
chains of ligand 8 occupy opposite cavities, respectively, (data not
shown) in comparison with the VDR-LBD/4 complex. This mode
is consistent with the results of ASMA. The increased activity of
H397A and H305A compared with the wild type (Supporting
Information Figure S2h) suggests that His397 does not form an
important hydrogen bond with ligand 8 and His305 exhibits crucial
steric repulsion from the ligand.
Docking analysis of ligand 10 demonstrated that its mode of
docking into the LBP is similar to that of compound 4. When
taking together with the results of ASMA, it appears that steric
repulsion between the i-propyl group of the side chain terminal
of 10 and the side chain of His305 explains the antagonistic
activity.
antagonists. On the basis of X-ray crystallographic analysis, we
identified a new class of ligands that modulate the pocket
structure of the VDR. This is the first report of a VDR antagonist
that generates a new cavity to alter the pocket structure of the
VDR, opening an interesting new perspective of ligand design
based on the alternative structural basis of VDR agonism and
antagonism.
Experimental Section
1H NMR and 13C NMR spectra were recorded in CDCl3 at 400
MHz, and chemical shifts are reported as δ units relative to
tetramethylsilane as an internal standard. High and low resolution
mass spectra were obtained at 70 eV. Relative intensities are given
in parentheses in low mass. IR spectra were recorded on a Shimazu
FTIR spectrometer. UV spectra were recorded on a BECKMAN
DU7500 spectrophotometer. All air and moisture sensitive reactions
were carried out under argon atmosphere.
22R-Butyl-(1r,3ꢀ)-chola-5,7-diene-1,3,24-triol (13). To a solu-
tion of 11 (50 mg, 0.087 mmol) in THF (1 mL) was added
diisobutyl-alminum hydride (DIBAL, 1.0 M solution in toluene,
1000 µL, 1 mmol) at 0 °C and the mixture was stirred for 2.5 h.
Then the mixture was allowed to come to room temperature and
was stirred for 10 h. The reaction mixture was poured into
ice-water, extracted with ethyl acetate, washed with water, dried
over Na2SO4, and evaporated. The residue was chromatographed
on silica gel (C-200, 4 g) with 3% EtOH-CH2Cl2 to give 13 (24
1
mg, 64%). H NMR δ 0.64 (3 H, s, H-18), 0.81 (3 H, d, J ) 6.9
Hz, H-21), 0.90 (3 H, t, J ) 6.4 Hz, CH3 of n-Bu), 0.95 (3 H, s,
H-19), 3.58 and 3.71 (each 1 H, m, H-24), 3.77 (1 H, m, H-1),
4.08 (1 H, m, H-3), 5.38 and 5.74 (each 1 H, m, H-6 and H-7). MS
m/z 430 (M+, 10), 412 (6), 394 (11), 371 (13), 55 (100). UV (EtOH)
λmax 272, 282, 294 nm.
22S-Butyl-(1r,3ꢀ)-chola-5,7-diene-1,3,24-triol (14). The com-
pound 14 was obtained from 12 (0.35 mmol) by the same procedure
1
as described for 13 (yield 75%). H NMR δ 0.63 (3 H, s, H-18),
0.83 (3 H, d, J ) 6.1 Hz, H-21), 0.90 (3 H, t, J ) 6.7 Hz, CH3 of
Bu), 0.95 (3 H, s, H-19), 3.62 and 3.68 (each 1 H, m, H-24), 3.77
(1 H, m, H-1), 4.07 (1 H, m, H-3), 5.38 and 5.74 (each 1 H, m,
H-6 and H-7). MS m/z 430 (M+, 14), 412 (8), 394 (15), 371 (20),
55 (100). UV (EtOH) λmax 272, 282, 294 nm.
22R-Butyl-1r,24-dihydroxy-24,25,26-trinorvitamin D3 (3). A
solution of provitamin D 13 (22 mg, 0.051 mmol) in benzene/EtOH
(130:50, 180 mL) was bubbled with argon at 0 °C for 15 min and
then irradiated with a 100 W high pressure mercury lamp through
a Vycor filter until most of the starting material 13 was consumed.
After removal of the solvent, the residue was chromatographed on
Sephadex LH-20 (15 g) with CHCl3/hexane/MeOH (70:30:1.5) to
give previtamin D. The previtamin D in 95% EtOH (5 mL) was
stored in the dark at room temperature. After 12 days, the solution
was evaporated and the residue was chromatographed on Sephadex
LH-20 (15 g) with CHCl3/hexane/MeOH (70:30:2) to give desired
vitamin D 3 (5.0 mg, 23%). The purity was proved to be more
than 99% by two different HPLC systems: (1) YMC Pack ODS-
AM 4.6 mm × 150 mm, H2O/MeOH (15:85), 1.0 mL/min; (2)
PEGASIL Silica SP100, 4.6 mm × 150 mm, hexane/CHCl3/MeOH
GEMINI (1b), which has two identical side chains branching
at C(20), induces structural rearrangement of the LBP.11 We
identified potent agonist 5 and potent antagonist 4, both of which
have a branched 22S-butyl group and are isomers at C(20). We
found that both ligands rearrange the pocket structure of the VDR
through an unusual docking mode that has not yet been reported.
Other ligands (3, 6-10) showed intermediate activities (weakly
agonistic and weakly antagonistic) and appeared to modify the LBP
structure moderately. It is interesting that although GEMINI (1b)
and our compound 4 are accommodated into the VDR-LBD
through a similar docking mode, GEMINI (1b) works as a VDR
agonist whereas compound 4 works as a VDR antagonist.
1
(100:25:10), 1.0 mL/min. H NMR δ 0.56 (3 H, s, H-18), 0.79 (3
H, d, J ) 6.7 Hz, H-21), 0.89 (3 H, t, J ) 6.7 Hz, CH3 of n-Bu),
3.58 and 3.72 (each 1 H, m, H-24), 4.23 (1 H, m, H-3), 4.43 (1 H,
m, H-1), 5.00 and 5.33 (each 1 H, s, H-19), 6.02 and 6.38 (each 1
H, d, J ) 11.0 Hz, H-7 and H-6, respectively). MS m/z 430 (M+,
3), 412 (7), 394 (5), 371 (2),134 (87), 55 (100). UV (EtOH) λmax
263 nm, λmin 228 nm. HRMS calcd for C28H46O3 430.3447, found
430.3436.
Conclusions
We synthesized 22-butyl-1,24-(OH)2D3 derivatives 3-10 and
evaluated their biological activities. Four compounds, 4, 5, 8,
and 10, showed significant VDR binding affinity but three of
them, 4, 8, and 10, showed little transactivation, cell differentia-
tion, and CYP24A1 gene expression potency. Instead, these
three compounds concentration-dependently inhibited biological
activities induced by 1,25-(OH)2D3, indicating that they are VDR
22S-Butyl-1r,24-dihydroxy-24,25,26-trinorvitamin D3 (4). The
compound 4 was obtained from 14 (0.022 mmol) by the same
procedure as described for 3 (yield 26%). The purity was proved
to be more than 99% by two different HPLC systems: (1) YMC
Pack ODS-AM 4.6 mm × 150 mm, H2O/MeOH (15:85), 1.0 mL/
min; (2) PEGASIL Silica SP100, 4.6 mm × 150 mm, hexane/
1
CHCl3/MeOH (100:25:10), 1.0 mL/min. H NMR δ 0.55 (3 H, s,

1446 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Inaba et al.
4.92 (1 H, m, H-3), 5.38 and 5.69 (each 1 H, m, H-6 and H-7). 13
H-18), 0.81 (3 H, d, J ) 6.7 Hz, H-21), 0.90 (3 H, t, J ) 7.4 Hz,
CH3 of Bu), 3.62 and 3.66 (each 1 H, m, H-24), 4.23 (1 H, m,
H-3), 4.43 (1 H, m, H-1), 5.00 and 5.33 (each 1 H, m, H-19), 6.02
and 6.38 (each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively).
MS m/z 430 (M+, 4), 412 (7), 394 (5), 371 (2),134 (65), 55 (100).
UV (EtOH) λmax 264 nm, λmin 228 nm. HRMS calcd for C28H46O3
430.3447, found 430.3438.
C
NMR δ 12.1, 13.5, 14.4, 16.3, 20.6, 23.1, 23.4, 28.3, 28.6, 31.0,
32.1, 35.5, 35.8, 36.6, 38.0, 38.2, 39.2, 41.4, 43.0, 53.3, 54.7, 54.9,
55.0, 62.0, 72.4, 78.6, 115.6, 122.4, 133.9, 141.4, 155.2, 155.3.
MS m/z (%): 546 (M+, 1), 470 (10), 394 (100), 251 (18), 224 (25),
209 (44), 197 (35), 167 (14), 157 (29), 149 (90), 141 (40), 129
(30), 115 (11), 105 (12). HRMS calcd for C30H46O4
(M+-CH3O(CO)OH) 470.3396, found 470.3385. IR (neat) 3335,
20S,22R-Butyl-(1r,3ꢀ)-chola-5,7-diene-1,3,24-triol (18). The
compound 18 was obtained from 16 (0.08 mmol) by the same
2955, 2937, 2872, 1747, 1443, 1283, 1271, 1254, 1148 cm-1
.
1
procedure as described for 13 (yield 62%). H NMR δ 0.63 (3 H,
1r,3ꢀ-Bis[(methoxycarbonyl)oxy]-22R-butyl-5,7-choladiene-
24-al (21). To a solution of oxalyl chloride (15.5 µL, 0.18 mmol)
in CH2Cl2 (0.8 mL) was added a solution of DMSO (27.4 µL, 0.39
mmol) in CH2Cl2 (0.2 mL) at -78 °C and the mixture was stirred
at that temperature for 15 min. To this solution was added a solution
of 19 (88 mg, 0.16 mmol) in CH2Cl2 (1.0 mL), and the mixture
was stirred for 15 min. Triethylamine (112 µL, 0.81 mmol) was
added to the reaction mixture at -78 °C, and then the mixture was
allowed to warm to room temperature. The reaction mixture was
diluted with CH2Cl2, washed with water, dried, and evaporated.
The residue was chromatographed on silica gel (8 g) with 2.4%
AcOEt-benzene to yield 21 (63 mg, 72%). 1H NMR δ 0.64 (3 H,
s, H-18), 0.82 (3 H, d, J ) 6.9 Hz, H-21), 0.89 (3 H, t, J ) 6.9 Hz,
CH3 of n-Bu), 1.01 (3 H, s, H-19), 3.78 and 3.79 (each 3 H, s,
CH3OCO-), 4.84 (1 H, m, H-1), 4.92 (1 H, m, H-3), 5.38 and
5.70 (each 1 H, m, H-6 and H-7), 9.76 (1 H, m, H-24).
s, H-18), 0.74 (3 H, d, J ) 6.1 Hz, H-21), 0.90 (3 H, t, J ) 6.7 Hz,
CH3 of Bu), 0.94 (3 H, s, H-19), 3.68 (2 H, m, H-24), 3.77 (1 H,
m, H-1), 4.06 (1 H, m, H-3), 5.38 and 5.73 (each 1 H, m, H-6 and
H-7). MS m/z 430 (M+, 13), 412 (7), 394 (12), 371 (18), 55 (100).
UV (EtOH) λmax 272, 282, 294 nm.
20S,22S-Butyl-(1r,3ꢀ)-chola-5,7-diene-1,3,24-triol (17). The
compound 17 was obtained from 15 (0.07 mmol) by the same
1
procedure as described for 13 (yield 59%). H NMR δ 0.60 (3 H,
s, H-18), 0.74 (3 H, d, J ) 6.7 Hz, H-21), 0.88 (3 H, t, J ) 7.3 Hz,
CH3 of Bu), 0.91 (3 H, s, H-19), 3.56 and 3.72 (each 1 H, m, H-24),
3.77 (1 H, m, H-1), 4.22 (1 H, m, H-3), 5.24 and 5.59 (each 1 H,
m, H-6 and H-7). MS m/z 430 (M+, 13), 412 (7), 394 (13), 371
(20), 55 (100). UV (EtOH) λmax 272, 282, 294 nm.
20S,22R-Butyl-1r,24-dihydroxy-24,25,26-trinorvitamin D3 (6).
The compound 6 was obtained from 18 (0.016 mmol) by the same
procedure as described for 3 (yield 16%). The purity was proved
to be more than 99% by two different HPLC systems: (1) YMC
Pack ODS-AM 4.6 mm × 150 mm, H2O/MeOH (15:85), 1.0 mL/
min; (2) PEGASIL Silica SP100, 4.6 mm × 150 mm, hexane/
1r,3ꢀ-Bis[(methoxycarbonyl)oxy]-22S-butyl-5,7-choladiene-
24-al (22). The compound 22 was obtained from 20 (0.09 mmol)
1
by the same procedure as described for 21 (yield 72%). H NMR
δ 0.59 (3 H, s, H-18), 0.82 (3 H, d, J ) 5.9 Hz, H-21), 0.90 (3 H,
t, J ) 6.9 Hz, CH3 of n-Bu), 1.01 (3 H, s, H-19), 3.78 and 3.79
(each 3 H, s, CH3OCO-), 4.84 (1 H, m, H-1), 4.92 (1 H, m, H-3),
5.38 and 5.69 (each 1 H, m, H-6 and H-7), 9.74 (1 H, dd, J ) 2.5,
1.5 Hz, H-24). 13C NMR δ 12.0, 13.4, 14.3, 16.2, 20.6, 23.1, 23.2,
27.8, 28.0, 30.6, 32.0, 35.1, 35.7, 37.9, 39.2, 39.6, 41.4, 43.0, 47.5,
53.3, 54.7, 54.8, 55.0, 72.4, 78.6, 115.7, 122.3, 134.0, 141.1, 155.17,
155.23, 203.7. MS m/z (%): 544 (M+, 1), 468 (10), 392 (100), 224
(24), 209 (40), 195 (13), 167 (23), 157 (22), 155 (29), 149 (45),
143 (10), 129 (12), 105 (10). HRMS calcd for C30H44O4
(M+-CH3O(CO)OH) 468.3240, found 468.3230. IR (neat) 2955,
1
CHCl3/MeOH (100:25:10), 1.0 mL/min. H NMR δ 0.53 (3 H, s,
H-18), 0.74 (3 H, d, J ) 6.7 Hz, H-21), 0.90 (3 H, t, J ) 6.7 Hz,
CH3 of Bu), 3.62 and 3.67 (each 1 H, m, H-24), 4.23 (1 H, m,
H-3), 4.43 (1 H, m, H-1), 5.00 and 5.33 (each 1 H, m, H-19), 6.02
and 6.38 (each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively).
MS m/z 430 (M+, 3), 412 (5), 394 (4), 134 (58), 55 (100). UV
(EtOH) λmax 264 nm, λmin 229 nm. HRMS calcd for C28H46O3
430.3447, found 430.3473.
20S,22S-Butyl-1r,24-dihydroxy-24,25,26-trinorvitamin D3 (5).
The compound 5 was obtained from 17 (0.013 mmol) by the same
procedure as described for 3 (yield 24%). The purity was proved
to be more than 99% by two different HPLC systems: (1) YMC
Pack ODS-AM 4.6 mm × 150 mm, H2O/MeOH (15:85), 1.0 mL/
min; (2) PEGASIL Silica SP100, 4.6 mm × 150 mm, hexane/
2858, 1744, 1441, 1271, 1252 cm-1
.
(1r,3ꢀ,22R,24R)-Bis[(methoxycarbonyl)oxy]-22-butylcholesta-
5,7-dien-24-ol (23) and (1r,3ꢀ,22R,24S)-Bis[(methoxycarbony-
l)oxy]-22-butylcholesta-5,7-dien-24-ol (24). To a solution of 21
(64 mg, 0.12 mmol) in THF (1 mL) was added isopropyl-
magnesium chloride (2.0 M solution in THF, 118 µL, 0.24 mmol)
at room temperature and the mixture was stirred for 25 min. Then,
the reaction was quenched with water. The mixture was extracted
with ethyl acetate, washed with water, dried over Na2SO4, and
evaporated. The residue was chromatographed on silica gel (C-
200, 6 g) with 2-6% AcOEt-benzene to give 23 (25 mg, 36%)
1
CHCl3/MeOH (100:25:10), 1.0 mL/min. H NMR δ 0.54 (3 H, s,
H-18), 0.72 (3 H, d, J ) 6.7 Hz, H-21), 0.89 (3 H, t, J ) 6.7 Hz,
CH3 of Bu), 3.63 and 3.72 (each 1 H, m, H-24), 4.23 (1 H, m,
H-3), 4.43 (1 H, m, H-1), 5.00 and 5.33 (each 1 H, m, H-19), 6.02
and 6.38 (each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively).
MS m/z 430 (M+, 4), 412 (7), 394 (4), 134 (58), 55 (100). UV
(EtOH) λmax 264 nm, λmin 228 nm. HRMS calcd for C28H46O3
430.3447, found 430.3434.
1
and 24 (29 mg, 42%) in this order. 23: H NMR δ 0.64 (3 H, s,
1r,3ꢀ-Bis[(methoxycarbonyl)oxy]-22R-butyl-5,7-choladiene-
24-ol (19). To a solution of 11 (200 mg, 0.35 mmol) in THF (3
mL) was added diisobutyl-aluminum hydride (DIBAL, 1.0 M
solution in toluene, 700 µL, 0.7 mmol) at -30 °C and the mixture
was stirred for 1 h. Then, the mixture was allowed to 0 °C and
was stirred for 3 h. The reaction mixture was poured into ice-water,
extracted with ethyl acetate, washed with water, dried over Na2SO4,
and evaporated. The residue was chromatographed on silica gel
(C-200, 12 g) with 0.5% EtOH-CH2Cl2 to give 19 (122 mg, 64%).
1H NMR δ 0.63 (3 H, s, H-18), 0.80 (3 H, d, J ) 6.9 Hz, H-21),
0.90 (3 H, t, J ) 6.9 Hz, CH3 of n-Bu), 1.01 (3 H, s, H-19), 3.60
and 3.72 (each 1 H, m, H-24), 3.78 and 3.79 (each 3 H, s,
CH3OCO-), 4.84 (1 H, m, H-1), 4.91 (1 H, m, H-3), 5.38 and
5.69 (each 1 H, m, H-6 and H-7).
H-18), 0.78 (3 H, d, J ) 6.7 Hz, H-21), 0.90 (3 H, t, J ) 6.8 Hz,
CH3 of n-Bu), 0.91 and 0.92 (each 3 H, d, J ) 6.7 Hz, H-26 and
-27), 1.02 (3 H, s, H-19), 3.35 (1 H, m, H-24), 3.77 and 3.79 (each
3 H, s, CH3OCO-), 4.84 (1 H, m, H-1), 4.92 (1 H, m, H-3), 5.38
1
and 5.69 (each 1 H, m, H-6 and H-7). 24: H NMR δ 0.63 (3 H,
s, H-18), 0.81 (3 H, d, J ) 6.7 Hz, H-21), 0.90 (3 H, t, J ) 6.7 Hz,
CH3 of n-Bu), 0.88 and 0.96 (each 3 H, d, J ) 6.7 Hz, H-26 and
-27), 1.02 (3 H, s, H-19), 3.48 (1 H, m, H-24), 3.77 and 3.79 (each
3 H, s, CH3OCO-), 4.84 (1 H, m, H-1), 4.90 (1 H, m, H-3), 5.38
and 5.68 (each 1 H, m, H-6 and H-7).
(1r,3ꢀ,22S,24R)-Bis[(methoxycarbonyl)oxy]-22-butylcholesta-
5,7-dien-24-ol (25) and (1r,3ꢀ,22S,24S)-Bis[(methoxycarbony-
l)oxy]-22-butylcholesta-5,7-dien-24-ol (26). The compounds 25
(yield 34%) and 26 (yield 51%) were obtained from 22 (0.061
1
1r,3ꢀ-Bis[(methoxycarbonyl)oxy]-22S-butyl-5,7-choladiene-
mmol) by the same procedure as described for 23 and 24. 25: H
24-ol (20). The compound 20 was obtained from 12 (0.26 mmol)
NMR δ 0.63 (3 H, s, H-18), 0.78 (3 H, d, J ) 5.9 Hz, H-21), 0.89
(3 H, t, J ) 6.9 Hz, CH3 of n-Bu), 0.93 (6H, d, J ) 6.4 Hz, H-26
and -27), 1.02 (3 H, s, H-19), 3.39 (1 H, m, H-24), 3.78 and 3.79
(each 3 H, s, CH3OCO-), 4.84 (1 H, m, H-1), 4.92 (1 H, m, H-3),
5.38 and 5.69 (each 1 H, m, H-6 and H-7). 13C NMR δ 12.1, 13.2,
1
by the same procedure as described for 19 (yield 62%). H NMR
δ 0.61 (3 H, s, H-18), 0.82 (3 H, d, J ) 5.4 Hz, H-21), 0.89 (3 H,
t, J ) 6.9 Hz, CH3 of n-Bu), 1.01 (3 H, s, H-19), 3.66 (2 H, m,
H-24), 3.78 and 3.79 (each 3 H, s, CH3OCO-), 4.84 (1 H, m, H-1),

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1447
14.4, 16.3, 17.7, 19.0, 20.6, 23.2, 23.5, 28.4, 28.7, 30.8, 32.1, 34.6,
35.8, 36.00, 36.02, 36.9, 38.0, 39.2, 41.4, 43.1, 53.3, 54.8, 54.9,
55.1, 72.4, 74.1, 78.7, 115.7, 122.4, 133.9, 141.4, 155.2, 155.3.
MS m/z (%): 588 (M+, 1), 512 (5), 436 (85), 279 (15), 251 (12),
224 (23), 209 (37), 197 (27), 157 (19), 155 (28), 149 (100), 141
(26). HRMS calcd for C33H52O4 (M+-CH3O(CO)OH) 512.3866,
found 512.3861. IR (neat) 3572, 2955, 2872, 1745, 1443, 1344,
Hz, H-26 and -27), 3.35 (1 H, m, H-24), 4.23 (1 H, m, H-3), 4.43
(1 H, m, H-1), 5.01 and 5.33 (each 1 H, m, H-19), 6.02 and 6.38
(each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively). MS m/z
472 (M+, 3), 454 (9), 436 (6), 152 (22),134 (72), 55 (100). UV
(EtOH) λmax 264 nm, λmin 229 nm. HRMS calcd for C31H52O3
472.3916, found 472.3904.
(1r,22R,24S)-22-Butyl-1,24-dihydroxyvitamin D3 (8). 1H NMR
δ 0.57 (3 H, s, H-18), 0.80 (3 H, d, J ) 6.8 Hz, H-21), 0.89 (3 H,
t, J ) 6.7 Hz, CH3 of n-Bu), 0.89 and 0.97 (each 3 H, d, J ) 6.7
Hz, H-26 and -27), 3.49 (1 H, m, H-24), 4.23 (1 H, m, H-3), 4.43
(1 H, m, H-1), 5.00 and 5.33 (each 1 H, m, H-19), 6.02 and 6.38
(each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively). MS m/z
472 (M+, 3), 454 (11), 436 (11), 152 (21),134 (77), 55 (100). UV
(EtOH) λmax 264 nm, λmin 229 nm. HRMS calcd for C31H52O3
472.3916, found 472.3942.
1
1283, 1254, 1148 cm-1. 26: H NMR δ 0.61 (3 H, s, H-18), 0.83
(3 H, d, J ) 6.4 Hz, H-21), 0.90 (3 H, t, J ) 6.9 Hz, CH3 of
n-Bu), 0.89 and 0.95 (each 3 H, d, J ) 6.9 Hz, H-26 and -27),
1.01 (3 H, s, H-19), 3.45 (1 H, m, H-24), 3.78 and 3.79 (each 3 H,
s, CH3OCO-), 4.84 (1 H, m, H-1), 4.92 (1 H, m, H-3), 5.38 and
5.69 (each 1 H, m, H-6 and H-7). 13C NMR δ 12.1, 13.7, 14.4,
16.2, 16.3, 19.5, 20.6, 23.2, 23.5, 28.2, 29.1, 31.3, 32.1, 33.2, 35.8,
37.5, 38.0, 38.3, 39.2, 39.7, 41.4, 43.0, 53.6, 54.7, 54.8, 55.0, 72.4,
76.1, 78.7, 115.6, 122.4, 133.9, 141.4, 155.2, 155.3. MS m/z (%):
588 (M+, 1), 512 (10), 436 (100), 251 (14), 249 (12), 223 (13),
210 (16), 197 (34), 183 (13), 157 (24), 155 (35), 143 (11), 141
(32), 129 (14). HRMS calcd for C33H52O4 (M+-CH3O(CO)OH)
512.3866, found 512.3862. IR (neat) 3695, 2957, 2872, 1745, 1443,
(1r,22S,24R)-22-Butyl-1,24-dihydroxyvitamin D3 (9). 1H NMR
δ 0.57 (3 H, s, H-18), 0.78 (3 H, d, J ) 5.5 Hz, H-21), 0.90 (3 H,
t, J ) 6.7 Hz, CH3 of n-Bu), 0.92 (6 H, d, J ) 6.7 Hz, H-26 and
-27), 3.38 (1 H, m, H-24), 4.23 (1 H, m, H-3), 4.43 (1 H, m,
H-1), 5.00 and 5.33 (each 1 H, s, m, H-19), 6.02 and 6.38 (each 1
H, d, J ) 11.0 Hz, H-7, H-6, respectively. MS m/z 472 (M+, 4),
454 (9), 436 (6), 152 (23),134 (76), 55 (100). UV (EtOH) λmax 265
nm, λmin 228 nm. HRMS calcd for C31H52O3 472.3916, found
472.3893.
1281, 1271, 1252 cm-1
.
(1r,3ꢀ,22R,24R)-22-butylcholesta-5,7-diene-1,3,24-triol (27).
To a solution of 23 (28 mg, 0.048 mmol) in CH2Cl2 (0.6 mL) was
added 5% KOH/MeOH (3.4 mL) and the mixture was stirred at 40
°C for 1.5 h. The reaction mixture was diluted with CHCl3, washed
with water, dried over Na2SO4, and evaporated. The residue was
chromatographed on silica gel with 65% AcOEt-CH2Cl2 to give
(1r,22S,24S)-22-Butyl-1,24-dihydroxyvitamin D3 (10). 1H
NMR δ 0.55 (3 H, s, H-18), 0.83 (3 H, d, J ) 5.4 Hz, H-21), 0.90
(3 H, t, J ) 7.4 Hz, CH3 of n-Bu), 0.89 and 0.95 (each 3 H, d, J
) 6.7 Hz, H-26 and -27), 3.45 (1 H, m, H-24), 4.23 (1 H, m, H-3),
4.43 (1 H, m, H-1), 5.01 and 5.33 (each 1 H, m, H-19), 6.02 and
6.38 (each 1 H, d, J ) 11.0 Hz, H-7 and H-6, respectively). MS
m/z 472 (M+, 3), 454 (9), 436 (5), 152 (22),134 (76), 55 (100).
UV (EtOH) λmax 265 nm, λmin 229 nm. HRMS calcd for C31H52O3
472.3916, found 472.3893.
1
27 (18 mg, 80%). H NMR δ 0.65 (3 H, s, H-18), 0.80 (3 H, d, J
) 6.7 Hz, H-21), 0.91 (3 H, t, J ) 6.7 Hz, CH3 of n-Bu), 0.91 and
0.93 (each 3 H, d, J ) 6.7 Hz, H-26 and -27), 0.95 (3 H, s, H-19),
3.34 (1 H, m, H-24), 3.77 (1 H, m, H-1), 4.10 (1 H, m, H-3), 5.38
and 5.73 (each 1 H, m, H-6 and H-7). MS m/z 472 (M+, 13), 454
(9), 436 (13), 413 (14), 55 (100). UV (EtOH) λmax 272, 282, 294
nm.
(3R,5R)-5-(1-{(1S,3R,10R,13R,17R)-1,3-bis[(methoxycarbony-
l)oxy]-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahy-
dro-1H-cyclopenta[a]phenanthren-17-yl}ethyl)-2-methylnonan-
3-yl (2S)-3,3,3-Trifluoro-2-methoxy-2-phenylpropanoate (31). To
a solution of compound 23 (6.1 mg, 0.010 mmol) in dry CH2Cl2
(100 µL) were added Et3N (30 µL, 0.21 mmol), a solution of
R-MTPACl (20 µL, 0.10 mmol) in dry CH2Cl2 (60 µL), and DMAP
(15.5 mg, 0.13 mmol), and the reaction mixture was stirred for 30
min at room temperature. The reaction was quenched with H2O at
0 °C and was extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (5 g, hexane/AcOEt ) 4/1) to give
Compounds 28-30. By the same procedure as described for
27, each triol 28 (yield 57%), 29 (yield 60%), and 30 (yield 57%)
was obtained from the corresponding precursors, 24 (0.043 mmol),
25 (0.026 mmol), and 26 (0.036 mmol), respectively.
(1r,3ꢀ,22R,24S)-22-butylcholesta-5,7-diene-1,3,24-triol (28).
1H NMR δ 0.65 (3 H, s, H-18), 0.82 (3 H, d, J ) 6.7 Hz, H-21),
0.90 (3 H, t, J ) 7.3 Hz, CH3 of n-Bu), 0.88 and 0.96 (each 3 H,
d, J ) 6.7 Hz, H-26 and -27), 0.95 (3 H, s, H-19), 3.49 (1 H, m,
H-24), 3.77 (1 H, m, H-1), 4.09 (1 H, m, H-3), 5.38 and 5.73 (each
1 H, m, H-6 and H-7). MS m/z 472 (M+, 14), 454 (10), 436 (15),
413 (19), 55 (100). UV (EtOH) λmax 272, 282, 294 nm.
(1r,3ꢀ,22S,24R)-22-butylcholesta-5,7-diene-1,3,24-triol (29).
1H NMR δ 0.64 (3 H, s, H-18), 0.80 (3 H, d, J ) 5.9 Hz, H-21),
0.90 (3 H, t, J ) 6.7 Hz, CH3 of n-Bu), 0.93 (6 H, d, J ) 6.7 Hz,
H-26 and -27), 0.96 (3 H, s, H-19), 3.40 (1 H, m, H-24), 3.78 (1
H, m, H-1), 4.08 (1 H, m, H-3), 5.39 and 5.74 (each 1 H, m, H-6
and H-7). MS m/z 472 (M+, 27), 454 (16), 436 (21), 413 (21), 55
(100). UV (EtOH) λmax 272, 282, 294 nm.
(1r,3ꢀ,22S,24S)-22-butylcholesta-5,7-diene-1,3,24-triol (30).
1H NMR δ 0.63 (3 H, s, H-18), 0.85 (3 H, d, J ) 6.7 Hz, H-21),
0.90 (3 H, t, J ) 6.7 Hz, CH3 of n-Bu), 0.89 and 0.95 (each 3 H,
d, J ) 6.7 Hz, H-26 and -27), 0.95 (3 H, s, H-19), 3.46 (1 H, m,
H-24), 3.77 (1 H, m, H-1), 4.07 (1 H, m, H-3), 5.39 and 5.73 (each
1 H, m, H-6 and H-7). MS m/z 472 (M+, 15), 454 (11), 436 (17),
413 (19), 55 (100). UV (EtOH) λmax 272, 282, 294 nm.
1
S-MTPA ester 31 (5.8 mg, 69.6%). 31: H NMR δ 0.57 (3 H, s,
H-18), 0.76 (3 H, d, J ) 6.7 Hz, H-21), 0.84 and 0.89 (each 3 H,
d, J ) 6.9 Hz, H-26, 27), 0.90 (3 H, t, J ) 7.2 Hz, CH3 of Bu),
1.01 (3 H, s, H-19), 3.49 (3 H, s, OCH3), 3.776 and 3.784 (each 3
H, s, O(CO)OCH3), 4.84 (1 H, m, H-1), 4.90 (1 H, m, H-3), 5.08
(1 H, dd, J ) 10.3, 1.7 Hz, H-24), 5.36 (1 H, dd, J ) 5.6, 2.9 Hz,
H-7), 5.69 (1 H, dd, J ) 5.6, 1.9 Hz, H-6), 7.40 (3 H, m, Ph-3, 4,
5), 7.53 (2 H, m, Ph-2, 6).
(3R,5R)-5-(1-{(1S,3R,10R,13R,17R)-1,3-bis[(methoxycarbonyl)-
oxy]-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahydro-
1H-cyclopenta[a]phenanthren-17-yl}ethyl)-2-methylnonan-3-yl (2R)-
3,3,3-Trifluoro-2-methoxy-2-phenylpropanoate (32). In a similar
manner to that for the synthesis of 31 from 23, a crude product
was obtained from 23 (14.4 mg, 0.024 mmol), Et3N (36 µL, 0.24
mmol), S-MTPACl (23 µL, 0.24 mmol), and DMAP (18.7 mg, 0.15
mmol) in dry CH2Cl2 at room temperature for 30 min and was
purified by chromatographed on silica gel (5 g, hexane/AcOEt )
Compounds 7-10. By the same procedure as described for 3,
compounds 7 (yield 21%), 8 (yield 26%), 9 (yield 28%), and 10
(yield 27%) were obtained from the corresponding precursors, 27
(0.01 mmol), 28 (0.016 mmol), 29 (0.017 mmol), and 30 (0.011
mmol), respectively. The purity of compounds 7-10 was proved
to be more than 99% by two different HPLC systems: (1) YMC
Pack ODS-AM 4.6 mm × 150 mm, H2O/MeOH (10:90), 1.0 mL/
min; (2) PEGASIL Silica SP100, 4.6 mm × 150 mm, hexane/
CHCl3/MeOH (100:25:8), 1.0 mL/min.
1
4/1) to give R-MTPA ester 32 (5.8 mg, 29.5%). 32: H NMR δ
0.53 (3 H, s, H-18), 0.72 (3 H, d, J ) 6.7 Hz, H-21), 0.92 (3 H, t,
J ) 7.1 Hz, CH3 of Bu), 0.93 (6 H, d, J ) 6.9 Hz, H-26, 27), 1.00
(3 H, s, H-19), 3.58 (3 H, s, OCH3), 3.776 and 3.779 (each 3 H, s,
O(CO)OCH3), 4.83 (1 H, m, H-1), 4.89 (1 H, m, H-3), 5.09 (1 H,
dd, J ) 9.6, 3.7 Hz, H-24), 5.35 (1 H, dd, J ) 5.6, 2.9 Hz, H-7),
5.69 (1 H, dd, J ) 5.6, 1.8 Hz, H-6), 7.39 (3 H, m, Ph-3, 4, 5),
7.55 (2 H, m, Ph-2, 6).
(1r,22R,24R)-22-Butyl-1,24-dihydroxyvitamin D3 (7). 1H NMR
δ 0.58 (3 H, s, H-18), 0.77 (3 H, d, J ) 6.7 Hz, H-21), 0.90 (3 H,
t, J ) 6.7 Hz, CH3 of n-Bu), 0.91 and 0.93 (each 3 H, d, J ) 6.7

1448 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5
Inaba et al.
(3S,5S)-5-(1-{(1S,3R,10R,13R,17R)-1,3-bis[(methoxycarbony-
l)oxy]-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahy-
dro-1H-cyclopenta[a]phenanthren-17-yl}ethyl)-2-methylnonan-
3-yl (2S)-3,3,3-Trifluoro-2-methoxy-2-phenylpropanoate (33).
Compound 33 was obtained from 26 (0.01 mmol) by the same
an NBT reduction assay method as previously reported.33,34 The
experiments were done in duplicate.
X-ray Crystallographic Analysis of the Complex Structure.
In this study, the method reported by Vanhooke et al.6 was used
with some modifications to prepare the crystals of VDR complexes.
The rat VDR-LBD (residues 116-423, ∆165-211) was cloned
as an N-terminal His6-tagged fusion protein into the pET14b
expression vector and overproduced in Escherichia coli C41. The
cells were grown at 37 °C in LB medium (including ampicilin 100
mg/L) and subsequently induced for 6 h with 15 µM isopropyl-ꢀ-
D-thiogalactopyranoside (IPTG) at 23 °C. The purification procedure
included affinity chromatography on a Ni-NTA column, followed
by dialysis and cation-exchange chromatography (SP-Sepharose).
After tag removal by thrombin digestion, protease was removed
by filtration through a HiTrap benzamidine column and the protein
was further purified by gel filtration on a Superdex200 column.
The purity and homogeneity of the rVDR-LBD were assessed by
SDS-PAGE.
1
procedure as described for 31 (yield 97.1%). 33: H NMR δ 0.59
(3 H, s, H-18), 0.83 (3 H, d, J ) 6.1 Hz, H-21), 0.90 (3 H, t, J )
6.8 Hz, CH3 of Bu), 0.91 and 0.93 (each 3 H, d, J ) 6.9 Hz, H-26,
27), 1.01 (3 H, s, H-19), 3.53 (3 H, s, OCH3), 3.78 and 3.79 (each
3 H, s, O(CO)OCH3), 4.85 (1 H, m, H-1), 4.90 (1 H, m, H-3), 5.04
(1 H, m, H-24), 5.37 (1 H, m, H-7), 5.69 (1 H, dd, J ) 5.6, 2.0 Hz,
H-6), 7.39 (3 H, m, Ph-3, 4, 5), 7.54 (2 H, m, Ph-2, 6).
(3S,5S)-5-(1-{(1S,3R,10R,13R,17R)-1,3-bis[(methoxycarbony-
l)oxy]-10,13-dimethyl-2,3,4,9,10,11,12,13,14,15,16,17-dodecahy-
dro-1H-cyclopenta[a]phenanthren-17-yl}ethyl)-2-methylnonan-
3-yl (2R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (34).
Compound 34 was obtained from 26 (0.01 mmol) by the same
1
procedure as described for 32 (yield 93.1%). 34: H NMR δ 0.61
(3 H, s, H-18), 0.83 (3 H, d, J ) 6.8 Hz, H-21), 0.85 and 0.88
(each 3 H, d, J ) 6.7 Hz, H-26, 27), 0.90 (3 H, t, J ) 6.8 Hz, CH3
of Bu), 1.01 (3 H, s, H-19), 3.56 (3 H, s, OCH3), 3.78 and 3.79
(each 3 H, s, O(CO)OCH3), 4.84 (1 H, m, H-1), 4.91 (1 H, m,
H-3), 5.05 (1 H, m, H-24), 5.37 (1 H, m, H-7), 5.69 (1 H, dd, J )
5.6, 1.9 Hz, H-6), 7.40 (3 H, m, Ph-3, 4, 5), 7.56 (2 H, m, Ph-2, 6).
Competitive Binding Assay, Bovine Thymus VDR. Binding
to bovine thymus VDR was evaluated according to the procedure
reported.20 Bovine thymus VDR was purchased from Yamasa
Biochemical (Choshi, Chiba, Japan) and dissolved in 0.05 M
phosphate buffer (pH 7.4) containing 0.3 M KCl and 5 mM
dithiothreitol just before use. The receptor solution (500 µL) in an
assay tube was incubated with 0.072 nM [3H]-1,25-(OH)2D3
together with graded amounts of each vitamin D analogue
(0.001-20 nM) or vehicle for 19 h at 4 °C. The bound and free
[3H]-1,25-(OH)2D3 were separated by treating with dextran-coated
charcoal for 20 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) supple-
mented 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 lipofec-
tion method as described previously.32 After 8 h of 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, Wisconsin). Transactivation measured
by the luciferase activity was normalized with the internal control.
All experiments were done in triplicate.
Quantitative Real-Time RT-PCR Analysis. Total RNAs from
samples were prepared with RNAgents Total RNA Isolation system
(Promega), and cDNAs were synthesized using the ImProm-II
reverse transcription system (Promega). Real-time PCR was
performed on the ABI PRISM 7300 sequence detection system
(Applied Biosystems, Foster City, CA) using SYBR Green Realtime
PCR Master Mix -Plus- (Toyobo, Osaka, Japan). Primers were as
follows: CYP24A1, 5′-TGA ACG TTG GCT TCA GGA GAA-3′
and 5′-AGG GTG CCT GAG TGT AGC ATC T-3′; actin, 5′-GAC
AGG ATG CAG AAG GAG AT-3′ and 5′-GAA GCA TTT GCG
GTG GAC GAT-3′. The RNA values were normalized to the level
of Actin mRNA and represent the mean ( SD of triplicate assays.
Differentiation of HL-60 Cells. Human promyelocytic leukemia
cells (HL-60) were plated at 2 × 105 cells/plate and were cultured
in Eagle’s modified medium as described previously.33 The cells
were incubated with each vitamin D compound (10-6, 10-7 and
10-8 M) for 4 days. The differentiation activity was determined by
Purified rVDR-LBD solution was concentrated to about 0.75
mg/mL by ultrafiltration. To an aliquot (800 µL) of the protein
solution was added a ligand (ca. 10 equiv), the solution was further
concentrated to about 1/8, and then a solution (25 mM Tris-HCl,
pH 8.0; 50 mM NaCl; 10 mM DTT; 0.02% NaN3) of coactivator
peptide (H2N-KNHPMLMNLLKDN-CONH2) derived from DRIP205
was added. This solution of VDR/ligand/peptide was allowed to
crystallize by the vapor diffusion method using a series of
precipitant solutions containing 0.1 M MOPS-NaOH (pH7.0),
0.1-0.4 M sodium formate, 12-22% (w/v) PEG4000, and 5% (v/
v) ethylene glycol. Droplets for crystallization were prepared by
mixing 2 µL of complex solution and 1 µL precipitant solution,
and droplets were equilibrated against 500 µL precipitant solution
at 20 °C. It took 1-2 days to obtain reproducible crystals with
X-ray diffraction quality for VDR complex with the ligand 5. On
the other hand, the VDR complex with the ligand 4 gave only
crystals of poor quality except the one used for the structure
determination, which was barely suitable for X-ray diffraction
experiment.
Prior to diffraction data collection, crystals were soaked in a
cryoprotectant solution containing 0.1 M MOPS-NaOH (pH 7.0),
0.1-0.4 M sodium formate, 15-20% PEG4000, and 17-20%
ethylene glycol. Diffraction data sets were collected at 100 K in a
stream of nitrogen gas at beamline BL-6A of KEK-PF (Tsukuba,
Japan). Reflections were recorded with an oscillation range per
image of 1.0°. Diffraction data were indexed, integrated, and scaled
using the program HKL2000.35 The structures of complex were
solved by molecular replacement with the program CNS36 using a
rat VDR-LBD coordinates (PDB code: 1RK3), and finalized sets
of atomic coordinates were obtained after iterative rounds of model
modification with the program XtalView37 and refinement with CNS
by rigid body refinement, simulated annealing, positional minimiza-
tion, water molecule identification, and individual isotropic B-value
refinement. The coordinates of the two complexes were deposited
in the Protein Data Bank with accession number 2ZXM for ligand
4 and 2ZXN for ligand 5.
Graphical Manipulations and Ligand Docking. Graphical
manipulations were performed using SYBYL 8.0 (Tripos, St. Louis).
The atomic coordinates of the crystal structure of zVDR-LBD
complexed with GEMINI (1b) were retrieved from Protein Data
Bank (PDB) (entry 2HCD).11 22-Butyl vitamin D analogues (3, 6,
7-10) were docked into the ligand-binding pocket using Surflex
Dock 2.0 (Tripos, St. Louis), and then the docking models were
modified to agree with the biological activities.
Acknowledgment. Part of this work was supported by a
Grant-in-Aid for Scientific Research (no. 20590108) from the
Ministry of Education, Culture, Sports, Science, and Technol-
ogy, Japan.
Supporting Information Available: Transcriptional activities
in HEK293 cells, ASMA, recruitment of RXR and SRC-1, and

New Class of Vitamin D Analogues
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 5 1449
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, 1997; pp30-31.
HPLC charts to prove purity of compounds 3-10. This material is
available free of charge via the Internet at http://pubs.acs.org.
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