2-Methylene 19-nor-25-dehydro-1α-hydroxyvitamin D3 26,23-lactones: Synthesis, biological activities and molecular basis of passive antagonism

Article abstract of DOI:10.1016/j.bmc.2007.09.017

To investigate the molecular mechanism of vitamin D receptor (VDR) antagonists having no structurally bulky group interfering with helix 12 of the ligand-binding domain of the VDR, we have synthesized four diastereomers at C(20) and C(23) of 19-nor-1α-hydroxyvitamin D₃ 25-methylene-26,23-lactone bearing a 2MD-type A-ring. All four analogs showed significant VDR affinity. Transactivation was tested by using Cos7 cells and HEK293 cells. In both types of cells, LAC67a showed little transactivation potency and inhibited the activation induced by the natural hormone concentration-dependently, indicating that LAC67a works as an antagonist for the VDR in these cells. LAC67b, LAC82a and LAC82b similarly acted as VDR antagonists in Cos7 cells, but in HEK293 cells they behaved as potent VDR agonists. Docking of four lactones into the VDR-LBD, followed by structural analysis, demonstrated that each lactone lacks the hydrophobic interaction with helix12 necessary for maintaining the active conformation of the VDR, indicating that these lactones are passive-type antagonists. Furthermore, each docking structure explained the characteristic transactivation profiles of the four lactones. On the basis of our present findings, we suggest that the ligand acts as an agonist if there are appropriate coactivators in the cells to bind to the looser VDR-ligand complex, and as an antagonist if there are no such appropriate coactivators. The molecular basis of the passive antagonism is discussed in detail.

Full text of DOI:10.1016/j.bmc.2007.09.017

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 457–473
2-Methylene 19-nor-25-dehydro-1a-hydroxyvitamin D3
26,23-lactones: Synthesis, biological activities and molecular
basis of passive antagonism
Nobuko Yoshimoto,a,c Yuka Inaba,a,b,c Sachiko Yamada,b,* Makoto Makishima,b
Masato Shimizua and Keiko Yamamotoc,*
aSchool of Biomedical Sciences, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
bDepartment of Biochemistry, Nihon University School of Medicine, 30-1 Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japan
cLaboratory of Drug Design and Medicinal Chemistry, Showa Pharmaceutical University, 3-3165 Higashi-tamagawagakuen,
Machida, Tokyo 194-8543, Japan
Received 21 August 2007; revised 10 September 2007; accepted 11 September 2007
Available online 14 September 2007
Abstract—To investigate the molecular mechanism of vitamin D receptor (VDR) antagonists having no structurally bulky group
interfering with helix 12 of the ligand-binding domain of the VDR, we have synthesized four diastereomers at C(20) and C(23)
of 19-nor-1a-hydroxyvitamin D3 25-methylene-26,23-lactone bearing a 2MD-type A-ring. All four analogs showed significant
VDR affinity. Transactivation was tested by using Cos7 cells and HEK293 cells. In both types of cells, LAC67a showed little trans-
activation potency and inhibited the activation induced by the natural hormone concentration-dependently, indicating that LAC67a
works as an antagonist for the VDR in these cells. LAC67b, LAC82a and LAC82b similarly acted as VDR antagonists in Cos7 cells,
but in HEK293 cells they behaved as potent VDR agonists. Docking of four lactones into the VDR–LBD, followed by structural
analysis, demonstrated that each lactone lacks the hydrophobic interaction with helix12 necessary for maintaining the active con-
formation of the VDR, indicating that these lactones are passive-type antagonists. Furthermore, each docking structure explained
the characteristic transactivation profiles of the four lactones. On the basis of our present findings, we suggest that the ligand acts as
an agonist if there are appropriate coactivators in the cells to bind to the looser VDR–ligand complex, and as an antagonist if there
are no such appropriate coactivators. The molecular basis of the passive antagonism is discussed in detail.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1a,25-Dihydroxyvitamin D3[1,25-(OH)2D3, 1] plays an
important role in regulation of calcium and bone metab-
olism, cellular differentiation and proliferation, and im-
mune responses.1 The majority of these actions are
mediated by the vitamin D receptor (VDR), which is a
member of the nuclear receptor superfamily and func-
tions as a ligand-dependent transcriptional factor.1,2
Upon ligand binding, the VDR undergoes conforma-
tional change to form the AF2 surface to allow binding
of a coactivator.3 In the absence of the ligand, a core-
pressor binds to the AF2 surface.3,4
Two types of nuclear receptor antagonists have been re-
ported. The selective estrogen receptor modulators
(SERM), 4-hydroxytamoxifen and raloxifene, constitute
the first group of antagonists, which have a bulky substi-
tuent that creates spatial restriction with residues on he-
lix 12 (H12) of estrogen receptor a in the active
conformation and prevents the H12 from occupying
the correct position on the AF2 surface.5,6 Ligands of
this type are called active antagonists. The other group
of antagonists has no such bulky substituent. For exam-
ple, progesterone works as an antagonist of the mineral-
ocorticoid receptor (MR), although this compound is
easily accommodated in the ligand-binding pocket
(LBP).7 Recently, Bledsoe et al. have solved and re-
ported the X-ray crystal structure of the MR ligand-
binding domain (LBD) complexed with progesterone,
in which progesterone interacts poorly with helix 3
(H3) and helix 10/11 (H10/11) of MR–LBD.8 This infe-
rior interaction results in insufficient interactions among
Keywords: Vitamin D receptor; Antagonist; Partial agonist; Transac-
tivation; Nuclear receptor; Vitamin D; Passive antagonist.
*
Corresponding authors. Tel./fax: +81 42 721 1580; e-mail addresses:
yamada.vd@image.ocn.ne.jp; yamamoto@ac.shoyaku.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.09.017

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N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
H3, helix 5 (H5), H10/11, and H12 mediated by the li-
gands, so that the correct AF2 surface cannot form,
and thus progesterone behaves as an antagonist.7,8 Li-
gands of this type are known as passive antagonists.
antagonist. These are analogs of TEI compounds having
methylene lactone on the side chain. The 2-methylene
19-nor structure of the A-ring was selected because the
19-nor structure is known to be more stable than the
conjugated triene structure of the hormone and is easily
synthesized. Insertion of a methylene group into C(2)
was expected to increase the VDR affinity and the bio-
logical activity, as in the case of 2MD compound.12
Since no VDR antagonist with a 20-epi-configuration
had yet been reported, LAC82a and LAC82b were also
designed to evaluate the effects of the stereochemistry
at C(20). In this report, we describe the synthesis and
biological activities of four diastereomers of vitamin
D3 derivatives having methylene lactone on the side
chain (LAC67a, LAC67b, LAC82a, and LAC82b), and
their interaction with the receptor. In addition, the
molecular basis of these lactones acting as passive antag-
onists was investigated by docking analysis.
Thousands of vitamin D analogues have been synthe-
sized to date. Most of them act as VDR agonists,
whereas only two known types act as VDR antagonists.
As active antagonists, ZK compounds such as
ZK168281 2 have been developed by the Schering group
(Chart 1).9 These compounds have a bulky ester group
at the side chain terminal that causes spatial restriction
with H12 of the active conformation of the VDR. The
other antagonists are TEI compounds, such as
TEI9647 3b and TET9648 3a, developed by the Teijin
group.10,11 They have a methylene lactone on the side
chain, and no bulky structure directed toward H12. In
the course of our studies to develop VDR antagonists
for treatment of metabolic bone disease such as Paget
disease11b and to investigate the molecular basis of the
VDR antagonism, we designed and synthesized four
diastereomers of 2-methylene 19-nor-25-dehydro-1a-
hydroxyvitamin D3 26,23-lactone (LAC67a, LAC67b,
LAC82a, and LAC82b) as the candidates of passive
1. Synthesis
Four methylene lactone compounds (LAC67a, LAC67b,
LAC82a, and LAC82b) were synthesized using the Wit-
tig–Horner reaction of Grundmann’s ketone derivative
9/10 with A-ring phosphine oxide 4g derived from (ꢀ)-
quinic acid, followed by 2-methylenation and subse-
quent methylene lactonization (Scheme 1).
OH
O
OEt
OH
Phosphine oxide 4g was derived from (ꢀ)-quinic acid in
59% overall yield by a modification of DeLuca’s meth-
od. Conversion of (ꢀ)-quinic acid to methyl ester 4a
and conversion of keto compound 4c to phosphine
oxide 4g have been reported by DeLuca’s group13a
and Shimizu’s group,13b respectively. We reduced
methyl ester 4a with NaBH4 instead of DIBAL14 to give
4b, which was oxidatively cleaved to give keto com-
pound 4c. Phosphine oxide 4g was combined with
Grundmann’s ketone derivative 9, derived from vitamin
D2, to give the 23-cyanide 11 at 80% yield. Removal of
the protecting group followed by oxidation of the 2-hy-
droxyl group of 13 afforded the corresponding 2-keto
compound 15, which was then treated with Wittig re-
agent to give the 2-methylene compound 17. The cyano
group of 17 was reduced by DIBAL to give the aldehyde
19. This aldehyde was reacted with an organo-chro-
mium complex, prepared from allylic bromide and
low-valent chromium (II) derived from CrCl3 by reduc-
tion with LiAlH4, to give the lactone derivative 21.15
This method is a one-step allylation–lactonization reac-
tion that was developed by Kittaka’s group.16 Methy-
lene lactone 21 was obtained as a 2:3 mixture in terms
of the stereochemistry at C23. Since treatment of lactone
21 with n-Bu4NF gave a complex mixture of various
compounds, TBDMS groups protecting the two hydro-
xyl groups were removed by acid-hydrolysis to give a
mixture of LAC67a and LAC67b, which were then sep-
arated by HPLC. Stereochemistry at C(23) of LAC67a
and LAC67b was determined by the Kusumi–Mosher
method17 described below.
HO
OH
HO
OH
1:1,25-(OH)2D3
2:ZK168281
O
O
O
O
HO
OH
HO
OH
3a:TEI9648
3b:TEI9647
O
O
O
O
HO
OH
HO
OH
LAC67a:20R,23R
LAC82a:20S,23R
LAC67b:20R,23S
LAC82b:20S,23S
LAC82a and LAC82b were synthesized by the same pro-
cedure as LAC67a and LAC67b (Scheme 1).
Chart 1. Structures of 1,25-(OH)2D3 and its analogs.

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
459
O
HOOC OH
HO OH
MeOOC OH
TBSO OTBS
HOH2C OH
TBSO OTBS
a
b
c
d
TBSO
OTBS
OH
OH
OH
OH
D-(-)-Quinic Acid
4a (2 steps 87%)
4b (93%)
4c (quant.)
COOMe
O
OH
e
f
TBSO
OTBS
OTMS
TBSO
OTBS
OTMS
TBSO
OTBS
OTMS
4d (99%)
4e (90%)
4f (99%)
g
O
PPh2
CN
TBSO
OTBS
OTMS
CN
OTs
CN
4g (3 steps 82%)
h
i
VD2
j
TBSO
OTBS
OX
OH
OH
O
11:20R (X=TMS; 80%)
12:20S (X=TMS; 75%)
13:20R (X=H; 86%)
14:20S (X=H; 83%)
7:20S (98%)
8:20R (99%)
5:20S
6:20R
9:20R (92%)
10:20S (96%)
k
CN
CHO
O
O
Br
COOMe
l
n
o
TBSO
OTBS
TBSO
OTBS
TBSO
OTBS
X
15:20R (X=O; 96%)
16:20S (X=O; 99%)
17:20R (X=CH2; 94%)
18:20S (X=CH2; 89%)
19:20R (90%)
20:20S (87%)
21:20R (83%)
22:20S (82%)
m
O
O
O
O
p
+
HO
OH
HO
OH
LAC67a:20R
LAC82a:20S
LAC67b:20R
LAC82b:20S
Scheme 1. Synthetic scheme of lactones. Reagents: (a) 1—MeOH, p-TsOH; 2—TBSCl, Et3N; (b) NaBH4; (c) NaIO4; (d) TMSCl, imidazole; (e)
Me3SiCH2CO2Me, LDA; (f) DIBAL-H; (g) 1—p-TsCl, n-BuLi, then Ph2PH, n-BuLi; 2—10% H2O2; (h) KCN, DMSO; (i) TPAP, NMO, Molecular
Sieves 4A; (j) n-BuLi; (k) AcOH; (l) (COCl)2, DMSO, Et3N; (m) MeP+Ph3Brꢀ, n-BuLi; (n) DIBAL-H; (o) CrCl3, LiAlH4; (p) CSA, MeOH.

460
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
2. Determination of stereochemistry at C(23)
by 1,25-(OH)2D3 (1) concentration-dependently, indi-
cating they can work as VDR antagonists. Interestingly,
both the EC50 and IC50 of LAC67b in the transactiva-
tion assay were lower than those of LAC67a, in agree-
ment with their VDR affinity.
Stereochemistry at C(23) of each lactone was determined
by the Kusumi–Mosher method (Scheme 2). Methylene
lactone 21 was reduced by DIBAL to give a mixture of
23,26-diols 23a and 23b, which were separable by silica
gel column chromatography. The primary alcohol of
each compound was selectively protected as the pivalate.
Then, the pivalate esters (25a and 25b) were converted to
the corresponding S- and R-MTPA esters of the 23-hy-
droxyl group (27a and 28a; 27b and 28b). Chemical shift
differences between the S-MTPA ester and the corre-
sponding R-MTPA ester are shown in Figure 1a and
b. This analysis clearly indicated that the 23,26-diol
23a has a 23R-configuration. This diol 23a was con-
verted to the corresponding lactone LAC67a by oxida-
tion with MnO2, followed by removal of the TBDMS
group. HPLC analysis showed that the methylene lac-
tone derived from 23a co-migrated with LAC67a. Based
on these analyses we determined that LAC67a and
LAC67b have a 23R- and 23S-configuration, respec-
tively. In 20-epi-compounds, each stereochemistry at
C(23) of LAC82a and LAC82b was determined to be
R and S, respectively, according to the same method
(Fig. 1c and d).
Interestingly, LAC82a and LAC82b showed significant
agonistic activity that is 34% and 43% efficacy compared
with the hormone (1), respectively (Fig. 3b), and their
EC50 values were 6 and 10 nM. These results clearly
indicate that LAC82a and LAC82b are also partial ago-
nists. Inversion of stereochemistry from 20R to 20S en-
hanced transactivation in terms of both efficacy and
potency (EC50). As expected, both compounds reduced
the transactivation induced by hormone (1) to their
own efficacy of maximal activation (Fig. 3d). LAC82b
with the 23S-configuration showed stronger antagonis-
tic activity than LAC82a with the 23R-configuration,
in agreement with LAC67b and TEI9647 3b with the
23S-configuration.10,21 We found that 20-epimerization
(from LAC67a to LAC82a; from LAC67b to LAC82b)
enhanced both agonistic and antagonistic activities.
To evaluate the effects of cell type, we performed the
same transactivation assay using HEK293 cells. As also
observed in Cos7 cells, LAC67a had little transactiva-
tion potency, whereas LAC67b showed significant ago-
nistic activity (Fig. 4a). LAC67a, but not LAC67b,
significantly reduced the transactivation stimulated by
1,25-(OH)2D3 (Fig. 4c). These results indicate that
LAC67a acts almost as an antagonist in HEK293 cells,
whereas LAC67b acts as a partial agonist with potent
activity. The 20-epimer lactones LAC82a and LAC82b
showed strong transactivation potency (Fig. 4b). As ex-
pected, they showed little antagonistic activity (Fig. 4d),
indicating that both behave almost as full agonists in
HEK293 cells.
3. Biological activities
Biological activities in vitro are summarized in Table 1.
Binding affinity for the VDR was evaluated by compet-
itive-binding assay using rat recombinant VDR–LBD
prepared in our laboratory (Fig. 2).18 The binding affin-
ity of LAC67b was 1/7 as potent as the natural hormone
1, while that of the 23R-isomer LAC67a was 1/200 as
potent as the hormone 1. Interestingly, both 20-epimers
LAC82a and LAC82b showed VDR-binding potency
intermediate (1/50) between LAC67a and LAC67b.
These results indicated that all four lactones are VDR
ligands having significant affinity. The finding that
LAC67b with a 23S-configuration showed stronger
VDR affinity than its 23R-isomer LAC67a is in agree-
ment with the properties of the TEI compounds,
TEI9647 3b with a 23S-configuration having stronger
affinity than its 23R-epimer TEI9648 3a.10,19 However,
this was not the case for synthetic 20-epi compounds be-
cause LAC82a and LAC82b had almost the same VDR
affinity.
4. Receptor interaction
Docking analysis was performed by the procedure de-
scribed in the Experimental section. Figure 5a shows
the X-ray crystal structure of VDR–LBD complexed
with 1,25-(OH)2D3.22 Figure 5c–f show the docking
models of LAC67a, LAC67b, LAC82a, and LAC82b
with the VDR–LBD, and their superposition is shown
in Figure 5b. As shown in Figure 5, all of these four ana-
logs, LAC67a, LAC67b, LAC82a, and LAC82b, were
accommodated in the VDR–LBP where their hydroxyl
groups at the 1a- and 3b-positions form pincer-type
hydrogen bonds with Ser237 and Arg274, and Tyr143
and Ser278, respectively. Furthermore, the carbonyl
group at C(26) forms a pincer-type hydrogen bond with
His305 and His397, as in the case of 1,25-(OH)2D3 1.
Differences from 1,25-(OH)2D3 1 were found in hydro-
phobic interactions between amino acid residues on
H12, including the tail of H11 and the ligand. While
the terminal C(26)-methyl group of 1,25-(OH)2D3 1 di-
rectly interacted with residues Val418, Phe422 on H12
and Tyr401 on H11 (Fig. 5a), the terminal methylene
group of LAC67a is rather too distant from these three
residues to interact directly (Fig. 5c). This docking mode
The ability of methylene lactones to induce transcription
of a vitamin D-responsive gene was tested using a rat
osteopontin luciferase reporter gene assay system in
Cos7 cells.20 It is well known that, in this assay, 1,25-
(OH)2D3 (1) increases luciferase activity in a concentra-
tion-dependent manner. As shown in Figure 3a,
LAC67a and LAC67b activated the VDR in a concen-
tration-dependent manner, but their efficacy of maximal
activation was only 14% and 11% of that of the hor-
mone (1), respectively, indicating that both lactones
are extremely weak partial VDR agonists. Antagonistic
activity was evaluated by inhibition of VDR stimulation
by 10 nM 1,25-(OH)2D3 (1). As shown in Figure 3c,
both compounds inhibited the transactivation induced

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
461
OPiv
OH
O
OH
c
(S)-MTPA
27a:20R (78%)
29a:20S (quant.)
b
OPiv
OH
25a:20R (27%)
26a:20S (45%)
TBSO
OTBS
d
OPiv
O
(R)-MTPA
23a:20R (27%)
24a:20S (32%)
28a:20R (85%)
30a:20S (42%)
a
21:20R
22:20S
+
OH
OH
OPiv
O
c
(S)-MTPA
b
OPiv
27b:20R (71%)
29b:20S (quant.)
OH
25b:20R (44%)
26b:20S (24%)
TBSO
OTBS
d
OPiv
O
(R)-MTPA
23b:20R (23%)
24b:20S (30%)
28b:20R (79%)
30b:20S (47%)
e
e
f
23a
21a:23R
(83%)
LAC67a
(65%)
f
24b
22b:23S
LAC82b
(87%)
(51%)
Scheme 2. Synthesis of MTPA esters. Reagents: (a) DIBAL-H; (b) PivCl, pyridine; (c) R-MTPACl, Et3N, DMAP; (d) S-MTPACl, Et3N, DMAP; (e)
MnO2; (f) CSA, MeOH.
-0.04
-0.04
a
+0.09
b
-0.04
O
-0.01
O
+0.13
+0.01
OPiv
OPiv
+0.08
-0.03
+0.17
+0.17
-0.07
-0.07
MTPA
MTPA
+0.02
MTPA plane
MTPA plane
27b-28b
27a-28a
c
+0.04
+0.04
OPiv
d
-0.1
+0.03
O
OPiv
+0.04
-0.15
-0.16
-0.15
+0.07
+0.06
O
MTPA
MTPA
-0.02
+0.02
MTPA plane
MTPA plane
29a-30a
29b-30b
Figure 1. Determination of stereochemistry at C(23). Chemical shift differences.

462
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
Table 1. Biological activities of synthetic lactone compounds
VDR bindinga EC50 (M)
Cos7b
Transact.d EC50 (M)
HEK293c
Inhibit.e IC50 (M)
Transact.d EC50 (M)
Inhibit.e IC50 (M)
1,25-(OH)2D3 (1)
LAC67a
LAC67b
1 · 10ꢀ10
2 · 10ꢀ8
7 · 10ꢀ10
5 · 10ꢀ9
5 · 10ꢀ9
5 · 10ꢀ10
—
1 · 10ꢀ9
2 · 10ꢀ7
1 · 10ꢀ9
2 · 10ꢀ8
1.5 · 10ꢀ8
—
1.5 · 10ꢀ7
2.5 · 10ꢀ8
6 · 10ꢀ9
1 · 10ꢀ6
3 · 10ꢀ7
3 · 10ꢀ7
1 · 10ꢀ7
3 · 10ꢀ7
ND
ND
LAC82a
LAC82b
1 · 10ꢀ8
ND
a Competitive binding of 1,25-(OH)2D3 (1) and synthetic lactones to the rat vitamin D receptor. The EC50 values are derived from dose–response
curves and represent the analog concentration required for 50% displacement of the radio-labeled 1,25-(OH)2D3 from the receptor protein. The
experiments were carried out in duplicate.
b Transactivation was 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.20
c Transactivation potencies were evaluated using a full-length hVDR expression plasmid (pCMX-hVDR) and a reporter plasmid containing three
copies of the mouse osteopontin VDRE (SPPx3-TK-Luc) in HEK293 cells as described previously.31
d Agonistic activity. The EC50 values are derived from dose–response curves and represent the analog concentration capable of inducing 50%
maximal transactivation response. All experiments were carried out in triplicate.
e Antagonistic activity. The IC50 values are derived from dose–response curves and represent the analog concentration capable of reducing 50%
maximal transactivation response.
chain and Val418, Phe422, and Tyr401 at the C-terminal
of the VDR is within 4.7 A.22,26–30 These results indicate
˚
120
100
1,25D3
that intimate interaction is needed in order to form a
transcriptionally active conformation of the VDR.
Docking of synthetic lactones into the VDR–LBD and
structural analysis yielded insights into the molecular
basis of VDR passive antagonism.
LAC67a
LAC67b
LAC82a
LAC82b
80
60
40
20
As shown in Figure 5, the hydroxyl groups at the 1a-
and 3b-positions of the four lactones form pincer-type
hydrogen bonds with Ser237 and Arg274, and Tyr143
and Ser278, respectively, and the carbonyl group at
C(26) forms a pincer-type hydrogen bond with His305
and His397, as is the case for 1,25-(OH)2 D3 1. Con-
struction of these complete six hydrogen bonds is one
of the most important reasons why lactones can bind
to the VDR. However, docking models of the four lac-
tones demonstrated that hydrophobic interaction with
the C-terminal of the VDR is not enough for full agonist
action. In the case of LAC67a, all of the three residues
Tyr401, Val418, and Phe422 are far from the side chain
of this ligand, as shown in Figure 5c, indicating the loos-
er packing of H12 including the tail of H11. We con-
cluded that the lack of these hydrophobic interactions
is the reason why LAC67a works as an antagonist both
in Cos7 as well as HEK293 cells. The docking model of
LAC67b indicates that LAC67b has, in addition to six
complete hydrogen bonds, appropriate hydrophobic
interactions with Tyr401 and Val418, but not Phe422.
This proper interaction with Tyr401 and Val418 would
be one reason why LAC67b works as a potent agonist
in HEK293 cells. HEK293 cells, but not Cos7 cells,
might have an appropriate coactivator that can bind
to the AF2 surface of the VDR docked with LAC67b.
0
10-11 10-10 10-9 10-8 10-7 10-6 10-5
Concentration (M)
Figure 2. Competitive-binding assay of synthetic lactones.
suggests that LAC67a can bind to VDR but cannot
form a transcriptionally active conformation in which
H12 folds back ligand-dependently to form the AF2-
surface. In the complex of LAC67b, Tyr401 and
Val418 occupy the appropriate positions, but Phe422
is located far from the ligand (Fig. 5d).
20-epi-Compounds, LAC82a and LAC82b, also showed
moderate contacts with Tyr401 and Val418 and weak
contact with Phe422 (Fig. 5e and f). Compared with
LAC67a and LAC67b, C(22)H2 of the 20-epi-lactones
LAC82a and LAC82b interacts more intimately with
Val300. This close contact, similar to that observed in
the complex of 20-epi-1,25-(OH)2D3, might be the rea-
son why 20-epi-compounds have more potent activity
than 20-normal compounds.23–25
This implies that LAC67b might work as a tissue- or
cell-selective agonist for the VDR. Recently, we have
clarified the level of CYP24A1 mRNA in several cell
types treated with LAC67a and LAC67b by real time
PCR, and suggested that LAC67b might act as a selec-
tive VDR modulator (SVDRM).31 SVDRMs would
5. Discussion
Almost all of the X-ray crystal structures of the VDR–
LBD complexed with a ligand revealed that the distance
between the terminal alkyl group of the vitamin D side

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
463
1.2
1
a
b
3
0.8
0.6
0.4
0.2
0
2
1
0
D
8
vehicle
10
9
8
7
6
5
10
9
8
7
6 5
vehicle D 10
9
8
7
6
5
10
9
8
7
6 5
8
Lac67a
-log [M]
Lac82a
Lac67b
Lac82b
-log [M]
1.2
1
c
d
3
2
0.8
0.6
0.4
1
0.2
0
0
D
8
D
8
vehicle
10
9
8
7
6
5
10
9
8
7
6
5
vehicle
10
9
8
7
6
5
10
9
8
7
6 5
Lac82a
Lac82b
Lac67a
Lac67b
+
+
1,25-(OH)2D3
1,25-(OH)2D3
-log [M]
-log [M]
Figure 3. Transactivation of synthetic lactones in Cos7 cells. The activities 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.20,25 (a) agonistic activity of
LAC67a and LAC67b, (b) agonistic activity of LAC82a and LAC82b, (c) antagonistic activity of LAC67a and LAC67b in the presence of 10 nM 1,25-
(OH)2D3, (d) antagonistic activity of LAC82a and LAC82b in the presence of 10 nM 1,25-(OH)2D3.
act like as SERMs. Certain constituents in each cell are
important for determining cell specificity. For example,
the antagonistic function of ZK159222 has been shown
to depend on the cell-specific ratio between VDR and
RXR proteins.32 Kato’s group have reported that deple-
tion of serum from the culture medium converted
TEI9647 3b from an antagonist to an agonist of VDR-
mediated transactivation, whereas it retained antagonis-
tic activity in the presence of serum.33 Taken together
with our previous report, these findings indicate that
LAC67a is a VDR antagonist while LAC67b might be
a SVDRM.31
The mechanism of the antagonistic activity of the meth-
ylene lactone compound TEI9647 3b has been reported
by the Ishizuka and Norman group. They reported that
the importance of Cys403 and Cys410 of human VDR
for expression of the antagonistic activity of TEI9647
3b suggests a mechanism involving Michael-type addi-
tion of these cysteines to the methylene lactone.34,35
On the other hand, the same group, using a ligand ex-
change assay, demonstrated that TEI9647 3b bound to
VDR is freely exchanged with 1,25-(OH)2D3
1
in vitro.36 Since LAC67b has completely the same struc-
ture at the side chain as TEI9647 3b, the mechanism of
its antagonistic action is thought to be quite similar to
that of the latter. LAC67b acts as a VDR antagonist
in Cos7 cells transfected with human VDR, whereas this
compound functions as a potent VDR agonist in
HEK293 cells transfected with human VDR. The latter
fact that LAC67b can function as a potent VDR agonist
suggests that this compound does not form a covalent
bond with the human VDR. Therefore, we suggest that
methylene lactones such as LAC67b and TEI9647 3b in-
hibit the transactivation as passive antagonists, and not
via covalent bond formation.
Docking models of LAC82a and LAC82b show six com-
plete hydrogen bonds and moderate interactions with
Tyr401 and Val418, but not Phe422. Since these hydro-
phobic interactions are insufficient, the activity of these
compounds might depend on the cellular environment.
Therefore, the ligand would act as an agonist if there
are appropriate coactivators in that cell, whereas it
would act as an antagonist if proper proteins are lack-
ing. These observations demonstrate that the four syn-
thetic lactones are passive antagonists.

464
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
50
a
b
3000
2000
1000
40
30
20
10
0
0
vehicle
D
8
9
8
7
6
9
8
7
6
vehicle
D
8
9
8
7
6
9
8
7
6
Lac67a
-log [M]
Lac82a
-log [M]
Lac67b
Lac82b
1.2
1
c
d
40
30
20
0.8
0.6
0.4
10
0
0.2
0
vehicle
D
8
8
7
6
8
7
6
vehicle
D
8
8
7
6
8
7
6
Lac67a
Lac82a
Lac67b
Lac82b
+
+
1,25-(OH)2D3
1,25-(OH)2D3
-log [M]
-log [M]
Figure 4. Transactivation of four methylene lactones in HEK293 cells. Transactivation potencies were evaluated using a full-length hVDR expression
plasmid (pCMX-hVDR) and a reporter plasmid containing three copies of the mouse osteopontin VDRE (SPPx3-TK-Luc) in HEK293 cells as
described previously.31 (a) agonistic activity of LAC67a and LAC67b, (b) agonistic activity of LAC82a and LAC82b, (c) antagonistic activity of
LAC67a and LAC67b in the presence of 10 nM 1,25-(OH)2D3, (d) antagonistic activity of LAC82a and LAC82b in the presence of 10 nM 1,25-
(OH)2D3.
6. Conclusions
were recorded on a JASCO FT/IR-300E spectrometer.
UV spectra were recorded on a BECKMAN DU7500
spectrophotometer. All air and moisture sensitive reac-
tions were carried out under argon atmosphere.
We synthesized four methylene lactone analogs and
evaluated their affinity for the VDR as well as their ago-
nistic and antagonistic activities. Based on data for their
biological activities and a docking study, we concluded
that these methylene lactones are passive antagonists
lacking hydrophobic interactions with H12 of the
VDR. We have already synthesized and reported an-
other type of antagonist having a bulky substituent of
the adamantine ring at the side chain. Further experi-
ments are now in progress to study the molecular mech-
anism of VDR antagonism using these two types of
synthetic antagonist.
7.1. (3R,5R)-3,5-Bis{tert-butyl(dimethyl)silyl]oxy}-1-
(hydroxymethyl)-1,4-cyclohexanediol (4b)
To a solution of methyl ester 4a (10.4 g, 24 mmol) in
EtOH (60 mL) was added NaBH4 (2.8 g, 75 mmol),
and the mixture was stirred at 0 ꢁC for 5 h. The reaction
was quenched with H2O and brine at 0 ꢁC and was ex-
tracted with AcOEt. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (40 g, hexane/
AcOEt = 17:3) to give alcohol 4b (9.1 g, 93.3%) as a col-
orless solid.
7. Experimental
1H NMR spectra were recorded in CDCl3 at 400 MHz
and chemical shifts are reported as d units relative to tet-
ramethylsilane or solvent signal as an internal standard.
13C NMR spectra were recorded at 100 MHz. High and
low resolution mass spectra were obtained on a JEOL
JMS-AX505HA spectrometer at 70 eV. Relative intensi-
ties are given in parentheses in low mass. IR spectra
4b 1H NMR d 0.11 and 0.12 and 0.15 and 0.16 (each 3H,
s, SiMe), 0.90 and 0.91 (each 9H, s, t-Bu), 1.34 (1H, dd,
J = 12.9, 10.9 Hz), 1.50 (1H, dd, J = 14.5, 2.4 Hz), 2.00
(2H, m), 2.12 (1H, dd, J = 8.2, 4.7 Hz), 2.28 (1H, d,
J = 2.9 Hz), 3.33 (2H, m, CH2OH), 3.41 (1H, dd,
J = 11.0, 4.7 Hz), 4.11 (1H, ddd, J = 10.7, 9.0, 4.7 Hz),
4.32 (1H, m), 4.53 (1H, br s, OH).

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
465
Figure 5. Docking models of LAC67a, LAC67b, LAC82a and LAC82b into the VDR–LBD. Hydrogen bonds and hydrophobic interactions are
depicted as dotted red and green lines, respectively. (a) 1,25-(OH)2D3 and VDR–LBD complex. 26-Methyl group of 1,25-(OH)2D3 makes intimate
van der Waals contacts with Tyr401 (Y401), Val418 (V418), and Phe422 (F422). (b) Superposition of four docking models. LAC67a, LAC67b,
LAC82a and LAC82b in the VDR–LBP are drawn in blue, cyan, green, and yellow color, respectively. (c) LAC67a and VDR–LBD complex. (d)
LAC67b and VDR–LBD complex. (e) LAC82a and VDR–LBD complex. f. LAC82b and VDR–LBD complex.
7.2. (3R,5R)-3,5-Bis{tert-butyl(dimethyl)silyl]oxy}-4-
hydroxycyclohexanone (4c)
brine, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (30 g, hexane/
AcOEt = 23:2) to give ketone 4c (900.0 mg, quant.) as
a colorless solid.
To a solution of alcohol 4b (951.6 mg, 2.34 mmol) in
MeOH (30 mL) was added sodium periodate-saturated
water (8 mL), and the mixture was stirred at 0 ꢁC for
3 h. The reaction was poured into brine and was ex-
tracted with AcOEt. The organic layer was washed with
4c 1H NMR d 0.06 (6H, s, SiMe · 2), 0.08 and 0.09 (each
3H, s, SiMe), 0.85 and 0.90 (each 9H, s, t-Bu), 2.25 (1H,
m), 2.45 (1H, m), 2.60 (1H, m), 2.77 (1H, dd, J = 14.4,

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N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
3.3 Hz), 3.80 (1H, m, H-4), 4.27 (2H, m, H-3, 5). 13C
NMR d ꢀ5.1, ꢀ4.94, ꢀ4.88, ꢀ4.7, 17.9, 18.0, 25.6 (3
carbons), 25.7 (3 carbons), 44.3, 46.0, 69.4, 70.0, 72.6,
207.3.
7.6. (3S)-3-[(7aR)-7a-Methyl-4-oxooctahydro-1H-inden-
1-yl]butanenitrile (10)
Compound 10 was obtained from 8 by the same proce-
dure as described for 9 (yield 95.8%) as a colorless solid.
7.3. (3R)-3-[(4S,7aR)-4-Hydroxy-7a-methyloctahydro-1H-
inden-1-yl]butanenitrile (7)
10 1H NMR d 0.62 (3H, s, H-18), 1.08 (3H, d,
J = 6.6 Hz, H-21), 2.49 (1H, dd, J = 11.6, 7.7 Hz, H-
14). 13C NMR d 12.9, 19.0, 19.7, 24.0, 24.4, 27.1, 32.0,
38.6, 40.8, 49.5, 54.8, 61.6, 118.5, 211.2. MS m/z (%):
219 (M+, 65), 204 (100), 191 (28), 176 (80), 163 (45),
124 (58), 96 (65).
To a solution of tosylate 5 (130.1 mg, 0.36 mmol) in dry
DMSO (500 lL) was added KCN (46.5 mg, 0.71 mmol),
and the mixture was stirred at 70 ꢁC for 1.5 h. The reac-
tion 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 = 1:1) to give cyanide 7 (78.1 mg, 98.2%) as a
colorless solid.
7.7. (3R)-3-{(1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)silyl]-
oxy}-2-[(trimethylsilyl)oxy]-9,10-secoestra-5,7-dien-17-
yl}butanenitrile (11)
To a solution of phosphine oxide 4 (1.18 g, 1.80 mmol, a
2:3 mixture of diastereomers at C(4)) in dry THF
(10 mL) at ꢀ78 ꢁC was added slowly n-BuLi (1.59 M
hexane solution, 1.1 mL, 1.79 mmol), and the resulting
dark orange solution was stirred for 15 min. To this col-
ored solution was added a solution of ketone 9
(258.7 mg, 1.18 mmol) in dry THF (5 mL), the reaction
mixture was stirred for 1.5 h at same temperature, and
then the mixture was allowed to warm to 0 ꢁC for 2 h.
The reaction was quenched with saturated NH4Cl aq
solution at 0 ꢁC and was extracted with AcOEt. The or-
ganic layer was washed with brine, dried over MgSO4,
and evaporated. The residue was chromatographed on
silica gel (20 g, hexane/AcOEt = 19:1) to give compound
11 (683.7 mg, 79.8%, a 2:3 mixture of C(2) epimers) as
colorless oil.
7 1H NMR d 0.96 (3H, s, H-18), 1.15 (3H, d, J = 6.6 Hz,
H-21), 2.25 (1H, dd, J = 16.7, 6.9 Hz, H-22), 2.35 (1H,
dd, J = 16.7, 3.8 Hz, H-22), 4.09 (1H, m, H-8). 13C
NMR d 13.8, 17.5, 19.3, 22.6, 24.8, 27.2, 33.2, 33.7,
40.2, 42.1, 52.5, 55.3, 69.1, 119.1. MS m/z (%): 221
(M+, 15), 206 (45), 188 (15), 163 (15), 135 (20), 125
(20), 111 (100).
7.4. (3S)-3-[(4S,7aR)-4-Hydroxy-7a-methyloctahydro-1H-
inden-1-yl]butanenitrile (8)
Compound 8 was obtained from 6 by the same proce-
dure as described for 7 (yield 98.5%) as a colorless
solid.
8 1H NMR d 0.94 (3H, s, H-18), 1.07 (3H, d, J = 6.7 Hz,
H-21), 2.43 (2H, m, H-22), 4.09 (1H, m, H-8). 13C NMR
d 14.0, 17.5, 19.7, 22.4, 24.2, 27.1, 31.9, 33.6, 40.1, 41.8,
52.4, 55.0, 69.1, 118.9. IR (neat) 3493, 2930, 2871, 2248,
1454, 1167, 991, 954 cmꢀ1. MS m/z (%): 221 (M+, 20),
206 (60), 188 (15), 163 (15), 135 (35), 125 (25), 111
(100), 93 (30). HRMS calcd for C14H23ON 221.1780,
found 221.1781.
1
11 (a 2:3 mixture of C(2) epimers) H NMR d 0.0–0.1
(12H, s, SiMe · 4), 0.120, 0.125 (2:3) (9H, s, TMS),
0.56, 0.57 (2:3) (3H, s, H-18), 0.854, 0.867 (2:3) and
0.873, 0.891 (3:2) (each 9H, s, t-Bu), 1.17 (3H, d,
J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.54, 3.59 (3:2)
(1H, m, H-2), 3.80 (1H, m, H-3), 3.88, 3.92 (3:2) (1H,
m, H-1), 5.79, 5.82 (2:3) (1H, d, J = 11.0 Hz, H-7),
6.09, 6.12 (3:2) (1H, d, J = 11.0 Hz, H-6). IR (neat)
2953, 2856, 2245, 1620, 1471, 1251, 1095, 837,
775 cmꢀ1. MS m/z (%): 659 (M+, 20), 528 (30), 527
(70), 470 (90), 424 (80), 380 (50), 306 (30), 147 (30), 73
(60). HRMS calcd for C37H69O3NSi3 659.4585, found
659.4597.
7.5. (3R)-3-[(7aR)-7a-Methyl-4-oxooctahydro-1H-inden-
1-yl]butanenitrile (9)
To a solution of cyanide 7 (286.6 mg, 1.30 mmol) in dry
CH2Cl2 (3 mL) were added Molecular Sieves 4A
(150 mg) and N-methylmorpholine N-oxide (NMO,
1.26 g, 10.8 mmol) at room temperature. After 10 min,
tetrapropylammonium perruthenate (TPAP, 24.6 mg,
0.070 mmol) was added to the mixture, and the reaction
mixture was stirred for 2 h at same temperature. The
mixture was chromatographed on silica gel (20 g, hex-
ane/AcOEt = 1:1) to give ketone 9 (260.5 mg, 91.5%)
as a colorless solid.
7.8. (3S)-3-{(1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-[(trimethylsilyl)oxy]-9,10-secoestra-5,7-dien-17-yl}
butanenitrile (12)
Compound 12 was obtained from 10 by the same proce-
dure as described for 11 (yield 74.7%, a 2:3 mixture of
C(2) epimers) as colorless oil.
1
9 1H NMR d 0.67 (3H, s, H-18), 1.21 (3H, d, J = 6.6 Hz,
H-21), 2.52 (1H, dd, J = 11.7, 7.6 Hz, H-14). 13C NMR
d 12.6, 19.1, 19.4, 23.9, 24.8, 27.4, 33.2, 38.6, 40.8, 49.7,
55.1, 61.6, 118.6, 211.2. IR (neat) 2957, 2875, 2244,
1710, 1463, 1383, 1233 cmꢀ1. MS m/z (%): 219 (M+,
70), 204 (80), 179 (25), 176 (90), 163 (60), 124 (40), 96
(45).
12 (a 2:3 mixture of C(2) epimers) H NMR d ꢀ0.1–0.1
(12H, s, SiMe · l4), 0.119, 0.123 (2:3) (9H, s, TMS),
0.54, 0.55 (2:3) (3H, s, H-18), 0.85, 0.86 (2:3) and 0.87,
0.89 (3:2) (each 9H, s, t-Bu), 1.09 (3H, d, J = 6.6 Hz,
H-21), 2.80 (1H, m, H-9), 3.54, 3.59 (3:2) (1H, m, H-
2), 3.80 (1H, m, H-3), 3.88, 3.92 (3:2) (1H, m, H-1),
5.79, 5.82 (2:3) (1H, d, J = 11.1 Hz, H-7), 6.08, 6.11

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
467
(3:2) (1H, d, J = 11.1 Hz, H-6). IR (neat) 2954, 2928,
2856, 2247, 1729, 1471, 1251, 1095, 837 cmꢀ1. MS m/z
(%): 659 (M+, 10), 602 (8), 527 (35), 470 (40), 424 (38),
309 (58), 256 (52), 236 (55), 138 (60), 75 (100). HRMS
calcd for C37H69O3NSi3 659.4585, found 659.4579.
pound 13 (308.1 mg, 0.53 mmol, a 2:3 mixture of C(2)
epimers) in dry CH2Cl2 (2.5 mL) was added to the mix-
ture, and the reaction mixture was stirred for 15 min at
same temperature. Then, to this solution was added
Et3N (363 lL, 2.62 mmol), and mixture was stirred at
ꢀ78 ꢁC for 30 min and at 0 ꢁC for 1 h. The reaction
was quenched with H2O at 0 ꢁC and was extracted with
CH2Cl2. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was chro-
matographed on silica gel (10 g, hexane/AcOEt = 9:1)
to give ketone 15 (294.8 mg, 96.0%) as a colorless oil.
7.9. (3R)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-hydroxy-9,10-secoestra-5,7-dien-17-yl)butaneni-
trile (13)
A solution of compound 11 (532.6 mg, 0.81 mmol, a 2:3
mixture of C(2) epimers) in AcOH/THF/H2O (8:8:1,
17 mL) was stirred at room temperature for 22.5 h.
The reaction was quenched with 5% NaHCO3 aq solu-
tion 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 (20 g, hexane/AcOEt = 49:1 then 19:1) to give com-
pound 11 (38.4 mg, 7.2%, a 2:3 mixture of C(2) epimers)
and compound 13 (colorless oil, 405.6 mg, 85.5%, a 2:3
mixture of C(2) epimers).
1
15 H NMR d 0.05 and 0.08 (each 3H, s, SiMe), 0.06
(6H, s, SiMe · 2), 0.57 (3H, s, H-18), 0.86 and 0.88 (each
9H, s, t-Bu), 1.18 (3H, d, J = 6.6 Hz, H-21), 2.83 (1H, m,
H-9), 4.35 (1H, dd, J = 6.4, 4.2 Hz, H-3), 4.53 (1H, dd,
J = 8.7, 5.5 Hz, H-1), 5.81 (1H, d, J = 11.1 Hz, H-7),
6.33 (1H, d, J = 11.1 Hz, H-6). 13C NMR d ꢀ5.00,
ꢀ4.95, ꢀ4.7, ꢀ4.6, 12.3, 18.3, 18.5, 19.6, 22.3, 23.4,
25.0, 25.9 (3 carbons), 26.0 (3 carbons), 27.7, 28.9,
37.7, 40.3, 41.6, 45.9, 46.7, 55.3, 56.2, 74.5, 74.8, 116.4,
119.1, 124.5, 129.5, 142.3, 208.7. IR (neat) 2952, 2929,
2856, 2245, 1738, 1470, 1254, 1098, 836 cmꢀ1. MS m/z
(%): 585 (M+, 2), 570 (3), 528 (100), 396 (62), 230 (40),
75 (62), 73 (32). HRMS calcd for C34H59O3NSi2
585.4033, found 585.4055.
1
13 (a 2:3 mixture of C(2) epimers) H NMR d 0.0–0.2
(12H, s, SiMe · 4), 0.55, 0.56 (2:3) (3H, s, H-18), 0.84,
0.86 (2:3) and 0.87, 0.89 (3:2) (each 9H, s, t-Bu), 1.17
(3H, d, J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.51,
3.58 (3:2) (1H, m, H-2), 3.91, 3.99 (3:2) (1H, m, H-3),
3.99 (1H, m, H-1), 5.79 (1H, d, J = 11.2 Hz, H-7),
6.14, 6.17 (3:2) (1H, d, J = 11.2 Hz, H-6). IR (neat)
3481, 2953, 2930, 2857, 2246, 1717, 1471, 1254, 1090,
837, 779 cmꢀ1. MS m/z (%): 587 (M+, 5), 530 (7), 438
(5), 398 (100), 306 (15), 165 (10), 129 (10), 75 (40), 73
(18).
7.12. (3S)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-oxo-9,10-secoestra-5,7-dien-17-yl)butanenitrile
(16)
Compound 16 was obtained from 14 by the same proce-
dure as described for 15 (yield 99.2%) as a colorless oil.
1
7.10. (3S)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-hydroxy-9,10-secoestra-5,7-dien-17-yl)butaneni-
trile (14)
16 H NMR d 0.04 and 0.08 (each 3H, s, SiMe), 0.05
(6H, s, SiMe · 2), 0.55 (3H, s, H-18), 0.86 and 0.88 (each
9H, s, t-Bu), 1.09 (3H, d, J = 6.7 Hz, H-21), 2.82 (1H, m,
H-9), 4.35 (1H, dd, J = 6.4, 4.2 Hz, H-3), 4.53 (1H, dd,
J = 8.6, 5.5 Hz, H-1), 5.81 (1H, d, J = 11.2 Hz, H-7),
6.33 (1H, d, J = 11.2 Hz, H-6). 13C NMR d ꢀ5.1,
ꢀ5.0, ꢀ4.8, ꢀ4.7, 12.3, 18.2, 18.4, 19.6, 22.2, 23.4,
24.9, 25.81 (3 carbons), 25.85 (3 carbons), 27.6, 28.8,
34.0, 37.6,40.2, 45.8, 46.6, 55.2, 56.1, 74.4, 74.7, 116.3,
119.0, 124.5, 129.4, 142.2, 208.5. MS m/z (%): 528
(M+ꢀC(CH3)3, 100), 510 (4), 396 (42), 378 (12), 325
(15), 259 (10), 147 (12), 73 (45).
Compound 14 was obtained from 12 by the same proce-
dure as described for 13 (yield 82.6%, a 2:3 mixture of
C(2) epimers) as colorless oil.
1
14 (a 2:3 mixture of C(2) epimers) H NMR d ꢀ0.1–0.2
(12H, s, SiMe · 4), 0.53, 0.55 (2:3) (3H, s, H-18), 0.85,
0.86 (2:3) and 0.87, 0.89 (3:2) (each 9H, s, t-Bu), 1.09
(3H, d, J = 6.6 Hz, H-21), 2.80 (1H, m, H-9), 3.51,
3.58 (3:2) (1H, m, H-2), 3.91, 3.99 (3:2) (1H, m, H-3),
3.99 (1H, m, H-1), 5.80 (1H, d, J = 11.2 Hz, H-7),
6.13, 6.16 (3:2) (1H, d, J = 11.2 Hz, H-6). IR (neat)
7.13. (3R)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)
silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)
butanenitrile (17)
3555, 2952, 2855, 2246, 1462, 1254, 1085, 836 cmꢀ1
.
MS m/z (%): 587 (M+, 3), 530 (8), 438 (10), 398 (100),
380 (10), 306 (20), 257 (10), 236 (15), 129 (15), 73 (25).
HRMS calcd for C34H61O3NSi2 587.4190, found
587.4196.
To a suspension of methyltriphenylphosphonium bro-
mide (527.3 mg, 1.48 mmol) in dry THF (1.5 mL) at
0 ꢁC was added slowly n-BuLi (1.59 M hexane solution,
923 lL, 1.47 mmol), and the resulting yellow solution
was stirred for 1 h. To this colored solution was added
a solution of ketone 15 (285.9 mg, 0.49 mmol) in dry
THF (2 mL), the reaction mixture was stirred at 0 ꢁC
for 1 h, and then at room temperature for 1 h. The reac-
tion 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 (10 g, hexane/
7.11. (3R)-3-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]
oxy}-2-oxo-9,10-secoestra-5,7-dien-17-yl)butanenitrile
(15)
To a solution of oxalyl chloride (2.0 M CH2Cl2 solution,
315 lL, 0.63 mmol) in dry CH2Cl2 (1 mL) was added a
solution of DMSO (90 lL, 1.27 mmol) in dry CH2Cl2
(500 lL) at ꢀ78 ꢁC. After 10 min, a solution of com-

468
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
AcOEt = 19:1) to give 2-methylene 17 (268.4 mg, 94.2%)
as a colorless oil.
7.16. (3S)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)sil-
yl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butanal
(20)
17 1H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each 3H,
s, SiMe), 0.57 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-
Bu), 1.18 (3H, d, J = 6.6 Hz, H-21), 2.82 (1H, m, H-9),
4.43 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,
–C@CH2), 5.84 (1H, d, J = 11.2 Hz, H-7), 6.21 (1 H,
d, J = 11.2 Hz, H-6). 13C NMR d ꢀ4.9, ꢀ4.71, ꢀ4.66,
ꢀ4.64, 12.4, 18.36, 18.44, 19.7, 22.3, 23.4, 25.0, 25.98
(3 carbons), 26.04 (3 carbons), 27.8, 28.8, 34.1, 38.8,
40.4, 45.8, 47.8, 55.4, 56.2, 71.8, 72.7, 106.6, 116.7,
119.2, 122.4, 133.5, 140.4, 153.1. IR (neat) 2952, 2891,
Compound 20 was obtained from 18 by the same proce-
dure as described for 19 (yield 87.3%) as a colorless oil.
20 1H NMR d 0.02 and 0.04 and 0.06 and 0.07 (each 3H,
s, SiMe), 0.58 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-
Bu), 0.95 (3H, d, J = 6.6 Hz, H-21), 2.26 (1H, ddd,
J = 15.9, 9.6, 3.4 Hz, H-22), 2.67 (1H, dd, J = 15.9, 3.1
Hz, H-22), 2.82 (1H, m, H-9), 4.42 (2H, m, H-3, 1),
4.92 and 4.97 (each 1H, s, –C@CH2), 5.85 (1H, d,
J = 11.2 Hz, H-7), 6.20 (1H, d, J = 11.2 Hz, H-6), 9.75
(1 H, dd, J = 3.4, 1.0 Hz, H-23). 13C NMR d ꢀ4.9,
ꢀ4.7, ꢀ4.6 (2 carbons), 12.7, 18.4, 18.5, 20.0, 22.2,
23.5, 26.0 (3 carbons), 26.1 (3 carbons), 27.4, 28.8,
31.1, 38.8, 40.7, 45.8, 47.8, 50.3, 56.2, 56.3, 71.8, 72.7,
106.5, 116.7, 122.5, 133.4, 140.6, 153.1, 203.4.
2856, 2246, 1472, 1256 cmꢀ1
.
7.14. (3S)-3-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)sil-
yl] oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butaneni-
trile (18)
Compound 18 was obtained from 16 by the same proce-
dure as described for 17 (yield 88.5%) as a colorless oil.
7.17. 5-[(2R)-2-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)-
silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-
pyl]-3-methylenedihydro-2(3H)-furanone (21)
1
18 H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.89 (each
9H, s, t-Bu), 1.10 (3H, d, J = 6.7 Hz, H-21), 2.83 (1H,
m, H-9), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each
1H, s, –C@CH2), 5.85 (1H, d, J = 11.2 Hz, H-7),
6.20 (1H, d, J = 11.2 Hz, H-6). 13C NMR d ꢀ4.9,
ꢀ4.7, ꢀ4.6 (2 carbons), 12.6, 18.37, 18.45, 19.9,
22.2, 23.5, 24.5, 25.99 (3 carbons), 26.04 (3 carbons),
27.5, 28.7, 32.8, 38.8, 40.3, 45.5, 47.8, 55.0, 56.1,
71.8, 72.7, 106.6, 116.8, 119.0, 122.4, 133.5, 140.3,
153.1. IR (neat) 2934, 2885, 2857, 2247, 1472,
To a suspension of CrCl3 (74.8 mg, 0.47 mmol) in dry
THF (1 mL) was added slowly LiAlH4 (1.0 M solution
in THF, 235 lL, 0.24 mmol) at 0 ꢁC, and the mixture
was stirred at room temperature for 45 min. To the mix-
ture were added a solution of methyl 2-(bromo-
methyl)acrylate (34 lL, 0.28 mmol) in dry THF
(300 lL) and a solution of aldehyde 19 (68.9 mg,
0.12 mmol) in dry THF (750 lL) at room temperature,
and the resulting mixture was stirred at the same tem-
perature for 30 min. The reaction was quenched with
H2O and 1N HCl at 0 ꢁC and was extracted with
AcOEt. The organic layer was washed with brine, dried
over MgSO4, and evaporated. The residue was chro-
matographed on silica gel (5 g, hexane/AcOEt = 49:1)
to give methylene lactone 21 (amorphous solid,
63.6 mg, 82.7%, a 2:3 mixture of diastereomers at
C(23)) and 23-alcohol (a colorless oil, 4 mg, 5.8%).
1257 cmꢀ1
.
7.15. (3R)-3-((1R,3R,7E)-1,3-Bis{tert -butyl(dimethyl)sil-
yl] oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)butanal
(19)
To a solution of 2-methylene 17 (113.3 mg, 0.19 mmol)
in dry CH2Cl2 (1 mL) was added a solution of DI-
BAL-H (1.01 M solution in toluene, 252 lL, 0.25 mmol)
at 0 ꢁC for 1.5 h. The reaction was quenched with 10%
potassium sodium tartrate aq solution at 0 ꢁC and was
extracted with CH2Cl2. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The res-
idue was chromatographed on silica gel (5 g, hexane/
AcOEt = 49:1) to give aldehyde 19 (102.3 mg, 90.0%)
as a colorless oil.
21 (a 2:3 mixture of C(23) epimers) 1H NMR d ꢀ0.0–0.1
(12H, s, SiMe · 4), 0.557, 0.562 (2:3) (3H, s, H-18), 0.86
and 0.890, 0.894 (2:3) (each 9H, s, t-Bu), 1.02, 1.03 (3:2)
(3H, d, J = 6.3 Hz, H-21), 2.82 (1H, m), 3.06 (1H, m),
4.42 (2H, m, H-3, 1), 4.63 (1H, m, H-23), 4.91 and
4.97 (each 1H, s, –C@CH2), 5.62 (1H, t, J = 2.4 Hz,
H-27), 5.83 (1H, d, J = 11.2 Hz, H-7), 6.21 (1H, d,
J = 11.2 Hz, H-6), 6.22 (1H, m, H-27). MS m/z (%):
654 (M+, 12), 597 (8), 522 (78), 465 (18), 366 (35), 251
(18), 197 (15), 75 (100), 73 (38).
19 1H NMR d 0.02 and 0.04 and 0.06 and 0.07 (each 3H,
s, SiMe), 0.59 (3H, s, H-18), 0.86 and 0.89 (each 9H, s, t-
Bu), 1.02 (3H, d, J = 6.5 Hz, H-21), 2.81 (1H, m, H-9),
4.42 (2H, m, H-3, 1), 4.91 and 4.97 (each 1H, s,
–C@CH2), 5.83 (1H, d, J = 11.2 Hz, H-7), 6.20 (1H, d,
J = 11.2 Hz, H-6), 9.76 (1H, dd, J = 3.3, 1.2 Hz, H-23).
13C NMR d ꢀ4.9, ꢀ4.7, ꢀ4.6 (2 carbons), 12.3, 18.37,
18.45, 20.3, 22.4, 23.5, 25.98 (3 carbons), 26.04 (3 car-
bons), 28.1, 28.8, 32.1, 38.8, 40.6, 45.9, 47.8, 51.0,
56.40, 56.43, 71.8, 72.7, 106.5, 116.6, 122.5, 133.3,
140.8, 153.1, 203.6. MS m/z (%): 586 (M+, 12), 529
(10), 454 (100), 366 (34), 322 (12), 234 (12), 75 (32), 73
(30).
7.18. 5-[(2S)-2-((1R,3R,7E)-1,3-Bis{tert-butyl(dimethyl)-
silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-
pyl]-3-methylenedihydro-2(3H)-furanone (22)
Compound 22 was obtained from 20 by the same proce-
dure as described for 21 (yield 81.8%, 2:3 mixture of
C(23) epimers) as amorphous solid.
22 (a 2:3 mixture of C(23) epimers) 1H NMR d ꢀ0.1–0.1
(12H, s, SiMe · 4), 0.54, 0.55 (2:3) (3H, s, H-18), 0.859,

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
469
0.861 (3:2) and 0.89 (each 9H, s, t-Bu), 0.92, 0.95 (2:3)
(3H, d, J = 6.7 Hz, H-21), 2.81 (1H, m), 3.06 (1 H, m),
4.42 (2H, m, H-3, 1), 4.63 (1H, m, H-23), 4.92 and
4.97 (each 1H, s, –C@CH2), 5.66 (1H, m, H-27), 5.84
(1H, d, J = 11.2 Hz, H-7), 6.18 (1H, d, J = 11.2 Hz, H-
6), 6.27 (1H, m, H-27).
–C@CH2), 5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,
J = 11.3 Hz, H-7), 6.23 (1H, t, J = 2.7 Hz, H-27), 6.36
(1H, d, J = 11.3 Hz, H-6). IR (neat) 3421, 2929, 2875,
1762 cmꢀ1. MS m/z (%): 426 (M+, 30), 390 (70), 375
(20), 285 (25), 251 (100), 197 (50), 157 (40), 105 (58).
HRMS calcd for C27H38O4 426.2770, found 426.2787.
UV (95% EtOH): kmax 245, 253, 263 nm.
7.19. 5-{(2R)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-methylene-
9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylenedihy-
dro-2(3H)-furanone (LAC67a, b)
LAC82b (less polar) 1H NMR d 0.57 (3H, s, H-18), 0.95
(3H, d, J = 6.6 Hz, H-21), 4.49 (2H, m, H-3, 1), 4.64
(1H, m, H-23), 5.09 and 5.12 (each 1H, s, –C@CH2),
5.62 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,
J = 11.2 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.34
(1H, d, J = 11.2 Hz, H-6). IR (neat) 3421, 2936, 2875,
1759 cmꢀ1. MS m/z (%): 426 (M+, 20), 408 (28), 390
(90), 375 (28), 285 (30), 251 (100), 197 (55), 157 (30),
105 (55). HRMS calcd for C27H38O4 426.2770, found
426.2759. UV (95% EtOH): kmax 245, 253, 263 nm.
To a solution of methylene lactone 21 (26.0 mg,
0.040 mmol, 2:3 mixture of C(23) epimers) in MeOH
(1 mL) was added CSA (20.4 mg, 0.088 mmol) at room
temperature for 1.3 h. The reaction was quenched with
5% NaHCO3 at 0 ꢁC and was extracted with AcOEt.
The organic layer was washed with brine, dried over
MgSO4, and evaporated. The residue was chromato-
graphed on silica gel (5 g, AcOEt) to give lactone ana-
logue LAC67 (5.7 mg, 54.0%, a 2:3 mixture of C(23)
epimers) as a colorless solid. The mixture was separated
by HPLC (LiChrosorb Si 60, Hibar RT 250-10, 7 lm,
hexane/AcOEt = 7:3, 5 mL/min) to give LAC67a
(2.7 mg) and LAC67b (2.1 mg). The purity of LAC67a
and LAC67b was proved to be about 100% by HPLC.
7.21. (6R)-6-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]-
oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)-2-meth-
ylene-1,4-heptanediol (23a, b)
To a solution of methylene lactone 21 (21.0 mg,
0.032 mmol) in dry toluene (1 mL) was added a solution
of DIBAL-H (1.01 M solution in toluene, 137 lL,
0.14 mmol) at 0 ꢁC for 1 h. The reaction was quenched
with 10% potassium sodium tartrate aq solution at
0 ꢁC and was extracted with AcOEt. The organic layer
was washed with brine, dried over MgSO4, and evapo-
rated. The residue was chromatographed on silica gel
(7 g, hexane/AcOEt = 4:1) to give 23,26-diol 23a (a col-
orless oil, 5.7 mg, 27.0%, less polar) and diol 23b (a col-
orless oil, 4.9 mg, 23.2%, more polar).
LAC67a (less polar) 1H NMR d 0.58 (3H, s, H-18), 1.02
(3H, d, J = 6.5 Hz, H-21), 4.51 (2H, m, H-3, 1), 4.65
(1H, m, H-23), 5.09 and 5.12 (each 1H, s, –C@CH2),
5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,
J = 11.2 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.35
(1H, d, J = 11.2 Hz, H-6). IR (neat) 3384, 2942, 2823,
1748 cmꢀ1. MS m/z (%): 426 (M+, 10), 390 (50), 375
(12), 285 (12), 252 (100), 250 (60), 197 (32), 105 (25).
HRMS calcd for C27H38O4 426.2770, found 426.2794.
UV (95%EtOH): kmax 245, 254, 263 nm.
23a (less polar) 1H NMR d 0.02 and 0.05 and 0.06 and 0.08
(each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.85 and 0.90 (each
9H, s, t-Bu), 0.99 (3H, d, J = 6.5 Hz, H-21), 2.83 (1H, m,
H-9), 3.87 (1H, m, H-23), 4.12 (2H, s, H-26), 4.42 (2H, m,
H-3, 1), 4.92 and 4.97 (each 1H, s, –C@CH2), 4.98 and
5.15 (each 1H, s, H-27), 5.84 (1H, d, J = 11.1 Hz, H-7),
6.22 (1H, d, J = 11.1 Hz, H-6). MS m/z (%): 658 (M+,
8), 640 (17), 526 (32), 508 (100), 454 (50), 366 (56), 234
(23), 147 (17), 75 (75), 73 (68).
1
LAC67b (more polar) H NMR d 0.57 (3H, s, H-18),
1.04 (3H, d, J = 6.3 Hz, H-21), 4.50 (2H, m, H-3, 1),
4.60 (1H, m, H-23), 5.10 and 5.12 (each 1H, s,
–C@CH2), 5.63 (1H, t, J = 2.4 Hz, H-27), 5.89 (1H, d,
J = 11.5 Hz, H-7), 6.23 (1H, t, J = 2.8 Hz, H-27), 6.36
(1H, d, J = 11.5 Hz, H-6). IR (neat) 3452, 2923, 2853,
1759 cmꢀ1. MS m/z (%): 426 (M+, 10), 390 (80), 375
(20), 285 (20), 251 (100), 197 (42), 105 (40). HRMS calcd
for C27H38O4 426.2770, found 426.2750. UV (95%
EtOH): kmax 245, 253, 263 nm.
23b (more polar) 1H NMR d 0.02 and 0.05 and 0.06 and
0.08 (each 3H, s, SiMe), 0.56 (3H, s, H-18), 0.86 and
0.89 (each 9H, s, t-Bu), 1.01 (3H, d, J = 6.4 Hz, H-21),
2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.13 (2H, s, H-
26), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,
–C@CH2), 5.00 and 5.17 (each 1H, s, H-27), 5.84 (1H,
d, J = 11.2 Hz, H-7), 6.21 (1H, d, J = 11.2 Hz, H-6).
MS m/z (%): 658 (M+, 10), 640 (18), 526 (45), 508
(100), 454 (63), 366 (65), 236 (32), 234 (30), 147 (22),
138 (21), 75 (92), 73 (90).
7.20. 5-{(2S)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-methylene-
9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylenedihy-
dro-2(3H)-furanone (LAC82a, b)
LAC82 was obtained from 22 by the same procedure as
described for LAC67 (yield 99.1%) as a colorless solid.
The mixture was separated by HPLC (LiChrosorb Si
60, Hibar RT 250-10, 7 lm, hexane/AcOEt = 7:3,
5 mL/min) to give LAC82a and LAC82b. The purity
of LAC82a and LAC82b was proved to be about
100% by HPLC.
7.22. (6S)-6-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)silyl]
oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)-2-meth-
ylene-1,4-heptanediol (24a, b)
1
LAC82a (more polar) H NMR d 0.56 (3H, s, H-18),
0.94 (3H, d, J = 6.4 Hz, H-21), 4.49 (2H, m, H-3, 1),
4.61 (1H, m, H-23), 5.10 and 5.11 (each 1H, s,
Compound 24 was obtained from 22 by the same proce-
dure as described for 23a and 23b (24a 31.8% more po-
lar, 24b 29.7% less polar) as colorless oil, respectively.

470
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
24a (more polar) 1H NMR d 0.02 and 0.05 and 0.06 and
0.08 (each 3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and
0.89 (each 9H, s, t-Bu), 0.91 (3H, d, J = 6.7 Hz, H-21),
2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.12 (2H, s, H-
26), 4.42 (2H, m, H-3, 1), 4.92 and 4.97 (each 1H, s,
–C@CH2), 5.00 and 5.16 (each 1H, s, H-27), 5.84 (1H,
d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6).
592 (45), 508 (42), 454 (92), 366 (95), 234 (30), 197
(20), 147 (22), 73 (100).
7.25. 2-((2R,4S)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl
(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-
dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-
tyl)-2-propenyl pivalate (26a)
1
24b (less polar) H NMR d 0.02 and 0.05 and 0.06 and
Compound 26a was obtained from 24a by the same pro-
cedure as described for 25a (yield 45.3%) as a yellow oil,
with the recovered 24a (34.7%).
0.08 (each 3H, s, SiMe), 0.58 (3H, s, H-18), 0.86 and
0.89 (each 9H, s, t-Bu), 0.91 (3H, d, J = 6.9 Hz, H-21),
2.82 (1H, m, H-9), 3.86 (1H, m, H-23), 4.11 (2H, s, H-
26), 4.42 (2H, m, H-3, 1), 4.92 (1H, s, –C@CH2), 4.969
and 4.975 (each 1H, s, H-27, –C@CH2), 5.14 (1H, s,
H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,
J = 11.1 Hz, H-6).
1
26a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.90 (each
9H, s, t-Bu), 1.23 (9H, s, –COC(CH3)3), 2.82 (1H, m,
H-9), 3.85 (1H, m, H-23), 4.43 (2H, m, H-3, 1), 4.53
and 4.58 (each 1H, d, J = 13.6 Hz, H-26), 4.92 and
4.97 (each 1H, s, –C@CH2), 5.06 and 5.17 (each 1H, s,
H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,
J = 11.1 Hz, H-6).
7.23. 2-((2R,4R)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-
butyl(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)
ethylidene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxy-
pentyl)-2-propenyl pivalate (25a)
7.26. 2-((2S,4S)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl
(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-
dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-
tyl)-2-propenyl pivalate (26b)
To a solution of 23,26-diol 23a (18.4 mg, 0.028 mmol)
in dry CH2Cl2 (1 mL) were added dry pyridine
(9.5 lL, 0.12 mmol) and PivCl (4.5 lL, 0.036 mmol),
and the mixture was stirred at 0 ꢁC for 22.5 h. The
reaction was quenched with H2O at 0 ꢁC and was ex-
tracted with CH2Cl2. The organic layer was washed
with brine, dried over MgSO4, and evaporated. The
residue was chromatographed on silica gel (5 g, hex-
ane/AcOEt = 49:1 then 4:1) to give pivalate ester 25a
(a yellow oil, 5.5 mg, 26.5%) and 23,26-diol 23a
(5.6 mg, 30.4%).
Compound 26b was obtained from 24b by the same pro-
cedure as described for 25a (yield 23.8%) as a yellow oil,
with the recovered 24b (45.4%).
1
26b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.59 (3H, s, H-18), 0.86 and 0.90 (each
9H, s, t-Bu), 1.23 (9H, s, –COC(CH3)3), 2.82 (1H, m,
H-9), 3.88 (1H, m, H-23), 4.42 (2H, m, H-3, 1), 4.54
(2H, s, H-26), 4.92 and 4.97 (each 1H, s, –C@CH2),
5.04 and 5.15 (each 1H, s, H-27), 5.84 (1H, d,
J = 11.0 Hz, H-7), 6.21 (1H, d, J = 11.0 Hz, H-6).
1
25a H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.58 (3H, s, H-18), 0.85 and 0.90 (each
9H, s, t-Bu), 0.98 (3H, d, J = 6.4 Hz, H-21), 1.23 (9H,
s, –COC(CH3)3), 2.82 (1H, m, H-9), 3.88 (1H, m, H-
23), 4.42 (2H, m, H-3, 1), 4.55 (2H, s, H-26), 4.91 and
4.97 (each 1H, s, –C@CH2), 5.04 and 5.14 (each 1H, s,
H-27), 5.84 (1H, d, J = 11.1 Hz, H-7), 6.22 (1H, d,
J = 11.1 Hz, H-6). MS m/z (%): 742 (M+, 20), 724 (3),
640 (5), 610 (100), 592 (42), 508 (40), 454 (92), 366
(90), 234 (25), 197 (20), 147 (22), 73 (98).
7.27. (1R)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-
methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)
propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl
(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (27a)
To
0.0054 mmol) in dry CH2Cl2 (100 lL) were added
Et3N (7.5 lL, 0.054 mmol), DMAP (6.2 mg,
a
solution of pivalate ester 25a (4.0 mg,
7.24. 2-((2S,4R)-4-{(4E)-4-[2-((3R,5R)-3,5-Bis{[tert-butyl
(dimethyl)silyl]oxy}-4-methylenecyclohexylidene)ethyli-
dene]-7a-methyloctahydro-1H-inden-1-yl}-2-hydroxypen-
tyl)-2-propenyl pivalate (25b)
0.051 mmol), and a solution of R-MTPACl (5.1 lL,
0.027 mmol) in dry CH2Cl2 (40 lL), the reaction mix-
ture was stirred for 15 min at room temperature. The
reaction was quenched with H2O at 0 ꢁC and was ex-
tracted with CH2Cl2. The organic layer was washed with
brine, dried over MgSO4, and evaporated. The residue
was chromatographed on silica gel (5 g, hexane/
AcOEt = 9:1) to give S-MTPA ester 27a (3.8 mg,
78.0%) as a colorless oil.
Compound 25b was obtained from 23b by the same pro-
cedure as described for 25a (yield 43.7%) as a yellow oil,
with the recovered 23b (8.2%).
1
25b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.55 (3H, s, H-18), 0.86 and 0.89 (each
9H, s, t-Bu), 1.00 (3H, d, J = 6.1 Hz, H-21), 1.23 (9H,
s, –COC(CH3)3), 2.82 (1H, m, H-9), 3.85 (1H, m, H-
23), 4.43 (2H, m, H-3, 1), 4.53 and 4.58 (each 1H, d,
J = 13.7 Hz, H-26), 4.92 and 4.97 (each 1H, s,
–C@CH2), 5.06 and 5.17 (each 1H, s, H-27), 5.83 (1H,
d, J = 11.1 Hz, H-7), 6.21 (1H, d, J = 11.1 Hz, H-6).
MS m/z (%): 742 (M+, 20), 724 (5), 640 (3), 610 (92),
1
27a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.48 (3H, s, H-18), 0.86 and 0.89 (each
9H, s, t-Bu), 0.98 (3H, d, J = 6.3 Hz, H-21), 1.23 (9H,
s, –COC(CH3)3), 2.82 (1H, m, H-9), 3.51 (3H, s,
–OCH3), 4.41 (2H, m, H-3, 1), 4.48 and 4.53 (each 1H,
d, J = 13.7 Hz, H-26), 4.92 and 4.97 (each 1H, s,
–C@CH2), 4.93 and 5.05 (each 1H, s, H-27), 5.38 (1H,

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
471
m, H-23), 5.83 (1H, d, J = 11.1 Hz, H-7), 6.21 (1H, d,
J = 11.1 Hz, H-6), 7.38 (3H, m, Ph-3, 4, 5), 7.53 (2H,
m, Ph-2, 6).
m, H-23), 5.83 (1H, d, J = 11.0 Hz, H-7), 6.21 (1H,
d, J = 11.0 Hz, H-6), 7.38 (3 H, m, Ph-3, 4, 5), 7.52
(2H, m, Ph-2, 6).
7.28. (1R)-1-[(2R)-2-((1R,3R,7R)-1,3-Bis{[tert-butyl
(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-
17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-
butenyl (2R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate
(28a)
7.31. (1R)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl
(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-
17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-
butenyl (2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropano-
ate (29a)
In a similar manner to that for the synthesis of 27a from
25a, a crude product, which was obtained from 25a
(4.5 mg, 0.0061 mmol), Et3N (8.4 lL, 0.061 mmol),
DMAP (6.7 mg, 0.055 mmol), and S-MTPACl (5.7 lL,
0.030 mmol) in dry CH2Cl2 at room temperature for
15 min, was purified by chromatographed on silica gel
(5 g, hexane/AcOEt = 9:1) to give R-MTPA ester 28a
(5.0 mg, 84.8%) as a colorless oil.
Compound 29a was obtained from 26a by the same pro-
cedure as described for 27a (yield quant.) as a colorless
oil.
29a 1H NMR d 0.03 and 0.05 and 0.06 and 0.08
(each 3H, s, SiMe), 0.53 (3H, s, H-18), 0.86 and
0.90 (each 9H, s, t-Bu), 1.21 (9H, s, –COC(CH3)3),
2.83 (1H, m, H-9), 3.54 (3H, s, –OCH3), 4.42 (4
H, m, H-26, 3, 1), 4.85 (1H, s, H-27), 4.93 (1H,
s, –C@CH2), 4.98 (2H, s, H-27, –C@CH2), 5.34
(1H, m, H-23), 5.84 (1H, d, J = 11.0 Hz, H-7), 6.21
(1H, d, J = 11.0 Hz, H-6), 7.38 (3H, m, Ph-3, 4,
5), 7.53 (2H, m, Ph-2, 6).
1
28a H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.40 (3H, s, H-18), 0.87 and 0.89 (each
9H, s, t-Bu), 1.23 (9H, s, –COC(CH3)3), 2.80 (1H, m,
H-9), 3.53 (3H, s, –OCH3), 4.41 (2H, m, H-3, 1), 4.52
and 4.57 (each 1H, d, J = 13.7 Hz, H-26), 4.92 and
4.97 (each 1H, s, –C@CH2), 5.00 and 5.12 (each 1H, s,
H-27), 5.42 (1H, m, H-23), 5.81 (1H, d, J = 11.0 Hz,
H-7), 6.20 (1H, d, J = 11.0 Hz, H-6), 7.37 (3H, m, Ph-
3, 4, 5), 7.53 (2H, m, Ph-2, 6).
7.32. (1R)-1-[(2S)-2-((1R,3R,7R)-1,3-Bis{[tert-butyl
(dimethyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-
17-yl)propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-
butenyl (2R)-3,3,3-trifluoro-2-methoxy-2-phenylpropano-
ate (30a)
7.29. (1S)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)
silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-
pyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl
(2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (27b)
Compound 30a was obtained from 26a by the same pro-
cedure as described for 28a (yield 41.9%) as a colorless
oil.
1
Compound 27b was obtained from 25b by the same pro-
cedure as described for 27a (yield 70.6%) as a colorless
oil.
30a H NMR d 0.02 and 0.05 and 0.06 and 0.07 (each
3H, s, SiMe), 0.49 (3H, s, H-18), 0.86 and 0.89 (each
9H, s, t-Bu), 1.21 (9H, s, –COC(CH3)3), 2.81 (1H, m,
H-9), 3.52 (3H, s, –OCH3), 4.42 (2H, m, H-3, 1), 4.52
(2H, s, H-26), 4.92 and 4.97 (each 1H, s, –C@CH2),
5.01 and 5.13 (each 1H, s, H-27), 5.34 (1H, m, H-23),
5.82 (1H, d, J = 11.0 Hz, H-7), 6.20 (1H, d,
J = 11.0 Hz, H-6), 7.38 (3 H, m, Ph-3, 4, 5), 7.52 (2H,
m, Ph-2, 6).
1
27b H NMR d 0.02 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.51 (3H, s, H-18), 0.86 and 0.89 (each
9H, s, t-Bu), 1.22 (9H, s, –COC(CH3)3), 2.82 (1H, m,
H-9), 3.52 (3H, s, –OCH3), 4.43 (2H, m, H-3, 1), 4.52
and 4.56 (each 1H, d, J = 13.7 Hz, H-26), 4.92 and
4.97 (each 1H, s, –C@CH2), 5.01 and 5.14 (each 1H, s,
H-27), 5.32 (1H, m, H-23), 5.82 (1H, d, J = 11.1 Hz,
H-7), 6.21 (1H, d, J = 11.1 Hz, H-6), 7.39 (3H, m, Ph-
3, 4, 5), 7.53 (2H, m, Ph-2, 6).
7.33. (1S)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-
methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)
propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-bute-
nyl (2S)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate
(29b)
7.30. (1S)-1-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(dimethyl)-
silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)pro-
pyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl
(2R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (28b)
Compound 29b was obtained from 26b by the same pro-
cedure as described for 27a (yield 99.8%) as a colorless
oil.
Compound 28b was obtained from 25b by the same pro-
cedure as described for 28a (yield 79.2%) as a colorless
oil.
1
29b H NMR d 0.04 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.29 (3H, s, H-18), 0.88 and 0.89 (each
9H, s, t-Bu), 1.23 (9H, s, –COC(CH3)3), 2.79 (1H, m,
H-9), 3.54 (3H, s, –OCH3), 4.42 (2 H, m, H-3, 1), 4.53
and 4.58 (each 1H, d, J = 13.6 Hz, H-26), 4.93 and
4.97 (each 1H, s, –C@CH2), 5.00 and 5.12 (each 1H, s,
H-27), 5.40 (1H, m, H-23), 5.80 (1H, d, J = 11.2 Hz,
H-7), 6.19 (1H, d, J = 11.2 Hz, H-6), 7.36 (3H, m, Ph-
3, 4, 5), 7.53 (2H, m, Ph-2, 6).
1
28b H NMR d 0.03 and 0.05 and 0.06 and 0.08 (each
3H, s, SiMe), 0.54 (3H, s, H-18), 0.86 and 0.90 (each
9H, s, t-Bu), 1.21 (9H, s, –COC(CH3)3), 2.82 (1H, m,
H-9), 3.53 (3H, s, –OCH3), 4.42 (2H, m, H-3, 1), 4.43
(2H, s, H-26), 4.84 (1H, s, H-27), 4.92 (1H, s,
–C@CH2), 4.97 (2H, s, H-27, –C@CH2), 5.33 (1H,

472
N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
7.34. (1S)-1-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-
methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-yl)
propyl]-3-{[(2,2-dimethylpropanoyl)oxy]methyl}-3-butenyl
(2R)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate (30b)
7.38. (5S)-5-{(2S)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-meth-
ylene-9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylene-
dihydro-2(3H)-furanone (LAC82 b)
LAC82b was obtained from 22a by the same procedure
as described for LAC67 (yield 51.1%). The compound
was identified as LAC82b by HPLC analysis; Lichro-
spher Si 60, 5 lm, hexane/AcOEt = 1:1, flow rate
2 mL/min, retention time 8.9 min.
Compound 30b was obtained from 26b by the same pro-
cedure as described for 28a (yield 46.5%) as a colorless oil.
1
30b H NMR d 0.03 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.44 (3H, s, H-18), 0.87 and 0.90 (each
9H, s, t-Bu), 1.23 (9H, s, –COC(CH3)3), 2.81 (1H, m,
H-9), 3.48 (3H, s, –OCH3), 4.42 (2H, m, H-3, 1), 4.49
and 4.54 (each 1H, d, J = 13.5 Hz, H-26), 4.93 (2H, s,
H-27, –C@CH2), 4.97 (1H, s, –C@CH2), 5.06 (1H, s,
H-27), 5.37 (1H, m, H-23), 5.83 (1H, d, J = 11.1 Hz,
H-7), 6.20 (1H, d, J = 11.1 Hz, H-6), 7.39 (3H, m, Ph-
3, 4, 5), 7.53 (2H, m, Ph-2, 6).
7.38.1. Competitive-binding assay, rat VDR. The rat
VDR–LBD (amino acids 115–423) was expressed as an
amino-terminal His-tagged protein in Escherichia coli
BL21 (DE3) pLys S (Novagen).37 The cells were lysed
by sonication and the supernatants were diluted 1000
times in 50 mM Tris buffer (100 mM KCl, 5 mM
DTT, 0.5% Chaps, pH 7.5) containing bovine serum
albumin (100 lg/mL). This solution of crude rVDR–
LBD was pipetted into glass culture tubes. A solution
containing an increasing amount of 1a,25-(OH)2D3 or
the synthetic analogs in 15 lL of EtOH was added to
the receptor solution in each tube and the mixture was
vortexed 2–3 times. The mixture was incubated for 1 h
at room temperature. [3H]-1,25-(OH)2D3 (specific
activity, 6.62 TBq/mmol, ca. 5000 dpm) in 15 lL of
EtOH was added, vortexed 2–3 times, and the whole
mixture was then allowed to stand at 4 ꢁC for 18 h.
At the end of the second incubation, 200 lL of dex-
tran-coated charcoal suspension (purchased from Ya-
masa Shoyu) was added to bind any free ligands (or
to remove free ligands) and the sample was vortexed.
After 30 min at 4 ꢁC, bound and free [3H]-1,25-
(OH)2D3 were separated by centrifugation at
3000 rpm for 15 min at 4 ꢁC. Aliquots (500 lL) of
the supernatant were mixed with 9.5 mL of ACS-II
scintillation fluid (Amersham, Buckinghamshire,
U.K.) and submitted for radioactivity counting. Each
assay was performed at least twice in duplicate.
7.35. (5R)-5-[(2R)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-
methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-
17-yl)propyl]-3-methylenedihydro-2(3H)-furanone (21a)
To a solution of diol 23a (11.4 mg, 0.017 mmol) in dry
CH2Cl2 (2 mL) was added MnO2 (77.7 mg, 0.89 mmol),
and the mixture was stirred at room temperature for
76 h. The mixture was chromatographed on silica gel
(5 g, hexane/AcOEt = 4:1) to give methylene lactone
21a (9.4 mg, 83.1%).
1
21a H NMR d 0.02 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.57 (3H, s, H-18), 0.86 and 0.90 (each
9H, s, t-Bu), 1.02 (3H, d, J = 6.5 Hz, H-21), 4.43 (2H,
m, H-3, 1), 4.65 (1H, m, H-23), 4.91 and 4.97 (each
1H, s, –C@CH2), 5.62 (1H, t, J = 2.4 Hz, H-27), 5.84
(1H, d, J = 11.2 Hz, H-7), 6.22 (1H, d, J = 11.2 Hz, H-
6), 6.23 (1H, t, J = 2.7 Hz, H-27).
7.36. (5S)-5-[(2S)-2-((1R,3R,7E)-1,3-Bis{[tert-butyl(di-
methyl)silyl]oxy}-2-methylene-9,10-secoestra-5,7-dien-17-
yl) propyl]-3-methylenedihydro-2(3H)-furanone (22b)
7.38.2. Transfection and transactivation assay. Cos7 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-GGTTCAcgaGGTTCA,
SPPx3-TK-Luc), a wild-type hVDR expression plasmid
(pCMX-hVDR), and the internal control plasmid con-
taining sea pansy luciferase expression constructs
(pRL-CMV) by the lipofection method as described
previously.18,23,24 After 4 h incubation, the medium
was replaced with fresh DMEM containing 5% char-
coal-treated FCS (HyClone, UT, USA). The next day,
the cells were treated with either the ligand or ethanol
vehicle and cultured for 24 h. Cells in each well were
harvested with a cell lysis buffer, and the luciferase activ-
ity was measured with a luciferase assay kit (Toyo Ink,
Inc., Japan). Transactivation measured by the luciferase
activity was normalized with the internal control. All
experiments were done in triplicate.
Compound 22b was obtained from 24b by the same pro-
cedure as described for 21a (yield 86.9%).
1
22b H NMR d 0.02 and 0.05 and 0.07 and 0.08 (each
3H, s, SiMe), 0.56 (3H, s, H-18), 0.86 and 0.90 (each
9H, s, t-Bu), 0.95 (3H, d, J = 6.5 Hz, H-21), 4.42 (2H,
m, H-3, 1), 4.64 (1H, m, H-23), 4.92 and 4.97 (each
1H, s, –C@CH2), 5.62 (1H, t, J = 2.4 Hz, H-27), 5.84
(1H, d, J = 11.2 Hz, H-7), 6.21 (1H, d, J = 11.2 Hz, H-
6), 6.23 (1H, t, J = 2.8 Hz, H-27). MS m/z (%): 654
(M+, 3), 522 (10), 454 (12), 440 (12), 366 (15), 313
(40), 147 (20), 75 (100), 73 (70).
7.37. (5R)-5-{(2R)-2-[(1R,3R,7E)-1,3-Dihydroxy-2-meth-
ylene-9,10-secoestra-5,7-dien-17-yl]propyl}-3-methylen-
edihydro-2(3H)-furanone (LAC67a)
LAC67a was obtained from 21a by the same procedure
as described for LAC67 (yield 64.9%). The compound
was identified as LAC67a by HPLC analysis; Lichrosorb
Si 60, 5 lm, Hexane/AcOEt = 7:3, flow rate 2 mL/min,
retention time 34.2 min.
7.38.3. Cell culture and cotransfection assay. Human
embryonic kidney (HEK) 293 cells were cultured in

N. Yoshimoto et al. / Bioorg. Med. Chem. 16 (2008) 457–473
473
(b) Shimizu, M.; Iwasaki, Y.; Shibamoto, Y.; Sato, M.; DeLuca,
H. F.; Yamada, S. Bioorg. Med. Chem. Lett. 2003, 13, 809.
14. Ono, K.; Yoshida, A.; Saito, N.; Fujishima, T.; Honzawa,
S.; Suhara, Y.; Kishimoto, S.; Sugiura, T.; Waku, K.;
Takayama, H.; Kittaka, A. J. Org. Chem. 2003, 68, 7407.
15. Okuda, Y.; Nakatsukasa, S.; Oshima, K.; Nozaki, H.
Chem. Lett. 1985, 481.
16. Saito, N.; Saito, H.; Anzai, M.; Yoshida, A.; Fujishima,
T.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama,
H.; Kittaka, A. Org. Lett. 2003, 5, 4859.
17. Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H.
J. Am. Chem. Soc. 1991, 113, 4092.
18. Shimizu, M.; Miyamoto, Y.; Kobayashi, E.; Shimazaki,
M.; Yamamoto, K.; Reischl, W.; Yamada, S. Bioorg. Med.
Chem. 2006, 14, 4277.
DMEM containing 5% FBS, 100 U/mL penicillin, and
100 lg/mL streptomycin at 37 ꢁC in a humidified atmo-
sphere containing 5% CO2. Transfections in HEK293
cells were performed by the calcium phosphate coprecip-
itation assay as described previously.31,38 Eight hours
after transfection, compounds were added. Cells were
harvested after 16–24 h and were assayed for luciferase
and b-galactosidase activities using a luminometer and a
microplate reader (Molecular Devices, Sunnyvale, CA).
Luciferase data were normalized to the internal b-galacto-
sidase control and represent the means SD of triplicate
assays.
7.38.4. Graphical manipulations and ligand docking.
Graphical manipulations were performed using SYBYL
7.3 (Tripos, St. Louis). The atomic coordinates of the
human VDR–LBD (residues 118–427 D166–216) crystal
structure were retrieved from Protein Data Bank
(code: 2a-methyl-1,25-(OH)2D3,29 2HB8; 20-epi-1,25-
(OH)2D3,26 1IE9). LAC67a and LAC67b were docked
into the ligand-binding pocket of the VDR–LBD29 man-
ually by superposition with the 2a-methyl-1,25-(OH)2D3
at the A- to D-ring. LAC67a and LAC67b in the LBP of
the VDR–LBD were minimized on Tripos force field
with 100 times iterations.
19. Saito, N.; Matsunaga, T.; Fujishima, T.; Anzai, M.; Saito,
H.; Takenouchi, K.; Miura, D.; Ishizuka, S.; Takayama,
H.; Kittaka, A. Org. Biomol. Chem. 2003, 1, 4396.
20. Yamamoto, K.; Masuno, H.; Choi, M.; Nakashima, K.;
Taga, T.; Ooizumi, H.; Umesono, K.; Sicinska, W.;
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