Potent antagonist for the vitamin D receptor: Vitamin D analogues with simple side chain structure

Article abstract of DOI:10.1021/jm100649d

We previously reported that 22S-butyl-25,26,27-trinor-1α,24- dihydroxyvitamin D₃ 2 was a potent VDR antagonist. The X-ray crystal structure of the ligand binding domain of VDR complexed with 2 indicated that this ligand induces an extra cavity within the ligand-binding pocket. The structure also showed that the ligand forms only poor hydrophobic interactions with helix 12 of the protein. Here, to study the effects of the induction of the extra cavity and of insufficient interactions with helix 12 on antagonism, we designed and synthesized a series of vitamin D₃ analogues with or without a 22-alkyl substituent and evaluated their biological potency. We found that the 22-butyl analogues 3c and 5c act as full antagonists, the 22-ethyl analogues 3b, 4b, 5b, and 22-butyl analogue 4c act as partial agonists, and the others (3a, 4a, 5a, 6a, 6b, and 6c) act as full agonists for VDR. It is intriguing that 6c is a potent agonist for VDR, whereas its 26,27-dinor analogue 5c is a potent antagonist. Analogue 6c recruited coactivator SRC-1 well, but 5c did not. These results indicate that a combination of induction of the extra cavity and insufficient hydrophobic interactions with helix 12 is important for VDR antagonism in this class of ligands.

Full text of DOI:10.1021/jm100649d

J. Med. Chem. 2010, 53, 5813–5826 5813
DOI: 10.1021/jm100649d
Potent Antagonist for the Vitamin D Receptor: Vitamin D Analogues with Simple Side Chain Structure
Yuta Sakamaki,†,‡ Yuka Inaba,† Nobuko Yoshimoto,† and Keiko Yamamoto*,†
†Laboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida,
Tokyo 194-8543, Japan, and ‡Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
Received May 29, 2010
We previously reported that 22S-butyl-25,26,27-trinor-1R,24-dihydroxyvitamin D3 2 was a potent
VDR antagonist. The X-ray crystal structure of the ligand binding domain of VDR complexed with 2
indicated that this ligand induces an extra cavity within the ligand-binding pocket. The structure also
showed that the ligand forms only poor hydrophobic interactions with helix 12 of the protein. Here, to
study the effects of the induction of the extra cavity and of insufficient interactions with helix 12 on
antagonism, we designed and synthesized a series of vitamin D3 analogues with or without a 22-alkyl
substituent and evaluated their biological potency. We found that the 22-butyl analogues 3c and 5c act
as full antagonists, the 22-ethyl analogues 3b, 4b, 5b, and 22-butyl analogue 4c act as partial agonists,
and the others (3a, 4a, 5a, 6a, 6b, and 6c) act as full agonists for VDR. It is intriguing that 6c is a potent
agonist for VDR, whereas its 26,27-dinor analogue 5c is a potent antagonist. Analogue 6c recruited
coactivator SRC-1 well, but 5c did not. These results indicate that a combination of induction of the
extra cavity and insufficient hydrophobic interactions with helix 12 is important for VDR antagonism in
this class of ligands.
The discovery of vitamin D in the 1920s-1930s provided a
powerful weapon for the treatment of rickets and osteomala-
cia. Thereafter, it was found that vitamin D is hydroxylated at
the 25-position in the liver and then at the 1R-position in the
kidney to produce 1R,25-dihydroxyvitamin D3 [1,25-(OH)2-
D3,a 1], which is the active form of vitamin D3. It is now well-
known that 1,25-(OH)2D3 1 plays an important role in
calcium homeostasis, cell differentiation and proliferation,
and immunomodulation.1 1,25-(OH)2D3 (1) and its active an-
alogues exert these effects by binding to the vitamin D re-
ceptor (VDR), which belongs to the nuclear receptor super-
family.2 VDR is a ligand-dependent transcription factor that
forms a heterodimer with another nuclear receptor, retinoid X
receptor (RXR).3 Upon ligand binding, VDR undergoes a
conformational change that promotes RXR-VDR heter-
odimerization.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
transcription.
VDR antagonists have been developed by several groups,
including ours. ZK compounds4 and adamantane com-
pounds5 are antagonists containing a bulky side chain. In
contrast, (23S)-25-dehydro-1R-hydroxyvitaminD3-26,23-lac-
tone (TEI9647)6 and its derivatives are antagonists lacking a
bulky side chain.6-9 VDR antagonism is thought to be due to
VDR adopting a not-fully active conformation upon ligand
binding. A VDR-ligand complex with a not fully active
conformation would inhibit heterodimerization with RXR
and/or recruitment of coactivators. However, the molecular
basis for VDR antagonism is not clearly understood, in part
because VDR bound to an antagonist is not stable, so there is
no crystal structure to help explain antagonistic activity. Re-
cently, we reported that 22S-butyl-25,26,27-trinor-1R,24-di-
hydroxyvitamin D3 2 is a potent VDR antagonist.10 The
crystal structure of the ligand binding domain (LBD) of
VDR complexed with 2 showed insufficient interactions bet-
ween the ligand and helix 12 of VDR and the formation of an
extra cavity.10 In addition, we found that 22S-butyl-1R,24R-
dihydroxyvitamin D3, which contains the three carbons lack-
ing on the side chain terminal of 2, recovered agonistic
activity, albeit weakly. Furthermore, the crystal structure of
the VDR-LBD/22S-butyl-1R,24R-dihydroxyvitamin D3 com-
plex showed adequate VDR-ligand interactions.11 In the
present study, to investigate the effects of the induction of
the extra cavity and of insufficient interactions with helix 12 on
VDR antagonism, we synthesized a series of vitamin D3
analogues with and without a 22-alkyl substituent and eval-
uated their biological activities.
Design and Synthesis
Design. The crystal structure of the VDR-LBD/2 complex
showed that ligand 2 induces an extra cavity in the ligand-
binding pocket (LBP) of the VDR to accommodate the
branched butyl group. To investigate the effects of the 22-
alkyl substituent, and of the length and hydrophobicity of
the side chain, we designed 12 vitamin D analogues, 3-6
(Chart 1). The 2-methylene-19-nor structure, instead of the
original A-ring structure, was selected because the 19-nor
structure is more stable than the conjugated triene structure
*To whom correspondence should be addressed. Phone: þ81 42 721
1580. Fax: þ81 42 721 1580. E-mail: yamamoto@ac.shoyaku.ac.jp.
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.
r
2010 American Chemical Society
Published on Web 07/07/2010
pubs.acs.org/jmc

5814 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Chart 1. Structures of 1,25-(OH)2D3 (1) and Vitamin D Analogues
Sakamaki et al.
with MeLi to provide 24,24-dimethylated compound 12,
which was then treated with CSA to provide 4a.
Synthesis of 22-Alkyl 1,24-Dihydroxyvitamin D3 Analo-
gues. As shown in Scheme 2, 22-butyl vitamin D derivatives
3c and 4c were also synthesized from 22-aldehyde 8. Com-
pound 8 was treated with ethyl diphenylphosphonoacetate at
-78 °C to afford Z-enoate 13 (70%) together with E-isomer
9 (14%).16 Upon treatment of Z-enoate 13 with n-Bu2CuLi
in the presence of HMPA and TMSCl, a diastereofacial-sele-
ctive conjugate addition of n-Bu2CuLi took place to provide
22S-butyl compound 14 (74%) together with its 22R-isomer
(7%). The configuration at C(22) was determined from the
stereochemistry of the starting enoate and the reaction con-
ditions based on our previous report.13 Compound 14 was
reduced with DIBAL to give 24-alcohol 16, which was then
treated with CSA to provide the desired 22S-butyl analogue
3c. On the other hand, 24,24-dimethylated analogue 4c was
obtained upon treatment of 14 with MeLi followed by CSA.
22-Ethyl analogues 3b and 4b were synthesized from Z-
enoate 13 by procedures analogous to those described above
for the synthesis of 22-butyl analogues 3c and 4c (Scheme 2).
Thus, treatment of 13 with Et2CuLi in the presence of
HMPA and TMSCl afforded diastereoselective conjugate
addition product 15 (55%). Reduction of the side chain ester
followed by deprotection of the A-ring alcohol provided the
desired compound 3b. Treatment of 15 with MeLi provided
24,24-dimethyl compound 19 that was then treated with
CSA to give the target compound 4b.
Synthesis of 1,25-Dihydroxyvitamin D3 Analogues. Synth-
esis of 1,25-dihydroxyvitamin D3 derivatives 5a and 6a was
conducted as shown in Scheme 3. The convenient synthetic
intermediate 75 was treated with NaI to provide iodide 23.
The iodide (23) reacted with sulfone 22, which was derived
from 3-bromo-1-propanol, to give coupling product 24. Sul-
fone 24 was reduced with Na-Hg and then deprotected to
provide target compound 5a. 2-Methylene-19-nor-1,25-di-
hydroxyvitamin D3 6a was obtained from tosylate 7 by a
coupling reaction with bromide 26 followed by deprotection
of the three alcohols.
Synthesis of 22-Alkyl 1,25-Dihydroxyvitamin D3 Analo-
gues. 22-Butyl compounds 5c and 6c were synthesized from
22-butyl alcohol 16 (Scheme 4). Tosylate 28 derived from 16
was treated with KCN to give cyanate 30, which was then
reduced with DIBAL to give aldehyde 32. The aldehyde (32)
was reduced to alcohol 34 and then deprotected to give 5c.
On the other hand, aldehyde 32 was converted to ester 36 in
good yield by alkaline iodine oxidation which was developed
by our group.17 Ester 36 was treated with MeLi to give 38 and
deprotected to provide the target compound 6c. 22-Ethyl
compounds 5b and 6b were synthesized from 22-ethyl alcohol
17 by procedures analogous to those described above for the
synthesis of the 22-butyl compounds 5c and 6c (Scheme 4).
of the hormone and is easily synthesized. In addition, inser-
tion of a methylene group into C(2) was expected to increase
the VDR affinity and the biological activity, as in the case of
the 2MD compound.12 22S-Butyl-2-methylene-19,25,26,27-
tetranor-1R,24-dihydroxyvitamin D3 3c is a 2-methylene-19-
nor type compound corresponding to the parent compound
2. 1,24-Dihydroxyvitamin D3 derivatives 3a-c and 4a-c
were designed to investigate the effects of the 22-alkyl
substituent and of the hydrophobicity of the original vitamin
D side chain. 1,25-Dihydroxyvitamin D3 derivatives 5a-c
and 6a-c were designed to investigate the effects of side
chain length in addition to the 22-alkyl substituent.
Synthesis of the 1,24-Dihydroxyvitamin D3 Analogues. As
shown in Scheme 1, 1,24-dihydroxyvitamin D3 derivatives 3a
and 4a were synthesized from aldehyde 8. The aldehyde (8),
prepared from the corresponding tosylate 7, is a convenient
synthetic intermediate for various vitamin D side-chain
analogues.5 E-Enoate 9 was obtained by Horner-Emmons
reaction using triethyl phosphonoacetate in 79% yield.13
Selective hydrogenation of R,β-unsaturated ester 9 was
achieved by reduction using Mg in MeOH to provide 10 in
excellent yield (81%).14,15 Compound 10 was reduced with
DIBAL to give 24-alcohol 11, which was then treated with
camphor sulfonic acid (CSA) to afford the desired compound
3a. 24,24-Dialkyl analogue 4a was obtained by treating 10
Biological Activities
VDR Binding. Binding affinity for the VDR was evaluated
by a competitive binding assay using recombinant human
VDR-LBD expressed as the C-terminal GST-tagged protein
using pGEX-VDR vector18 in Escherichia coli BL21. There
are only a few reports in which human VDR was used for
competitive binding assays.6,19 To date, chick, bovine, and
rat VDRs have generally been used. Our results are summari-
zed in Table 1. All synthetic compounds showed specific
binding to VDR, indicating they are ligands for VDR.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5815
Scheme 1. Synthesis of Analogues 3a and 4a
Scheme 2. Synthesis of Analogues 3b, 3c, 4b, and 4c
Compounds with the same side chain as that of 1,25-
(OH)2D3 (6a-c) showed particularly strong affinity. All
22-butyl analogues (3c, 4c, 5c, and 6c) also showed signifi-
cant affinity.
Transactivation. The ability of synthetic compounds 3-6
to induce transcription of a vitamin D-responsive gene was
tested using the mouse osteopontin luciferase reporter gene
assay system in Cos7 cells.10 The results are shown in Figure 1.
Compounds with a 1,25-(OH)2D3 side chain, 6a-c, showed
particularly strong activity. In addition, all compounds
lacking a 22-substituent (3a, 4a, 5a, and 6a) showed full
agonistic activity. In contrast, compounds 3c, 4c, and 5c,
bearing a 22S-butyl group, showed little transactivation
potency. Interestingly, 22S-ethyl analogues 3b, 4b, and 5b
showed intermediate activity between the corresponding 22-
hydrogen and 22-butyl analogues.

5816 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Scheme 3. Synthesis of Analogues 5a and 6a
Sakamaki et al.
As shown in Figure 2b, compounds 3c, 4c, and 5c con-
centration dependently inhibited the transactivation induced
by 1,25-(OH)2D3 (1). In particular, 3c and 5c, which have a
primary hydroxyl group on the side chain, completely in-
hibited the transactivation, indicating that these two analo-
gues are full VDR antagonists. 22-Ethyl analogues 3b, 4b,
and 5b inhibited the transactivation induced by 1 to their
transactivation potency (Figure 2a).
Recruitment of RXR and Coactivator Peptide. We evalu-
ated ligand-dependent recruitment of RXR and a coacti-
vator peptide, SRC-1, to VDR using a mammalian two-
hybrid assay similar to that reported previously.18 As shown
in Figure 3, compounds 3a, 4a, 5a, and 6a, as well as 6b and
6c, recruited RXR and SRC-1 concentration dependently.
Three 22-butyl analogues 3c, 4c, and 5c, very weakly re-
cruited RXR and SRC-1. 22-Ethyl analogues 3b, 4b, and 5b
recruited RXR and SRC-1 moderately.
similar tendency was observed for the 24-nor-compounds
4a-c, although their affinity is weaker than that of 6a-c.
For primary alcohols 3a-c and 5a-c, hVDR affinity in-
creased in the order: 22-hydrogen, 22-ethyl, and 22-butyl
substituent. We reported previously that a primary alcohol
side chain has insufficient hydrophobic interactions with helix
12 of VDR and that 22-butyl substituent induces an extra
cavity in the native ligand binding pocket.10 An increase in
binding affinity by a 22-alkyl substituent, observed in primary
alcohols 3a-c and 5a-c, is thought to be caused by newly
generated hydrophobic interactions between the 22-alkyl
group and amino acid residues lining the extra cavity of
VDR. In the case of tertiary alcohols 4a-c and 6a-c, hydro-
phobic interactions with helix 12 appear to be sufficient
regardless of the 22-alkyl substituent.
In primary 24-alcohols 3a-c, transactivation potency is in
inverse proportion to the affinity for VDR (Figure 1). Trans-
activation and inhibition experiments indicated that 3a, 3b,
and 3c is an agonist, a partial agonist, and an antagonist,
respectively, for VDR (Figures 1 and 2). In primary 25-alco-
hols 5a-c, similar results were obtained (Figures 1 and 2).
Tertiary 24-alcohols 4a-c and 25-alcohols 6a-c showed
increased agonistic activity compared to the corresponding
primary alcohol (Figure 1). In particular, 6a-c exhibited
completely restored agonistic activity. The results clearly
demonstrate that hydrophobicity of the side chain terminal
Discussion
All synthetic analogues with a 24- or 25-hydroxyl group in
the side chain showed specific binding for hVDR (Table 1).
Therefore, all compounds 3-6 synthesized here are true
ligands for hVDR. Compounds 6a-c, which have the same
side chain as the hormone (1), showed strong affinity for
hVDR regardless of the length of the 22-alkyl substituent. A

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5817
Scheme 4. Synthesis of Analogues 5b, 5c, 6b, and 6c
Table 1. VDR Binding Affinity of Synthetic Analogues 3-6 IC50 (nM)a
a Competitive binding of 1,25-(OH)2D3 (1) and synthetic compounds (3a-c, 4a-c, 5a-c, and 6a-c) to the human vitamin D receptor. The
experiments were carried out in duplicate. The IC50 values are derived from dose-response curves and represent the compound concentration required
for 50% displacement of radiolabeled 1,25-(OH)2D3 from the receptor protein.
interacting with helix 12 is an important factor for expression
of transcriptional activity. Additionally, effects of the alkyl
substituent at C(22) are obvious. In 3a-c, 4a-c, and 5a-c,
the increased volume of the substituent strengthens inhibitory
activity. However, 6a-c, which have the prototype side chain
of hormone 1, showed strong agonistic activity regardless of
the 22-substituent. 22-Ethyl compounds 3b-5b showed mod-
erate agonistic activity, in between 22-hydrogen compounds
3a-5a and 22-butyl compounds 3c-5c. Among the 22-ethyl
compounds, 3b-5bare partialagonistsand 6bis a full agonist.
It is striking that primary alcohol 5c inactivates VDR stro-
ngly, whereas corresponding tertiary alcohol 6c activates
VDR strongly. The potency of RXR and SRC-1 recruitments
by each ligand correlates well with the transactivation potency
of the corresponding ligand.
The above results, combined with our previous study, allow
us to suggest a molecular basis for VDR agonistic and anta-
gonistic behaviors of a series of our synthetic side chain ana-
logues as shown in Figure 4. Several research groups have
solved crystal structures of VDR-LBD complexed with a vari-
ety of ligands.20-32 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”,23,27
an analogue with two identical side chains,33,34 and the complex
of VDR-LBD with 22-butyl analogue 2.10 The zebrafish VDR-
LBD/GEMINI complex revealed the formation of a new cavity
extending the original LBP in order to accommodate the sec-
ond side chain as well as the VDR-LBD/2 complex. Figure 4a
(conformer A) shows the crystal structure of VDR-LBD/1

5818 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Sakamaki et al.
VDR-LBD. VDR-LBD complexed with a full agonist having
no 22-substituent, such as 3a, 4a, 5a, or 6a, seems to adopt this
conformation. Figure 4b (conformer B) shows a docking
model of the VDR-LBD/6c complex in which the extra cavity
is induced to accommodate the branched side chain, but helix
12still folds backtoformanactiveconformation. Thisfolding
back is apparently due to sufficient hydrophobic interactions
between helix 12 and the side chain terminal (tertiary alcohol)
of the ligand. Thus, it seems that hydrophobic interactions
overcome strain caused by the formation of the extra cavity.
VDR-LBD complexed with a full agonist such as 6b and 6c
seems to adopt this conformation. It should be noted that
GEMINI could also adopt this conformation.
Figure 4c and 4d (conformers C and D) show docking
models in which the extra cavity is induced by the branched
side chain, so that helix 12 cannot adopt an active conforma-
tion. In contrast to full agonists 6b and 6c, full antagonists 2,
3c, and 5c would adopt conformer C and/or D because of
insufficient hydrophobic interactions between the side chain
terminal (primary alcohol) of the ligand and helix 12. Further-
more, compounds 3b, 4b, and 5b moderately induce the extra
cavity and have moderate hydrophobic interactions with helix
12. Therefore, in addition to inactive conformer C and/or D,
some proportion of conformer B would also be present in the
population.
The 12 compounds synthesized here can form hydrogen
bonds between three hydroxyl groups and VDR, as observed
in most of the VDR-LBD/ligand complexes. Therefore, in
addition to inducing an extra cavity, insufficient hydrophobic
interactions would result in inactive VDR.
Figure 1. Transactivation of compounds 3-6 in Cos7 cells. Tran-
scriptional 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 contain-
ing sea pansy luciferase expression constructs (pRL-CMV) in Cos7
Conclusions
cells as described previously.10 Luciferase activity of 10-8
M
1,25-(OH)2D3 (1) was defined as 1. (a) Transcriptional activity of
1, 3a, 4a, 5a, and 6a. (b) Transcriptional activity of 1, 3b, 4b, 5b, and
6b. (c) Transcriptional activity of 1, 3c, 4c, 5c, and 6c.
We performed a systematic study on the synthesis of a series
ofvitamin D3 analogues withor without a 22-alkyl substituent
and evaluated their biological potency. The present study
demonstrated that both the induction of an extra cavity and
insufficient hydrophobic interactions with helix 12 are neces-
sary for VDR antagonism by our new class of ligands. A com-
bination of these two structural factors would cause structural
strain in VDR, resulting in the unfolding or mis-folding of
helix 12. This study showed great promise of ligand design
based on the alternative structural basis of VDR agonism and
antagonism.
Experimental Section
All reagents were purchased from commercial sources. Unless
otherwise stated, NMR spectra were recorded at 300 MHz for 1H
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 a JEOL
JMS D-300 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 or
100:25:10), flow rate 1.0 mL/min] and was >95% for all com-
pounds tested.
Figure 2. Inhibitory effect on the transactivation induced by
1,25-(OH)2D3 (1) was evaluated using the same assay method
described in Figure 1. (a) transcriptional activity of 3b, 4b, and 5b
in the presence of 10-8 M 1,25-(OH)2D3 1. (b) Transcriptional
activity of 3c, 4c, and 5c in the presence of 10-8 M 1,25-(OH)2D3 1.
All experiments were done in triplicate.
Ethyl (2E)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)-
silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]pent-2-
enoate (9). To a suspension of sodium hydride (60% oil suspen-
sion, 23.8 mg, 0.546 mmol) in THF (1 mL) at 0 °C was added
complex31 in which helix 12 adopts an active conformat-
ion. Conformer A is known to be an active conformation of

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5819
Figure 3. RXRR and SRC-1 recruitment to VDR by 1,25-(OH)2D3 (1) and synthetic compounds (3a-c, 4a-c, 5a-c, and 6a-c) in Cos7 cells.
The activities were evaluated by a dual luciferase assay using VP16-VDR expression plasmid (pCMX-VP16-hVDR), RXRR or SRC-1
expression plasmid (GAL4-RXRR or GAL4-SRC-1), a reporter plasmid containing four copies of GAL4 response element (MH100 ꢀ 4-TK-
Luc), and an internal control plasmid containing sea pansy luciferase expression construct (pRL-CMV). All experiments were carried out in
triplicate. Black bars indicate activities of 1, 3a, 4a, 5a, and 6a. White bars indicate activities of 3b, 4b, 5b, and 6b. Gray bars indicate activities of
3c, 4c, 5c, and 6c. (a) RXRR recruitment, (b) SRC-1 recruitment.
triethyl phosphonoacetate (117 μL, 0.585 mmol), and the mix-
ture was stirred for 10 min. To this solution aldehyde 8 (104 mg,
0.182 mmol) in THF (1 mL) was added, and the mixture was
stirred at 0 °C for 1.5 h. The reaction was quenched with water,
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/
99) to afford 9 (92.8 mg, 79%).
room temperature for 20 h. The reaction was quenched with 1N
HCl and extracted with AcOEt. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel (AcOEt/hexane = 1/9) to afford
10 (101.6 mg, 84%).
10. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.86, 0.89 (each 9 H, s, t-Bu), 0.93 (3 H, d, J = 6.2
Hz, H-21), 2.83 (1 H, m, H-9), 3.67 (3H, s, -COOMe), 4.43 (2 H,
m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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 -5.0,
-4.9, -4.8 (2 carbons), 12.0, 18.1, 18.2, 18.4, 22.2, 23.4, 25.7 (3
carbons), 25.8 (3 carbons), 27.5, 28.7, 30.9, 31.0, 35.7, 38.5, 40.5,
45.6, 47.6, 51.5, 56.2 (2 carbons), 71.6, 72.5, 106.2, 116.1, 122.4,
132.7, 140.9, 152.9, 174.7. MS (FAB) m/z (%): 631 (Mþ, 5).
HRMS (FAB) calcd for C37H67O4Si2 (Mþ þ H) 631.4577, found
631.4565.
9. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.58
(3 H, s, H-18), 0.86, 0.90 (each 9 H, s, t-Bu), 1.10 (3 H, d, J =
6.6 Hz, H-21), 1.29 (3H, t, J = 7.1 Hz), 2.83 (1 H, m, H-9), 4.18
(2 H, q, J = 7.1 Hz, -COOCH2-), 4.43 (2 H, m, H-1, 3), 4.93,
4.97 (each 1 H, s, -CdCH2), 5.75 (1 H, d, J = 15.6 Hz, H-23),
5.83 (1 H, d, J = 11.4 Hz, H-7), 6.21 (1 H, d, J = 11.4 Hz, H-6),
6.84 (1 H, dd, J = 15.6, 9.0 Hz, H-22). 13C NMR δ -5.0, -4.9,
-4.8 (2 carbons), 12.3, 14.2, 18.1, 18.2, 19.3, 22.2, 23.3, 25.7
(3 carbons), 25.8 (3 carbons), 27.5, 28.6, 38.6, 40.0, 40.4, 45.8,
47.6, 55.3, 56.0, 60.0, 71.6, 72.5, 106.3, 116.4, 119.1, 122.2, 133.0,
140.5, 152.9, 154.5, 166.5. MS (EI) m/z (%): 642 (Mþ, 5), 585
(10), 510 (100), 453 (15), 366 (23), 147 (16), 73 (81). HRMS (EI)
calcd for C38H66O4Si2 642.4500, found 642.4503. IR (neat)
3375, 2954, 2929, 2887, 2856, 1658, 1620, 1461, 1359, 1255,
1101, 1072, 935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254,
264 nm.
4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-
methylidene-9,10-secoestra-5,7-dien-17-yl]pentan-1-ol (11). Es-
ter 10 (16.2 mg, 0.0257 mmol) in CH2Cl2 (0.5 mL) was treated
with DIBALH (160 μL of 1.0 M toluene solution, 016 mmol) at
0 °C for 3 h. The reaction was quenched with water and
extracted with CH2Cl2. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The residue was chro-
matographed on silica gel (AcOEt/hexane = 1/9) to afford 11
(7.1 mg, 46%).
Methyl 4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]pentanoate (10).
A mixture of ester 9 (122.9 mg, 0.1914 mmol) and magnesium
turnings (93.1 mg, 3.83 mmol) in MeOH (2 mL) was stirred at
11. 1H NMR δ 0.02, 0.05, 0.06, 0.09 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.87, 0.89 (each 9 H, s, t-Bu), 0.94 (3 H, d, J =
6.4 Hz, H-21), 2.82(1 H, m, H-9), 3.62 (2 H, t, J = 6.4 Hz, H-24),

5820 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Sakamaki et al.
(96), 95 (100). HRMS (EI) calcd for C24H38O3 374.2811, found
374.2821. IR (neat) 3298, 2941, 2925, 2871, 1658, 1620, 1442,
1261, 1070, 1049, 865, 673 cm-1.UV(EtOH) λmax 245, 254, 263 nm.
5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-
methylidene-9,10-secoestra-5,7-dien-17-yl]-2-methylhexan-2-ol
(12). To a solution of ester 10 (64.4 mg, 0.102 mmol) in THF
(1 mL) at -78 °C was added MeLi (470 μL of 1.09 M Et2O solu-
tion, 0.512 mmol), and the mixture was stirred for 30 min. The
reaction was quenched with satd NH4Cl, and the mixture was
extracted with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (AcOEt/hexane = 5/95) to afford 12
(51.9 mg, 0.0824 mmol, 81%).
12. 1H NMR δ 0.02, 0.04, 0.07, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.86, 0.89 (each 9 H, s, t-Bu), 0.94 (3 H, d, J =
6.8 Hz, H-21), 1.20 (6 H, s, H-25, 26), 2.80 (1 H, m, H-9), 4.43
(2 H, m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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
-5.0, -4.9, -4.8 (2 carbons), 12.0, 18.1, 18.2, 18.9, 22.2, 23.4,
25.7 (3 carbons), 25.8 (3 carbons), 27.6, 28.7, 29.0, 29.3, 30.1,
36.2, 38.5, 40.0, 40.5, 45.6, 47.6, 56.2, 56.3, 71.1, 71.6, 72.5,
106.2, 116.1, 122.4, 132.7, 141.1, 152.9. MS (EI) m/z (%): 630
(Mþ, 10), 498 (100), 480 (16), 366 (31), 234 (12), 147(10), 73 (33).
HRMS (EI) calcd for C38H70O3Si2 630.4864, found 630.4861.
IR (neat) 3361, 2952, 2856, 1658, 1620, 1461, 1251, 1101, 1072,
835, 775 cm-1. UV (hexane) λmax 246, 254, 264 nm.
2-Methylidene-19,24-dinor-1r,25-dihydroxyvitamin D3 (4a).
In a similar manner to that for the synthesis of 3a from 11,
target compound 4a (28.2 mg, 0.0702 mmol) was obtained from
12 (51.9 mg, 0.0808 mmol) in 87% yield.
4a. 1H NMR δ 0.55 (3 H, s, H-18), 0.92 (3 H, d, J = 6.1 Hz,
H-21), 1.21 (6 H, s, H-25, 26), 4.46 (2 H, m, H-1, 3), 5.08, 5.10
(each 1 H, s, -CdCH2), 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 12.1, 18.3, 22.2, 23.4,
27.5, 28.9, 29.0, 29.3, 30.1, 36.1, 38.1, 39.9, 40.4, 45.7 (2 car-
bons), 56.1, 56.3, 70.6, 71.2, 71.8, 107.7, 115.3, 124.2, 130.5,
143.3, 152.0. MS (EI) m/z (%): 402 (Mþ, 65), 161 (61), 147(70),
135 (93), 81 (100). HRMS (EI) calcd for C26H42O3 402.3134,
found 402.3135. IR (neat) 3355, 2960, 2871, 1650, 1620, 1440,
1377, 1215, 1074, 1045, 908, 756 cm-1. UV (EtOH) λmax 245,
254, 263 nm.
Ethyl (2Z)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)-
silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]pent-2-
enoate (13). To a solution of ethyl diphenylphosphonoacetate
(109 μL, 132.8 mg, 0.415 mmol) in THF (1 mL) was added
sodium hydride (50% oil suspension, 18.1 mg, 0.377 mmol) at
0 °C, and the mixture was stirred for 10 min. The mixture was
cooled to -78 °C, and then aldehyde 8 (107.9 mg, 0.189 mmol) in
THF (1 mL) was added. The mixture was gradually warmed up
to -10 °C from -78 °C for 1.5 h. The reaction was quenched
with aqueous 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 = 1/99) to afford 13 (85.3 mg, 70%)
and E-isomer 9 (17.1 mg, 14%).
Figure 4. Conformation models of VDR-LBD complexed with
agonist and antagonist. (a) Crystal structure of VDR-LBD/1 comp-
lex, (b) docking model of VDR-LBD and 22S-butyl-2-methylidene-
19-nor-1R,25-dihydroxyvitamin D3 (6c), (c) docking model of
VDR-LBD and 22S-butyl-2-methylidene-19,26,27-trinor-1R,25-di-
hydroxyvitamin D3 (5c), (d) docking model of VDR-LBD and 22S-
butyl-2-methylidene-19,26,27-trinor-1R,25-dihydroxyvitamin D3 (5c).
4.43 (2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 5.82
(1 H, d, J = 11.3 Hz, H-7), 6.21 (1 H, d, J = 11.3 Hz, H-6). 13
C
NMR δ -5.0, -4.9, -4.8 (2 carbons), 12.0, 18.1, 18.2, 18.8, 22.2,
23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.7, 28.7, 29.4, 31.8,
35.9, 38.5, 40.5, 45.6, 47.6, 56.2, 56.4, 63.6, 71.6, 72.5, 106.2,
116.1, 122.4, 132.7, 141.1, 152.9. MS (EI) m/z (%): 602 (Mþ, 1),
470 (22), 366 (10), 279 (9), 167(26), 149 (83), 73 (100). HRMS
(FAB) calcd for C36H67O3Si2 (Mþ þ H) 603.4629, found
603.4627. IR (neat) 3344, 2952, 2927, 2833, 2856, 1658, 1620,
1461, 1255, 1101, 1072, 935, 896, 835, 775, 667 cm-1
.
2-Methylidene-19,25,26,27-tetranor-1r,24-dihydroxyvitamin
D3 (3a). A solution of 11 (17.1 mg, 0.02841 mmol) and camphor
sulfonic acid (19.8 mg, 0.08523 mmol) in MeOH (0.5 mL) was
stirred at room temperature for 1 h. Aqueous NaHCO3 was
added, and the mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and evapo-
rated. The residue was chromatographed on silica gel (AcOEt/
hexane = 1/1) to afford 3a (12.3 mg, quant).
13. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.62
(3 H, s, H-18), 0.86, 0.90 (each 9 H, s, t-Bu), 1.05 (3 H, d, J =
6.6 Hz, H-21), 1.29 (3 H, t, J = 7.1 Hz, -COOEt), 2.82(1 H, m,
H-9), 4.18 (2 H, q, J = 7.1 Hz, -COOCH2-), 4.43 (2 H, m, H-1,
3), 4.92, 4.96 (each 1 H, s, -CdCH2), 5.65 (1 H, d, J = 11.5 Hz,
H-23), 5.82 (1 H, d, J = 11.2 Hz, H-7), 5.98 (1 H, dd, J = 11.5,
10.7 Hz, H-22), 6.20 (1 H, d, J = 11.2 Hz, H-6. MS (EI) m/z (%):
642 (Mþ, 2), 510 (46), 366 (28), 147 (23), 73 (100). HRMS (EI)
calcd for C38H66O4Si2 642.4500, found 642.4501. IR (neat)
3394, 2952, 2927, 2887, 2856, 1720, 1641, 1461, 1251, 1180,
1099, 1070, 935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 255,
264 nm.
3a. 1H NMR (300 MHz, MeOD) δ 0.59 (3 H, s, H-18), 0.97
(3 H, d, J = 6.2 Hz, H-21), 2.83 (1 H, m, H-9), 3.51 (2 H, t, J =
6.4 Hz, H-24), 4.40 (2 H, m, H-1, 3), 5.04, 5.06 (each 1 H, s,
-CdCH2), 5.90 (1 H, d, J = 11.1 Hz, H-7), 6.26 (1 H, d, J =
11.1 Hz, H-6). 13C NMR (100 MHz, MeOD) δ 12.5, 19.4, 23.3,
24.6, 28.7, 29.9, 30.3, 33.2, 37.4, 39.0, 41.9, 46.8, 46.9, 57.5, 57.9,
63.6, 71.6, 72.5, 107.9, 117.2, 124.1, 133.1, 142.5, 153.9. MS (EI)
m/z (%): 374 (Mþ, 76), 289 (30), 269 (28), 220 (31), 147 (77), 135
Ethyl (3S)-3-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)-
silyl]oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}hep-
tanoate (14). A suspension of CuBr/Me2S (1.5 g, 7.27 mmol) in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5821
THF (6 mL) was cooled to -30 °C, and to this solution was
added n-BuLi (8.76 mL, 14.5 mmol, 1.66 M/hexane) and the
mixture was stirred for 15 min. To this solution was added
TMSCl (1.16 mL, 9.09 mmol), HMPA (1.59 mL, 9.09 mmol),
and a solution of enoate 13 (584 mg, 0.909 mmol) in THF (6 mL)
in this order. The mixture was stirred at -30 °C for 1.5 h, and the
reaction was quenched with satd NH4Cl. The mixture was
extracted with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (AcOEt/hexane = 5/995) to afford 14 (471
mg, 0.673 mmol, 74%) and its 22R-isomer (44.6 mg, 0.06371
mmol, 7.0%).
1658, 1620, 1444, 1379, 1263, 1072, 1045, 912, 738 cm-1. UV
(EtOH) λmax 246, 254, 263 nm.
(4S)-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}-2-met-
hyloctan-2-ol (18). In a similar manner to that for the synthesis of
12 from 10, target compound 18 (34.6 mg, 0.0504 mmol) was
obtained from 14 (85.3 mg, 0.122 mmol) in 64% yield.
18. 1H NMR δ 0.03, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.53
(3 H, s, H-18), 0.83 (3 H, d, J = 6.4 Hz, H-21), 0.86, 0.89 (each
9 H, s, t-Bu), 1.23 (6 H, s, H-25, 26), 2.82 (1 H, m, H-9), 4.43 (2 H,
m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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 -5.0,
-4.9, -4.8 (2 carbons), 12.0, 13.6, 14.2, 18.1, 18.2, 22.1, 23.2,
23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.5, 28.8, 29.3, 29.8,
30.5, 30.8, 36.1, 38.6, 40.1, 40.6, 45.6, 45.9, 47.5, 54.4, 56.2, 71.6,
71.9, 72.5, 106.2, 116.1, 122.4, 132.7, 141.2, 152.9. MS (EI) m/z
(%): 686 (Mþ, 1), 554 (28), 536 (7), 366 (24), 234 (12), 147 (12), 73
(100). HRMS (EI) calcd for C42H78O3Si2 686.5490, found
686.5478. IR (neat) 3440, 2954, 2927, 2856, 1658, 1620, 1461,
1255, 1101, 1072, 935, 896, 835, 775 cm-1. UV (hexane) λmax
246, 255, 264 nm.
14. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.52
(3 H, s, H-18), 0.81 (3 H, d, J = 6.1 Hz, H-21), 0.86, 0.89 (each
9 H, s, t-Bu), 1.26 (3 H, t, J = 7.1 Hz, -COOEt), 2.82 (1 H, m,
H-9), 4.13 (2 H, m, -COOEt), 4.43 (2 H, m, H-1, 3), 4.92, 4.97
(each 1 H, s, -CdCH2), 5.82 (1 H, d, J = 11.1 Hz, H-7), 6.21
(1 H, d, J = 11.1 Hz, H-6). 13C NMR δ -5.0, -4.9, -4.8 (2
carbons), 11.9, 13.2, 14.1, 14.3, 18.1, 18.2, 22.1, 23.0, 23.4, 25.7
(3 carbons), 25.8 (3 carbons), 27.3, 27.7, 28.8, 30.3, 37.3, 37.8,
38.5, 38.6, 40.7, 45.6, 47.6, 53.9, 56.3, 60.0, 71.6, 72.5, 106.2,
116.1, 122.4, 132.7, 141.2, 152.9, 173.9. MS (EI) m/z (%): 700
(Mþ, 1), 568 (44), 511 (11), 366 (34), 351 (11), 234 (17), 147(22),
73 (100). HRMS (EI) calcd for C42H76O4Si2 700.5282, found
700.5270. IR (neat) 2954, 2929, 2856, 1735, 1658, 1461, 1251,
1101, 1070, 935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254,
264 nm.
22S-Butyl-2-methylidene-19,24-dinor-1r,25-dihydroxyvitamin
D3 (4c). In a similar manner to that for the synthesis of 3a from
11, target compound 4c (16.2 mg, 0.0354 mmol) was obtained
from 18 (34 mg, 0.0496 mmol) in 71% yield.
4c. 1H NMR δ 0.54 (3 H, s, H-18), 0.82 (3 H, d, J = 6.3 Hz,
H-21), 0.90 (6 H, t, J = 6.8 Hz, CH3 of Bu), 1.22 (6 H, s, H-25,
26), 4.46 (2 H, m, H-1, 3), 5.08, 5.10 (each 1 H, s, -CdCH2), 5.88
1
22R-Isomer of 14. H NMR δ 0.03, 0.04, 0.07, 0.08 (each 3 H,
(1 H, d, J = 11.2 Hz, H-7), 6.35 (1 H, d, J = 11.2 Hz, H-6). 13
C
s, SiMe), 0.56 (3 H, s, H-18), 0.79 (3 H, d, J = 6.7 Hz, H-21),
0.86, 0.89 (each 9 H, s, t-Bu), 1.26 (3 H, t, J = 7.1 Hz, -COOEt),
2.85 (1 H, m, H-9), 4.13 (2 H, q, J = 7.1 Hz, -COOEt), 4.43
(2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 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
-5.0, -4.9, -4.8 (2 carbons), 11.9, 13.0, 14.0, 14.2, 18.1, 18.2,
22.1, 22.8, 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.5, 28.8,
29.6, 32.6, 34.8, 37.1, 37.3, 38.6, 40.6, 45.6, 47.5, 54.2, 56.3, 60.1,
71.6, 72.5, 106.2, 116.1, 122.4, 132.8, 140.9, 152.9, 174.5.
(3S)-3-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}heptan-
1-ol (16). In a similar manner to that for the synthesis of 11 from
10, target compound 16 (287 mg, 0.436 mmol) was obtained
from 14 (440 mg, 0.629 mmol) in 69% yield.
NMR δ 12.1, 13.6, 14.2, 22.1, 23.3, 23.5, 27.4, 29.0, 29.3, 29.9,
30.4, 30.8, 36.1, 38.1, 40.0, 40.5, 45.8 (2 carbons), 45.9, 54.4,
56.3, 70.6, 71.8, 71.9, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS
(EI) m/z (%): 458 (Mþ, 35), 161 (40), 135 (69), 69 (100). HRMS
(EI) calcd for C30H50O3 458.3760, found 458.3770. IR (neat)
3353, 2952, 2927, 2869, 1612, 1454, 1380, 1074, 1045, 910, 896,
742 cm-1. UV (EtOH) λmax 246, 254, 263 nm.
Ethyl (3S)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpenta-
noate (15). A suspension of CuBr/Me2S (491 mg, 2.31 mmol) in
THF (4 mL) was cooled to -30 °C, and to this solution was
added EtLi (9.2 mL, 4.6 mmol, 0.5 M solution in benzene/
cyclohexane = 90/10) and the mixture was stirred for 15 min. To
this solution was added TMSCl (249 μL, 1.95 mmol), HMPA
(341 μL, 1.95 mmol), and a solution of enoate 13 (178 mg, 0.278
mmol) in THF (2 mL) in this order. The mixture was stirred at
-30 °C for 3.5 h, and the reaction was quenched with satd
NH4Cl. The mixture was extracted with AcOEt. The organic
layer was washed with brine, dried over MgSO4, and evapo-
rated. The residue was chromatographed on silica gel (AcOEt/
hexane = 3/997) to afford 15 (103 mg, 0.153 mmol, 55%).
15. 1H NMR δ 0.03, 0.05, 0.06, 0.07 (each 3 H, s, SiMe), 0.52
(3 H, s, H-18), 0.81 (3 H, d, J = 6.2 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 1.25 (3 H, t, J = 7.1 Hz, -COOEt), 2.80 (1 H, m,
H-9), 4.13 (2 H, m, -COOEt), 4.43 (2 H, m, H-1, 3), 4.92, 4.97
(each 1 H, s, -CdCH2), 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
carbons), 11.9, 12.7, 13.2, 14.3, 18.1, 18.2, 20.7, 22.1, 23.4, 25.7
(3 carbons), 25.8 (3 carbons), 27.3, 28.7, 37.3, 38.5 (2 carbons),
39.4, 40.6, 45.6, 47.6, 53.8, 56.3, 60.0, 71.6, 72.5, 106.2, 116.2,
122.3, 132.8, 141.0, 152.9, 173.8. MS (EI) m/z (%): 672 (Mþ, 1),
540 (24), 366 (17), 234 (9), 147 (12), 129 (16) 73 (100). HRMS
(EI) calcd for C40H72O4Si2 672.4969, found 672.4959. IR (neat)
2954, 2929, 2883, 2856, 1735, 1658 1614, 1461, 1251, 1101,
1070, 935, 896, 835, 775, 696 cm-1. UV (hexane) λmax 246, 254,
264 nm.
16. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.80 (3 H, d, J = 6.2 Hz, H-21), 0.86, 0.89 (each
9 H, s, t-Bu), 2.80 (1 H, m, H-9), 3.66 (2 H, m, H-24), 4.43 (2 H,
m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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 -5.0,
-4.9, -4.8 (2 carbons), 11.9, 13.2, 14.2, 18.1, 18.2, 22.1, 23.2,
23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.8, 28.4, 28.7, 30.7,
35.3, 36.4, 38.0, 38.5, 40.6, 45.6, 47.6, 53.8, 56.3, 61.8, 71.6, 72.5,
106.2, 116.1, 122.4, 132.8, 141.1, 152.9. MS (EI) m/z (%):
658 (Mþ, 1), 526 (28), 366 (5), 279 (13), 167(36), 149 (99), 73
(100). HRMS (EI) calcd for C40H74O3Si2 658.5177, found
658.5170. IR (neat) 3301, 2952, 2927, 2893, 2856, 1461, 1380,
1255, 1101, 1072, 835, 775 cm-1. UV (hexane) λmax 246, 254,
264 nm.
22S-Butyl-2-methylidene-19,25,26,27-tetranor-1r,24-dihydro-
xyvitamin D3 (3c). In a similar manner to that for the synthesis of
3a from 11, target compound 3c (26.3 mg, 0.0609 mmol) was
obtained from 16 (49.6 mg, 0.0753 mmol) in 81% yield.
3c. 1H NMR δ 0.55 (3 H, s, H-18), 0.80 (3 H, d, J = 6.4 Hz,
H-21), 0.89 (3 H, t, J = 7.0 Hz, CH3 of Bu), 3.64 (2 H, m, H-24),
4.47 (2 H, m, H-1, 3), 5.08, 5.10 (each 1 H, s, -CdCH2), 5.88 (1
H, d, J = 11.3 Hz, H-7), 6.35 (1 H, d, J = 11.3 Hz, H-6). 13
C
NMR δ 11.0, 12.2, 13.2, 21.2, 22.2, 22.5, 26.7, 27.3, 27.9, 29.7,
34.2, 35.4, 36.9, 37.1, 39.5, 44.7 (2 carbons), 52.8, 55.3, 60.8,
69.6, 70.8, 106.7, 114.3, 123.2, 129.4, 142.3, 150.9. MS (EI) m/z
(%): 430 (Mþ, 34), 315 (15), 297 (19), 269 (14), 251 (15), 161 (40),
147 (51), 135 (97), 69 (100). HRMS (EI) calcd for C28H46O3
430.3447, found 430.3447. IR (neat) 3332, 2925, 2869, 1772,
(3S)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpentan-1-ol
(17). In a similar manner to that for the synthesis of 11 from 10,
target compound 17 (246 mg, 0.390 mmol) was obtained from 15
(307.2 mg, 0.457 mmol) in 85% yield.

5822 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Sakamaki et al.
17. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.81 (3 H, d, J = 6.3 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.81 (1 H, m, H-9), 3.67 (2H, m, H-24), 4.42 (2 H, m,
H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 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 -5.0,
-4.9, -4.8 (2 carbons), 11.9, 13.1, 13.3, 18.1, 18.2, 21.1, 22.1,
23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.8, 28.7, 34.7, 38.0,
38.5 (2 carbons), 40.6, 45.6, 47.6, 53.9, 56.3, 61.8, 71.6, 72.5,
106.2, 116.1, 122.3, 132.8, 141.1, 152.9. MS (EI) m/z (%): 630
(Mþ, 2), 498 (51), 366 (28), 351 (11), 234 (16), 147 (19), 73 (100).
HRMS (EI) calcd for C38H70O3Si2 630.4863, found 630.4863.
IR (neat) 3313, 2952, 2927, 2883, 2856, 1658 1612, 1461, 1251,
1101, 1070, 1004, 935, 896, 835, 779 cm-1. UV (hexane) λmax
246, 254, 264 nm.
dried over MgSO4, and evaporated. The residue was added to a
solution of sodium thiophenoxide (225 mg, 1.53 mmol) in EtOH
(1.7 mL), and the mixture was stirred for 1.5 h at room tem-
perature. The mixture was diluted with 1N NaOH and extracted
with benzene. The organic layer was washed with satd NH4Cl
and brine and evaporated. The residue was solved in CH2Cl2
(9 mL) and treated with m-chloroperbenzoic acid (195 mg, 2.87
mmol, 65%) for 2 h at room temperature. The mixture was
diluted with CH2Cl2 and washed with 1N NaOH, satd NH4Cl,
and brine. The organic layer was dried over MgSO4, and eva-
porated. The residue was chromatographed on silica gel (AcOEt/
hexane = 1/1) to afford 22 (211 mg, 0.865 mmol, 62% from
3-bromo-1-propanol).
20. 1H NMR (400 MHz, CDCl3) δ 2.13 (2 H, tt, J = 6.3,
6.0 Hz, H-2), 3.37 (3 H, s, -O-CH3), 3.53 (2 H, t, J = 6.3 Hz,
22S-Ethyl-2-methylidene-19,25,26,27-tetranor-1r,24-dihydro-
xyvitamin D3 (3b). In a similar manner to that for the synthesis of
3a from 11, target compound 3b (12.4 mg, 0.0307 mmol) was
obtained from 17 (28.2 mg, 0.0228 mmol) in 69% yield.
3b. 1H NMR δ 0.54 (3 H, s, H-18), 0.80 (3 H, d, J = 6.4 Hz,
H-21), 0.90 (4 H, m), 3.66 (2H, m, H-24), 4.46 (2 H, m, H-1, 3),
5.08, 5.10 (each 1 H, s, -CdCH2), 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 12.0, 13.1,
13.3, 21.2, 22.2, 23.5, 27.7, 28.9, 34.7, 37.9, 38.1, 38.5, 40.5, 45.7
(2 carbons), 53.9, 56.3, 61.8, 70.6, 71.8, 107.7, 115.3, 124.2,
130.5, 143.3, 151.9. MS (EI) m/z (%): 402 (Mþ, 55), 315 (20), 297
(27), 251 (26), 173 (30),161 (58), 147(75), 135 (100), 69 (92).
HRMS (EI) calcd for C26H42O3 402.3134, found 402.3150. IR
(neat) 3336, 2947, 2871, 1726, 1658, 1620, 1440, 1380, 1070,
1045, 1010, 754, 699 cm-1. UV (EtOH) λmax 245, 254, 263 nm.
(4S)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethyl-2-methyl-
hexan-2-ol (19). In a similar manner to that for the synthesis of
12 from 10, target compound 19 (31.6 mg, 0.0480 mmol) was
obtained from 15 (38.1 mg, 0.0567 mmol) in 85% yield.
19. 1H NMR δ 0.03, 0.04, 0.06, 0.07 (each 3 H, s, SiMe), 0.53
(3 H, s, H-18), 0.83 (3 H, d, J = 6.4 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 1.24 (6 H, s, H-25, 26), 2.82 (1 H, m, H-9), 4.43 (2 H,
m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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 carbons), 12.0, 13.2, 13.6, 18.1, 18.2, 22.1 (2
carbons), 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.5, 28.8,
29.8, 30.5, 38.2, 38.6, 40.2, 40.6, 45.4, 45.6, 47.5, 54.5, 56.3, 71.6,
71.9, 72.4, 106.2, 116.1, 122.4, 132.8, 141.1, 152.9. MS (EI) m/z
(%): 658 (Mþ, 3), 526 (48), 508 (13), 366 (35), 351 (12), 234 (18),
147 (20), 97 (40), 73 (100). HRMS (EI) calcd for C40H74O3Si2
658.5177, found 658.5196. IR (neat) 3394, 2954, 2927, 2885,
2856, 1658, 1620, 1469, 1461, 1251, 1101, 1070, 1004, 935, 896,
835, 779 cm-1. UV (hexane) λmax 246, 255, 264 nm.
H-3), 3.66 (2 H, t, J = 6.0 Hz, H-1), 4.63 (2 H, s, O-CH2-O). 13
C
NMR (100 MHz, CDCl3) δ 30.8 (C-2), 33.2 (C-3), 55.6 (-O-Me),
65.4 (C-1), 96.9 (-O-CH2-O-).
21. 1H NMR (400 MHz, CDCl3) δ 1.92 (2 H, tt, J = 7.1, 6.1
Hz, H-2), 3.03 (2H, t,J =7.1 Hz, H-3), 3.36 (3 H, s,-O-Me), 3.63
(2 H, t, J = 6.1 Hz, H-1), 4.61 (2 H, s, O-CH2-O), 7.17 (1 H, td,
J = 7.6, 1.2Hz, arom-H), 7.28 (2 H, dd, J = 7.6, 7.3 Hz, arom-H),
7.34 (2 H, dd, J = 7.3, 1.2 Hz, arom-H). 13C NMR (100 MHz,
CDCl3) δ 29.7 (C-2), 30.7 (C-3), 55.6 (-O-Me), 66.3 (C-1), 96.9
(-O-CH2-O-), 126.3, 129.3 (2 carbons), 129.6 (2 carbons), 136.8.
22. 1H NMR (400 MHz, CDCl3) δ 2.01 (2 H, m, H-2), 3.23
(2 H, t, J = 7.8 Hz, H-3), 3.30 (3 H, s, -O-Me), 3.57 (2 H, t, J =
6.0 Hz, H-1), 4.55 (2 H, s, O-CH2-O), 7.58 (2 H, dd, J = 6.9, 7.3
Hz, arom-H), 7.66 (1 H, t, J = 7.3 Hz, arom-H), 7.94 (2 H, d,
J = 6.9 Hz, arom-H). 13C NMR (100 MHz, CDCl3) δ 23.8 (C-
2), 53.9 (C-3), 55.7 (-O-Me), 65.7 (C-1), 96.8 (-O-CH2-O-),
128.4 (2 carbons), 129.7 (2 carbons), 134.1, 139.5.
(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-17-(1-
iodopropan-2-yl)-2-methylidene-9,10-secoestra-5,7-diene (23). A
mixture of tosylate 7 (102 mg, 0.141 mmol), sodium iodide (83.7
mg, 0.563 mmol), and lithium carbonate (43.7 mg, 0.591 mmol)
in acetone (3 mL) was refluxed for 4 h. The mixture was diluted
with water and 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 23 (94.2 mg, 98%).
1
23. H NMR (400 MHz, CDCl3) δ 0.02, 0.05, 0.06, 0.08 (each
3 H, s, SiMe), 0.58 (3 H, s, H-18), 0.86, 0.96 (each 9 H, s, t-Bu),
1.04 (3 H, d, J = 6.3 Hz, H-21), 2.82 (1 H, m, H-9), 3.18 (1 H, dd,
J = 9.7, 5.7 Hz, H-22), 3.35 (1 H, dd, J = 9.7, 2.5 Hz, H-22), 4.43
(2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 5.84 (1 H, d,
J = 11.3 Hz, H-7), 6.20 (1 H, d, J = 11.3 Hz, H-6). Mass m/z
(%): 684 (Mþ, 10), 552 (100), 424 (28), 366 (28), 73 (49). HRMS
calcd for C34H61O2Si2I 684.3255, found 684.3279. IR (neat)
22S-Ethyl-2-methylidene-19,24-dinor-1r,25-dihydroxyvitamin
D3 (4b). In a similar manner to that for the synthesis of 3a from
11, target compound 4b (20.1 mg, 0.0467 mmol) was obtained
from 19 (31.6 mg, 0.0480 mmol) in 97% yield.
2952, 2925, 2854, 1461, 1255, 1101, 1070, 835, 777 cm-1
.
(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-17-[6-
(methoxymethoxy)hexan-2-yl]-2-methylidene-9,10-secoestra-5,7-
diene (25). To a solution of sulfone 22 (67.2 mg, 0.275 mmol) and
diisopropylamine (195 μL, 1.39 mmol) in THF (0.5 mL) was
added n-BuLi (481 μL, 0.687 mmol, 1.43 M hexane) at -20 °C
and the mixture was stirred for 5 min. A solution of iodide 27
(94.2 mg, 0.138 mmol) and HMPA (479 μL, 2.76 mmol) in THF
(0.5 mL) was added, and the mixture was stirred for 1 h. The
reaction was quenched with satd NH4Cl, and the mixture was
extracted with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (AcOEt/hexane = 10/90) to afford 24 (62.2
mg, 56%). Compound 24 was solved in THF/MeOH (3:2, 1.25
mL) and NaHPO4 (223 mg, 1.57 mmol) and NaHg (368 mg, 1.64
mmol) were added at 0 °C. The mixture was stirred at room
temperature for 2 h. The reaction was quenched with 1N HCl
and extracted with AcOEt. The organic layer was washed with
aqueous NaHCO3 and brine, dried over MgSO4, and evapo-
rated. The residue was chromatographed on silica gel (AcOEt/
hexane = 10/90) to afford 25 (37.2 mg, 72%).
4b. 1H NMR δ 0.54 (3 H, s, H-18), 0.82 (3 H, d, J = 6.2 Hz,
H-21), 0.88 (4 H, m), 1.23 (6 H, s, H-25, 26), 4.45 (2 H, m, H-1, 3),
5.08, 5.10 (each 1 H, s, -CdCH2), 5.88 (1 H, d, J = 11.1 Hz,
H-7), 6.35 (1 H, d, J = 11.1 Hz, H-6). 13C NMR δ 12.1, 13.2,
13.6, 22.0, 22.1, 23.5, 27.4, 29.0, 29.8, 30.4, 38.1, 38.2, 40.1, 40.5,
45.4, 45.7 (2 carbons), 54.5, 56.3, 70.6, 71.8, 71.9, 107.7, 115.3,
124.2, 130.5, 143.3, 151.9. MS (EI) m/z (%): 430 (Mþ, 43), 161
(50), 135 (90), 97 (100). HRMS (EI) calcd for C28H46O3
430.3447, found 430.3462. IR (neat) 3373, 2958, 2871, 1726,
1649, 1612, 1461, 1379, 1074, 1045, 900, 865, 756, 669 cm-1. UV
(EtOH) λmax 246, 254, 263 nm.
{[3-(Methylperoxy)propyl]sulfonyl}benzene (22). To a solu-
tion of 3-bromo-1-propanol (125 μL, 1.39 mmol) and diisopro-
pylethylamine (725 μL, 4.16 mmol) in CH2Cl2 (0.5 mL) was
added methoxymethyl chloride (526 μL, 6.93 mmol) at 0 °C and
the mixture was stirred for 1 h. The reaction was quenched with
1N HCl, and the mixture was extracted with CH2Cl2. The
organic layer was washed with aqueous NaHCO3 and brine,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5823
1
25. H NMR (400 MHz, CDCl3) δ 0.02, 0.05, 0.07, 0.08 (each
3 H, s, SiMe), 0.54 (3 H, s, H-18), 0.86, 0.89 (each 9 H, s, t-Bu),
0.93 (3 H, d, J = 6.4 Hz, H-21), 2.81 (1 H, m, H-9), 3.36 (3 H, s,
-O-Me), 3.52 (2 H, t, J = 6.6 Hz, H-25), 4.42 (2 H, m, H-1, 3),
4.62 (2 H, s, -O-CH2-O), 4.91, 4.96 (each 1 H, s, -CdCH2 ꢀ 2),
5.83 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz, H-6).
Mass m/z (%): 660 (21), 528 (100), 366 (44), 75 (34), 73 (36).
HRMS calcd for C39H72O4Si2 660.4969, found 660.4953.
2-Methylidene-19,26,27-trinor-1r,25-dihydroxyvitamin D3 (5a).
A solution of 25 in 3%HCl-MeOH was stirred at room
temperature for 4 h. The mixture was evaporated, and the re-
sidue was chromatographed on silica gel (AcOEt/hexane = 80/
20) to afford 5a (8.3 mg, 53%).
(36), 251 (16), 73 (100). HRMS (EI) calcd for C47H80O5Si2
812.5265, found 812.5267. IR (neat) 2952, 2927, 2891, 2856,
1361, 1251, 1188, 1178, 1099, 1070, 935, 896, 835, 775, 667 cm-1
.
UV (hexane) λmax 246, 254, 264 nm.
(4S)-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}octanenitrile
(30). In a similar manner to that for the synthesis of 31 from 29,
target compound 30 (212 mg, 0.318 mmol) was obtained from 28
(284 mg, 0.351 mmol) in 91% yield.
30. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.79 (3 H, d, J = 6.4 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.83 (1 H, m, H-9), 4.42 (2 H, m, H-1, 3), 4.92, 4.97
(each 1 H, s, -CdCH2), 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 -5.0, -4.9, -4.8 (2
carbons), 12.0, 12.9, 14.1, 15.7, 18.1, 18.2, 22.1, 23.1, 23.4, 25.7
(3 carbons), 25.8 (3 carbons), 27.4, 27.7, 28.0, 28.7, 30.5, 36.8,
38.5, 39.3, 40.6, 45.6, 47.6, 53.5, 56.3, 71.6, 72.5, 106.2, 116.2,
119.8, 122.4, 133.0, 140.8, 152.9. MS (EI) m/z (%): 667 (Mþ, 1),
535 (21), 478 (33), 366 (17), 73 (100). HRMS (EI) calcd for
C41H73NO2Si2 667.5180, found 667.5151. IR (neat) 2952, 2927,
2883, 2856, 2245, 1461, 1251, 1101, 1072, 935, 896, 835, 775
cm-1. UV (hexane) λmax 246, 254, 264 nm.
5a. 1H NMR (400 MHz, CDCl3) δ 0.55 (3 H, s, H-18), 0.93
(3 H, d, J = 6.4 Hz, H-21), 3.65 (2 H, t, J = 6.5 Hz, H-25), 4.47
(2 H, m, H-1, 3), 5.09, 5.11 (each 1 H, s, -CdCH2 ꢀ 2), 5.88 (1
H, d, J = 11.5 Hz, H-7), 6.35 (1 H, d, J = 11.5 Hz, H-6). 13
C
NMR δ 12.1, 18.7, 22.2, 22.3, 23.5, 27.6, 28.9, 33.2, 35.6, 36.0,
38.1, 40.4, 45.8 (2 carbons), 56.3, 56.4, 63.1, 70.7, 71.8, 107.7,
115.3, 124.3, 130.3, 143.4, 151.9. MS (EI) m/z (%): 388 (100), 370
(23), 352 (26), 303 (34), 287 (47), 269 (44), 251 (49), 235 (29), 147
(47), 135 (39), 107 (44), 81 (36), 55 (36). HRMS (EI) calcd for
C25H40O3 388.2977, found 388.2978. IR (neat) 3346, 2993, 2869,
1436, 1375, 1072, 1043, 754 cm-1. UV (EtOH) λmax 246, 254,
263 nm.
(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-2-met-
hylidene-17-{6-methyl-6-[(triethylsilyl)oxy]heptan-2-yl}-9,10-sec-
oestra-5,7-diene (27). To a solution of 26 (631 mg, 2.25 mmol) in
THF (2.5 mL) was added Mg turnings (54.2 mg, 2.25 mmol),
and the mixture was stirred for 0.5 h. The resulting Grignard
reagent was added to a solution of CuBr/Me2S (11.3 mg, 0.0550
mmol) in THF (1 mL) at -10 °C, and the mixture was stirred for
15 min. To this solution was added 7 (36.7 mg, 0.0484 mmol) in
THF (1 mL), and the mixture was stirred at -10 °C for 1 h. The
reaction was quenched with satd NH4Cl and extracted with
AcOEt. The organic layer was washed with brine, dried over
MgSO4, and evaporated. The residue was chromatographed on
silica gel (AcOEt/hexane = 1/99) to afford 27 (31.5 mg, 0.0416
mmol, 86%).
(4S)-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}octanal
(32). In a similar manner to that for the synthesis of 33 from 31,
target compound 32 (166 mg, 0.248 mmol) was obtained from 30
(212 mg, 0.318 mmol) in 78% yield.
32. 1H NMR δ 0.02, 0.05, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.78 (3 H, d, J = 6.3 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.83 (1 H, m, H-9), 4.43 (2 H, m, H-1, 3), 4.92, 4.97
(each 1 H, s, -CdCH2), 5.83 (1 H, d, J = 11.0 Hz, H-7), 6.21 (1
H, d, J = 11.0 Hz, H-6) 9.78 (1 H, t, J = 1.9 Hz, -CHO). 13
C
NMR δ -5.0, -4.9, -4.8 (2 carbons), 11.9, 12.9, 14.2, 18.1, 18.2,
22.1, 23.2, 23.4, 24.0, 25.7 (3 carbons), 25.8 (3 carbons), 27.8,
28.5, 28.7, 30.8, 37.1, 38.5, 39.7, 40.6, 42.6, 45.6, 47.6, 53.7, 56.3,
71.6, 72.5, 106.2, 116.2, 122.4, 132.8, 141.0, 152.9, 203.1. MS
(EI) m/z (%): 670 (Mþ, 2), 538 (50), 366 (33), 149 (25), 73 (100).
HRMS (EI) calcd for C41H74O3Si2 670.5177, found 670.5151.
IR (neat) 2952, 2927, 2891, 2856, 1728, 1658, 1620, 1461, 1255,
1101, 1072, 935, 896, 835, 777 cm-1. UV (hexane) λmax 246, 254,
264 nm.
27. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.56 (6H, dt, J = 7.7, 8.0 Hz, -SiCH2-), 0.86,
0.89 (each 9 H, s, t-Bu), 0.92 (3 H, m, H-21), 0.95 (9H, dd, J =
7.7, 8.0 Hz, -SiCH2Me), 1.19 (6H, s, H-26, 27), 2.83 (1 H, m,
H-9), 4.42 (2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2),
5.83 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz, H-6).
2-Methylidene-19-nor-1r,25-dihydroxyvitamin D3 (6a). In a
similar manner to that for the synthesis of 3a from 11, target
compound 6a (14.6 mg, 0.0351 mmol) was obtained from 27
(31.1 mg, 0.0410 mmol) in 86% yield.
(4S)-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}octan-1-
ol (34). In a similar manner to that for the synthesis of 35 from
33, target compound 34 (43.1 mg, 0.0641 mmol) was obtained
from 32 (46.7 mg, 0.0697 mmol) in 92% yield.
34. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.78 (3 H, d, J = 5.6 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.83(1 H, m, H-9), 3.63 (2 H, dt, J = 1.7, 6.5 Hz,
H-25), 4.45 (2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2),
5.83 (1 H, d, J = 11.1 Hz, H-7), 6.22 (1 H, d, J = 11.1 Hz, H-6).
13C NMR δ -5.0, -4.9, -4.8 (2 carbons), 12.0, 13.0, 14.2, 18.1,
18.2, 22.2, 23.3, 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.7,
27.8, 28.7, 28.8, 30.9, 31.3, 37.2, 38.5, 39.8, 40.6, 45.6, 47.6, 53.8,
56.3, 63.5, 71.6, 72.5, 106.2, 116.1, 122.4, 132.7, 141.2, 152.9. MS
(EI) m/z (%): 672 (Mþ, 2), 540 (46), 366 (30), 147 (19), 73 (100).
HRMS (EI) calcd for C41H76O3Si2 672.5333, found 672.5326.
IR (neat) 3328, 2952, 2927, 2893, 2856, 1612, 1461, 1251, 1101,
1072, 935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254,
264 nm.
6a. 1H NMR δ 0.55 (3 H, s, H-18), 0.94 (3 H, d, J = 6.3 Hz,
H-21), 1.21 (6H, s, H-26, 27), 4.47 (2 H, m, H-1, 3), 5.09, 5.11
(each 1 H, s, -CdCH2), 5.88 (1 H, d, J = 11.2 Hz, H-7), 6.35 (1
H, d, J = 11.2 Hz, H-6). UV (EtOH) λmax 246, 254, 263 nm.
(3S)-3-{1-[(1R,3R,7E,17β)-1,3-bis{[tert-Butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}heptyl 4-
methylbenzenesulfonate (28). In a similar manner to that for the
synthesis of 29 from 17, target compound 28 (285 mg, 0.351
mmol) was obtained from 16 (287 mg, 0.436 mmol) in 80% yield.
28. 1H NMR δ 0.04, 0.05, 0.08, 0.09 (each 3 H, s, SiMe), 0.48
(3 H, s, H-18), 0.72 (3 H, d, J = 6.6 Hz, H-21), 0.88, 0.89 (each
9 H, s, t-Bu), 2.81 (1 H, m, H-9), 2.45 (3 H, s, Ph-Me), 2.80 (1 H,
m, H-9), 4.09 (2 H, m, H-24), 4.44 (2 H, m, H-1, 3), 4.93, 4.97
(each 1 H, s, -CdCH2), 5.83 (1 H, d, J = 11.1 Hz, H-7), 6.21 (1 H,
d, J = 11.1 Hz, H-6), 7.33 (2 H, d, J = 8.4 Hz, arom-H), 7.80 (2 H,
d, J = 8.4 Hz, arom-H). 13C NMR δ -5.0, -4.9, -4.8 (2 carbons),
11.9, 13.0, 14.1, 18.1, 18.2, 21.6, 22.1, 23.1, 23.4, 25.7 (3
carbons), 25.8 (3 carbons), 27.6, 28.0, 28.7, 30.5, 31.1, 36.2,
37.6, 38.6, 40.6, 45.6, 47.5, 53.6, 56.2, 69.6, 71.7, 72.3, 106.2,
116.2, 122.4, 127.8 (2 carbons), 129.7 (2 carbons), 132.8, 133.4,
140.9, 144.5, 152.9. MS (EI) m/z (%): 812 (Mþ, 1), 680 (10), 366
22S-Butyl-2-methylidene-19,26,27-trinor-1r,25-dihydroxyvi-
tamin D3 (5c). In a similar manner to that for the synthesis of 3a
from 11, target compound 5c (18.4 mg, 0.0414 mmol) was
obtained from 34 (43.1 mg, 0.0641 mmol) in 65% yield.
5c. 1H NMR δ 0.55 (3 H, s, H-18), 0.77 (3 H, d, J = 6.0 Hz,
H-21), 0.89 (3 H, t, J = 6.9 Hz, CH3 of Bu), 3.62 (2 H, dt, J =
1.6, 6.6 Hz, H-25), 4.46 (2 H, m, H-1, 3), 5.08, 5.10 (each 1 H, s,
-CdCH2), 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 12.0, 13.0, 14.2, 22.2, 23.3, 23.5, 27.7,
27.8, 28.7, 29.0, 30.9, 31.2, 37.2, 38.1, 39.8, 40.5, 45.7, 45.8, 53.8,

5824 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Sakamaki et al.
56.4, 63.4, 70.6, 71.8, 107.7, 115.3, 124.2, 130.4, 143.4, 152.0. MS
(EI) m/z (%): 444 (Mþ, 32), 315 (29), 297 (28), 161 (40), 147(52),
135 (100), 69 (89). HRMS (EI) calcd for C29H48O3 444.3603,
found 444.3589. IR (neat) 3346, 2927, 2864, 1448, 1380, 1072,
1045, 756 cm-1. UV (EtOH) λmax 246, 254, 263 nm.
4.09 (2 H, m, H-24), 4.43 (2 H, m, H-1, 3), 4.93, 4.96 (each 1 H, s,
-CdCH2), 5.85 (1 H, d, J = 11.1 Hz, H-7), 6.20 (1 H, d, J =
11.1 Hz, H-6), 7.33 (2 H, d, J = 8.4 Hz, arom-H), 7.80 (2 H, d,
J = 8.4 Hz, arom-H). 13C NMR δ -5.0, -4.9, -4.8 (2 carbons),
11.9, 12.8, 13.1, 18.1, 18.2, 21.0, 21.6, 22.1, 23.4, 25.82 (3
carbons), 25.84 (3 carbons), 27.5, 28.7, 30.6, 37.6, 38.3, 38.6,
40.6, 45.5, 47.5, 53.7, 56.3, 69.5, 71.7, 72.4, 106.2, 116.2, 122.4,
127.8 (2 carbons), 129.7 (2 carbons), 132.8, 133.4, 140.9, 144.5,
152.9. MS (EI) m/z (%): 784 (Mþ, 1), 652 (8), 366 (30), 349 (12),
251 (12), 147 (14), 73 (100). HRMS (EI) calcd for C45H76O5SSi2
784.4952, found 784.4974. IR (neat) 2952, 2927, 2883, 2856,
1658 1598, 1461, 1357, 1251, 1188, 1178, 1099, 1070, 935, 896,
835, 775, 665 cm-1. UV (hexane) λmax 246, 254, 264 nm.
(4S)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexanenitrile
(31). A solution of tosylate 29 (239 mg, 0.305 mmol) and KCN
(55.8 mg, 0.8579 mmol) in DMSO (7 mL) was stirred at 70 °C for
1 h. The reaction was quenched with water and the mixture was
extracted with AcOEt. The organic layer was washed with brine,
dried over MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (AcOEt/hexane = 5/95) to afford 31 (170
mg, 0.2665 mmol, 87%).
Methyl (4S)-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}oct-
anoate (36). In a similar manner to that for the synthesis of 37
from 33, target compound 36 (96.8 mg, 0.138 mmol) was
obtained from 32 (106.5 mg, 0.159 mmol) in 87% yield.
36. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.78 (3 H, d, J = 6.1 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.82 (1 H, m, H-9), 3.67 (3 H, m, -COOMe), 4.43
(2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 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
-5.0, -4.9, -4.8 (2 carbons), 11.9, 13.0, 14.2, 18.1, 18.2, 22.1,
23.2, 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.0, 27.7, 28.4,
28.7, 30.7, 32.8, 37.0, 38.5, 39.6, 40.6, 45.6, 47.6, 51.4, 53.7, 56.3,
71.6, 72.5, 106.2, 116.1, 122.4, 132.8, 141.2, 152.9, 174.5. MS
(EI) m/z (%): 700 (Mþ, 3), 568 (61), 366 (45), 147 (27), 73 (100).
HRMS (EI) calcd for C42H76O4Si2 700.5282, found 700.5270.
IR (neat) 2952, 2927, 2891, 2856, 1741, 1461, 1255, 1101, 1070,
935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254, 264 nm.
(5S)-5-{1-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]ethyl}-2-met-
hylnonan-2-ol (38). In a similar manner to that for the synthesis
of 12 from 10, target compound 38 (22.8 mg, 0.0326 mmol) was
obtained from 36 (40 mg, 0.0571 mmol) in 57% yield.
31. 1H NMR δ 0.02, 0.04, 0.06, 0.07 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.79 (3 H, d, J = 6.4 Hz, H-21), 0.86, 0.89 (each
9 H, s, t-Bu), 2.81 (1 H, m, H-9), 4.42 (2 H, m, H-1, 3), 4.92, 4.96
(each 1 H, s, -CdCH2), 5.83 (1 H, d, J = 11.1 Hz, H-7), 6.20 (1
H, d, J = 11.1 Hz, H-6). 13C NMR δ -5.0, -4.9, -4.8 (2
carbons), 11.9, 12.9, 13.0, 15.7, 18.1, 18.2, 20.9, 22.1, 23.4, 25.7
(3 carbons), 25.8 (3 carbons), 26.9, 27.7, 28.7, 36.8, 38.5, 40.6,
41.3, 45.6, 47.6, 53.6, 56.3, 71.6, 72.5, 106.2, 116.3, 119.9, 122.2,
132.9, 140.7, 152.9. MS (EI) m/z (%): 639 (Mþ, 1), 507 (33), 450
(51), 366 (19), 147 (16), 73 (100). HRMS (EI) calcd for
C39H69NO2Si2 639.4867, found 639.4865. IR (neat) 2954,
2929, 2885, 2856, 2244, 1658 1620, 1461, 1251, 1101, 1072,
935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254, 264 nm.
(4S)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexanal (33).
Cyanide 31 (170 mg, 0.267 mmol) in CH2Cl2 (3 mL) was treated
with DIBALH (400 μL of 1.0 M toluene solution, 0.400 mmol)
at -20 °C for 1 h. The reaction was quenched with aqueous
potassium sodium tartrate and extracted with CH2Cl2. 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 aldehyde 33 (132 mg, 0.205
mmol, 77%).
38. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.77 (3 H, d, J = 5.8 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 1.22 (6 H, s, H-26, 27), 2.82(1 H, m, H-9), 4.42 (2 H,
m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 5.82 (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 carbons), 12.0, 12.9, 14.2, 18.1, 18.2, 22.1, 23.3 (2
carbons), 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 26.4, 27.7,
28.8, 29.0, 29.3, 31.0, 37.2, 38.5, 40.5, 40.7, 42.3, 45.6, 47.6, 53.8,
56.3, 71.2, 71.6, 72.5, 106.2, 116.1, 122.4, 132.7, 141.2, 152.9. MS
(EI) m/z (%): 700 (Mþ, 1), 568 (21), 550 (11), 366 (21), 73 (100).
HRMS (EI) calcd for C43H80O3Si2 700.5646, found 700.5651.
IR (neat) 3381, 2954, 2927, 2894, 2856, 1658, 1616, 1461, 1251,
1101, 1072, 935, 896, 835, 775 cm-1. UV (hexane) λmax 246, 254,
264 nm.
22S-Butyl-2-methylidene-19-nor-1r,25-dihydroxyvitamin D3
(6c). In a similar manner to that for the synthesis of 3c from 11,
target compound 6c (12.6 mg, 0.02700 mmol) was obtained from
38 (22.8 mg, 0.0326 mmol) in 82% yield.
33. 1H NMR δ 0.02, 0.04, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.78 (3 H, d, J = 6.3 Hz, H-21), 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, -CdCH2), 5.82 (1 H, d, J = 11.1 Hz, H-7), 6.21
6c. 1H NMR δ 0.55 (3 H, s, H-18), 0.77 (3 H, d, J = 6.0 Hz,
H-21), 0.90 (3 H, t, J = 6.9 Hz, CH3 of Bu), 1.22 (6 H, s, H-26,
27), 4.47 (2 H, m, H-1, 3), 5.08, 5.10 (each 1 H, s, -CdCH2), 5.88
(1 H, d, J = 11.2 Hz, H-7), 6.35 (1 H, d, J = 11.2 Hz, H-6). 13
C
(1 H, d, J = 11.1 Hz, H-6) 9.78 (1 H, t, J = 1.8 Hz, -CHO). 13
C
NMR δ 12.0, 12.9, 14.2, 22.2, 23.3, 23.5, 26.3, 27.7, 28.8, 29.0,
29.1, 29.3, 31.0, 37.2, 38.1, 40.5 (2 carbons), 42.3, 45.7, 45.8,
53.8, 56.4, 70.6, 71.2, 71.9, 107.7, 115.3, 124.2, 130.4, 143.4,
152.0. MS (EI) m/z (%): 472 (Mþ, 29), 161 (36), 147 (42), 135
(78), 69 (100). HRMS (EI) calcd for C31H52O3 472.3916, found
472.3909. IR (neat) 3365, 2948, 2927, 2869, 1456, 1380, 1199,
1043, 669 cm-1. UV (EtOH) λmax 246, 254, 263 nm.
NMR δ -5.0, -4.9, -4.8 (2 carbons), 11.9, 13.0, 13.1, 18.1, 18.2,
21.3, 22.1, 23.4, 23.5, 25.7 (3 carbons), 25.8 (3 carbons), 27.7,
28.7, 37.1, 38.5, 40.6, 41.8, 42.5, 45.6, 47.6, 53.8, 56.3, 71.6, 72.5,
106.2, 116.2, 122.3, 132.8, 140.9, 152.9, 202.9. MS (EI) m/z (%):
642 (Mþ, 1), 510 (22), 366 (15), 147 (13), 73 (100). HRMS (EI)
calcd for C39H70O4Si2 642.4864, found 642.4890. IR (neat)
2954, 2929, 2885, 2856, 1726, 1620, 1461, 1251, 1101, 1072,
896, 835, 777, 669 cm-1. UV (hexane) λmax 246, 254, 264 nm.
(4S)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexan-1-ol
(35). To a solution of aldehyde 33 (36.7 mg, 0.0572 mmol) in
MeOH/CH2Cl2 (1:1, 2 mL) was added NaBH4 (12.0 mg, 0.317
mmol) at 0 °C, and the mixture was stirred for 1 h. To the
solution was added water 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 35 (36.5 mg,
0.0561 mmol, 98%).
(3S)-4-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-3-ethylpentyl 4-
Methylbenzenesulfonate (29). To a solution of alcohol 17 (222
mg, 0.352 mmol) in pyridine (1.5 mL) was added p-toluenesul-
fonyl chloride (229 mg, 2.07 mmol) at 0 °C, and the mixture was
stirred for 4 h. To this solution was added 1N HCl, 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/
99) to afford 29 (234 mg, 0.298 mmol, 85%).
29. 1H NMR δ 0.04 (6 H, s, SiMe), 0.07, 0.08 (each 3 H, s,
SiMe), 0.47 (3 H, s, H-18), 0.72 (3 H, d, J = 6.6 Hz, H-21), 0.86,
0.89 (each 9 H, s, t-Bu), 2.44 (3 H, s, Ph-Me), 2.79 (1 H, m, H-9),
35. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.77 (3 H, d, J =5.9Hz, H-21), 0.86, 0.89 (each9H,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5825
s, t-Bu), 2.80 (1 H, m, H-9), 3.64 (2 H, dt, J = 2.3, 6.6 Hz, H-25),
4.45 (2 H, m, H-1, 3), 4.92, 4.97 (each 1 H, s, -CdCH2), 5.83 (1
11, target compound 6b (12.3 mg, 0.0307 mmol) was obtained
from 39 (28.2 mg, 0.0448 mmol) in 69% yield.
H, d, J = 11.1 Hz, H-7), 6.21 (1 H, d, J = 11.1 Hz, H-6). 13
C
6b. 1H NMR δ 0.55 (3 H, s, H-18), 0.77 (3 H, d, J = 6.1 Hz,
H-21), 0.89 (4 H, m), 1.25 (6 H, s, H-26, 27), 4.46 (2 H, m, H-1, 3),
5.08, 5.10 (each 1 H, s, -CdCH2), 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 12.0, 13.0,
13.2, 21.6, 22.1, 23.5, 25.7, 27.6, 29.0, 29.1, 29.3, 37.2, 38.1, 40.5,
42.3, 42.5, 45.7, 45.8, 53.9, 56.4, 70.6, 71.2, 71.8, 107.7, 115.3,
124.2, 130.4, 143.4, 152.0. MS (EI) m/z (%): 444 (Mþ, 27), 161
(38), 147(45), 135 (85), 69 (100). HRMS (EI) calcd for C29H48O3
444.3603, found 444.3595. IR (neat) 3371, 2960, 2871, 1658,
1612, 1461, 1379, 1215, 1074, 1045, 910, 756 cm-1. UV (EtOH)
NMR δ -5.0, -4.9, -4.8 (2 carbons), 11.9, 13.0, 13.2, 18.1, 18.2,
21.5, 22.1, 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 27.3, 27.7,
28.7, 31.3, 37.2, 38.5, 40.7, 41.9, 45.6, 47.6, 53.9, 56.3, 63.5, 71.6,
72.5, 106.2, 116.3, 122.4, 132.8, 141.1, 152.9. MS (EI) m/z (%):
644 (Mþ, 1), 512 (24), 366 (20), 73 (100). HRMS (EI) calcd for
C39H72O3Si2 644.5020, found 644.5015. IR (neat) 3352, 2952,
2927, 2883, 2856, 1612, 1461, 1251, 1101, 1070, 835, 775 cm-1
.
UV (hexane) λmax 246, 254, 264 nm.
22S-Ethyl-2-methylidene-19,26,27-trinor-1r,25-dihydroxyvi-
tamin D3 (5b). In a similar manner to that for the synthesis of 3a
from 11, target compound 5b (15.1 mg, 0.0366 mmol) was
obtained from 35 (36.5 mg, 0.0561 mmol) in 65% yield.
5b. 1H NMR δ 0.55 (3 H, s, H-18), 0.77 (3 H, d, J = 6.1 Hz,
H-21), 0.88 (4 H, m), 3.63 (2H, m, H-25), 4.46 (2 H, m, H-1, 3),
5.08, 5.10 (each 1 H, s, -CdCH2), 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 12.0, 13.0,
13.2, 21.5, 22.2, 23.5, 27.3, 27.7, 29.0, 31.2, 37.2, 38.1, 40.5, 41.9,
45.7, 45.8, 53.9, 56.4, 63.4, 70.6, 71.8, 107.7, 115.3, 124.2, 130.4,
143.3, 151.9. MS (EI) m/z (%): 416 (Mþ, 41), 315 (30), 297 (28),
251 (19), 161 (45), 147(61), 135 (100), 69 (84). HRMS (EI) calcd
for C27H44O3 416.3291, found 416.3310. IR (neat) 3338, 2945,
2871, 1652, 1612, 1442, 1380, 1072, 1045, 912, 756 cm-1. UV
(EtOH) λmax 246, 254, 263 nm
Methyl (4S)-5-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylidene-9,10-secoestra-5,7-dien-17-yl]-4-ethylhexan-
oate (37). To a solution of aldehyde 33 (95 mg, 0.148 mmol) in
MeOH (10 mL) was added KOH (36 mg, 0.546 mmol) and then
iodine (55.5 mg, 0.219 mmol) at 0 °C, and the mixture was stirred
for 1 h. The reaction was quenched with 10%Na2SO3, 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 37 (67.2 mg, 0.1000 mmol, 68%).
λ
max 246, 254, 263 nm.
Competitive Binding Assay, Human VDR. The human recom-
binant VDR ligand-binding domain (LBD) was expressed as an
N-terminal GST-tagged protein in E. coli BL21 (DE3) pLys S
(Promega).18 The cells were lysed by sonication. The super-
natants were diluted approximately 500 times in 50 mM Tris
buffer (100 mM KCl, 5 mM DTT, 0.5%CHAPS, pH 7.5) con-
taining bovine serum albumin (100 μg/mL). Binding to GST-
hVDR-LBD was evaluated according to the procedure re-
ported.35 The receptor solution (570 μL) in an assay tube was
incubated with [3H]-1,25-(OH)2D3 (specific activity, 5.85 TBq/
mmol, ca. 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]-1,25-(OH)2D3 were separated by treating with
dextran-coated charcoal for 30 min at 4 °C. The assay tubes were
centrifuged at 1000gfor 10 min. The radioactivity of the supernatant
was counted. Nonspecific binding was subtracted. These experi-
ments 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 (50-GGTTCAc-
gaGGTTCA, SPPx3-TK-Luc), a wild-type 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.10
After 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). Transactivation measured by the luciferase activity was
normalized with the internal control. All experiments were
done in triplicate.
RXR and SRC-1 Recruitment. Recruitment of RXRR and
SRC-1 to VDR by synthetic vitamin D analogues and 1,25-
(OH)2D3 (1) were assessed using Cos7 cells. The activities were
evaluated by a dual luciferase assay using VP16-VDR expres-
sion plasmid (pCMX-VP16-hVDR), RXRR or SRC-1 expres-
sion plasmid (GAL4-RXRR or GAL4-SRC-1), a reporter
plasmid containing four copies of GAL4 response element
(MH100 ꢀ 4-TK-Luc), and the internal control plasmid con-
taining sea pansy luciferase expression construct (pRL-CMV).
All experiments were carried out in triplicate.
Graphical Manipulations and Ligand Docking. Graphical
manipulations were performed using SYBYL 8.0 (Tripos, St.
Louis, MO). The atomic coordinates of the crystal structure of
rVDR-LBD complexed with 1 were retrieved from Protein Data
Bank (PDB) (entry 2ZLC).31 22-Butyl vitamin D analogues (6c,
5c) were docked into the ligand-binding pocket using Surflex
Dock 2.0 (Tripos, St. Louis).
37. 1H NMR δ 0.02, 0.04, 0.06, 0.08 (each 3 H, s, SiMe), 0.54
(3 H, s, H-18), 0.78 (3 H, d, J = 6.2 Hz, H-21), 0.86, 0.89 (each 9
H, s, t-Bu), 2.82 (1 H, m, H-9), 3.67 (3 H, m, -COOMe), 4.43 (2
H, m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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
-5.0, -4.9, -4.8 (2 carbons), 11.9, 13.0 (2 carbons), 18.1, 18.2,
21.3, 22.1, 23.4, 25.7 (3 carbons), 25.8 (3 carbons), 26.4, 27.7,
28.7, 32.8, 37.0, 38.5, 40.6, 41.8, 45.6, 47.6, 51.4, 53.8, 56.3, 71.6,
72.5, 106.2, 116.1, 122.3, 132.8, 141.0, 152.9, 174.5. MS (EI) m/z
(%): 672 (Mþ, 4), 540 (61), 366 (39), 234 (18), 147 (26), 73 (100).
HRMS (EI) calcd for C40H72O4Si2 672.4969, found 672.4974.
IR (neat) 2952, 2927, 2885, 2856, 1741, 1658, 1620, 1461, 1251,
1101, 1070, 935, 896, 835, 775, 667 cm-1. UV (hexane) λmax 246,
254, 264 nm.
(5S)-6-[(1R,3R,7E,17β)-1,3-Bis{[tert-butyl(dimethyl)silyl]oxy}-
2-methylidene-9,10-secoestra-5,7-dien-17-yl]-5-ethyl-2-methyl-
heptan-2-ol (39). In a similar manner to that for the synthesis of
12 from 10, target compound 39 (29.8 mg, 0.0443 mmol) was
obtained from 37 (33.9 mg, 0.0505 mmol) in 88% yield.
39. 1H NMR δ 0.03, 0.05, 0.07, 0.08 (each 3 H, s, SiMe), 0.55
(3 H, s, H-18), 0.77 (3 H, d, J = 5.9 Hz, H-21), 0.86, 0.89 (each
9 H, s, t-Bu), 1.22 (6 H, s, H-26, 27), 2.80 (1 H, m, H-9), 4.42 (2 H,
m, H-1, 3), 4.92, 4.96 (each 1 H, s, -CdCH2), 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 -5.0,
-4.9, -4.8 (2 carbons), 11.9, 13.0, 13.3, 18.1, 18.2, 21.6, 22.1,
23.4, 25.7(3 carbons), 25.8 (4 carbons), 27.7, 28.8, 29.1, 29.3, 37.3,
38.5, 40.7, 42.3, 42.5, 45.6, 47.6, 53.9, 56.3, 71.2, 71.6, 72.5, 106.2,
116.1, 122.4, 132.7, 141.2, 152.9. MS (EI) m/z (%): 672 (Mþ, 1),
540 (24), 522 (12), 366 (20), 234 (13), 147(13), 73 (100). HRMS
(EI) calcd for C41H76O3Si2 672.5333, found 672.5341. IR (neat)
3357, 2954, 2927, 2891, 2856, 1650, 1612, 1461, 1373, 1251, 1101,
1072, 835, 775 cm-1. UV (hexane) λmax 246, 254, 264 nm.
22S-Ethyl-2-methylidene-19-nor-1r,25-dihydroxyvitamin D3
(6b). In a similar manner to that for the synthesis of 3a from
Acknowledgment. We are grateful to Prof. Hirokazu Ta-
mamura of Tokyo Medical and Dental University for support
of this project. Part of this work was supported by a Grant-
in-Aid for Scientific Research (no. 20590108) from the Ministry

5826 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Sakamaki et al.
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