Analogs of 1α,25-dihydroxyvitamin D3 with high potency in induction of osteoclastogenesis and prevention of dendritic cell differentiation: Synthesis and biological evaluation of 2-substituted 19-norvitamin D analogs

Article abstract of DOI:10.1016/j.bmc.2006.02.015

In our previous papers, we found that introduction of a substituent at C(2) into 1α,25-dihydroxy-19-norvitamin D₃ (2a) caused dramatic changes in binding affinity for the vitamin D receptor (VDR) and in transcriptional activity compared with th

Full text of DOI:10.1016/j.bmc.2006.02.015

Bioorganic & Medicinal Chemistry 14 (2006) 4645–4656
Analogs of 1a,25-dihydroxyvitamin D3 with high potency
in induction of osteoclastogenesis and prevention of dendritic
cell differentiation: Synthesis and biological evaluation
of 2-substituted 19-norvitamin D analogs
Mika Shimazaki,a,* Yukiko Miyamoto,a Keiko Yamamoto,a Sachiko Yamada,a
Masamichi Takami,b Toshimasa Shinki,b Nobuyuki Udagawac and Masato Shimizud,*
aInstitute of Biomaterials and Bioengineering Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai,
Chiyoda-ku, Tokyo 101-0062, Japan
bLaboratory of Biochemistry at School of Dentistry, Syowa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
cDepartment of Biochemistry, Matsumoto Dental University, 1780 Gobara, Hiro-oka, Shiojiri, Nagano 399-0781, Japan
dLaboratory of Medicinal Chemistry, School of Biomedical Science, Tokyo Medical and Dental University,
2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Received 5 January 2006; revised 6 February 2006; accepted 7 February 2006
Available online 2 March 2006
Abstract—In our previous papers, we found that introduction of a substituent at C(2) into 1a,25-dihydroxy-19-norvitamin D3 (2a)
caused dramatic changes in binding affinity for the vitamin D receptor (VDR) and in transcriptional activity compared with the
parent compound. To investigate the broad biological activity of 2-substituted 19-norvitamin D analogs, we synthesized two
new (20S)-2-hydroxyethylidene-19-norvitamin D derivatives (3b and 4b) and a total of 16 A-ring-modified analogs including 3b
and 4b were tested for the following in vitro and in vivo biological activities: (1) affinity for the VDR, (2) transcriptional activity,
(3) osteoclast formation, (4) bone calcium mobilization in rats, and (5) effects on differentiation of dendritic cells (DCs). The bio-
logical effects of the analogs were compared with those of 1a,25-dihydroxyvitamin D3 (1a) and 2MD, which is being developed for
the treatment of osteoporosis. The efficacy of the (20S)-19-norvitamin D analogs with 2-hydroxyethylidene, 2-hydroxyethoxy, and
2-methyl moieties (3b, 5b, 6b, and 9b) was more than 10-fold stronger than that of 1a with respect to transcriptional activity, ability
to induce osteoclast formation, and ability to inhibit CD86 expression, a marker of mature DCs, and was similar to that of 2MD.
The (20S)-2b-hydroxyethoxy derivative 6b was 2 orders of magnitude more active than 1a and approximately twice as potent
as 2MD in preventing CD86 production. The 2-epoxy derivatives 7 and 8 were relatively poor ligands for the VDR and exhibited
activity lower than that of the natural hormone 1a.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
of cells, including cells of the immune system.1 In addi-
tion to the roles described above, recent evidence
suggests that 1,25-(OH)2D3 functions in diverse physio-
logical processes, such as hair follicle cycling2,3 and
blood pressure regulation.4,5
1a,25-Dihydroxyvitamin D3 [1,25-(OH)2D3, 1a] is a
seco-steroid hormone having flexible structural features.
1,25-(OH)2D3 exerts its hormonal effects predominantly
on intestine, bone, and kidney, where it plays a crucial
role in calcium and phosphorus homeostasis and bone
mineralization.1 The active vitamin D hormone also reg-
ulates the differentiation and function of various types
The biological effects of 1,25-(OH)2D3 are mediated by
the vitamin D receptor (VDR), which is a member of
a superfamily of nuclear hormone receptors functioning
as ligand-activated transcription factors.6 Ligand bind-
ing allows the VDR to heterodimerize with the retinoid
X receptor (RXR), which is the nuclear receptor for
9-cis-retinoic acid, and the VDR/RXR heterodimer
complex binds with high affinity to vitamin D responsive
elements (VDREs) located in the promoter regions of
Keywords: 2-Substituted 19-norvitamin D analog; Osteoclast forma-
tion; Dendritic cell; Immune tolerance.
*
Corresponding authors. Tel.: +81 03 5280 8117; fax: +81 03 5280
8005; e-mail: shimizu.mr@tmd.ac.jp
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.015

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M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
target genes. Once bound to DNA, the VDR/RXR
dimer recruits coactivators such as SRC-1 and DRIP,
and initiates gene transcription. The newly synthesized
proteins elicit the physiologic responses characteristic
of vitamin D.
The VDR is found in various cells of the immune sys-
tem. The immune effects of 1,25-(OH)2D3 are mainly
mediated through its action on dendritic cells (DCs).28
Recent studies have demonstrated that 1,25-(OH)2D3
inhibits the differentiation and maturation of DCs,
which are critical antigen-presenting cells in the induc-
tion of T-cell-mediated immune responses.29 In vitro,
treatment of DCs with 1,25-(OH)2D3 leads to down-reg-
ulated expression of the costimulatory molecules CD40,
CD80, and CD86 and to decreased IL-12 and enhanced
IL-10 production, resulting in decreased T cell activa-
tion.30 This combination of functional effects of 1,25-
(OH)2D3 in DCs is, in part, linked to induction of
DCs with tolerogenic properties.
1,25-(OH)2D3 has been widely used as a therapeutic
agent in the treatment of osteoporosis, rickets, second-
ary hyperparathyroidism, and psoriasis, but it has a
narrow therapeutic window limited by the development
of critical hypercalcemia.1 In attempts to overcome this
limitation, more than three thousand 1,25-(OH)2D3
analogs have been synthesized in a search for com-
pounds with the required cellular effects but lacking
the calcemic side effects.7 Currently, six synthetic ana-
logs of 1,25-(OH)2D3 are marketed for the treatment
of secondary hyperparathyroidism and psoriasis. Addi-
tionally, at least three analogs are now undergoing clin-
ical trials for the chemotherapy of osteoporosis8,9 and
various cancers.10,11 These studies have shown that
deletion of the 19-exomethylene moiety results in
considerable suppression of the calcemic activity of
1,25-(OH)2D3 but preserves its cell differentiation activ-
ities.12,13 19-Nor-1a,25-dihydroxyvitamin D2 (paricalci-
tol) has been approved for the treatment of secondary
hyperparathyroidism and does not cause significant
hypercalcemia. Shevde et al. found that another
In our investigation of the structure-activity relation-
ships (SAR) of a series of 19-norvitamin D analogs,
we have focused on modification of the A-ring at C(2).
Modification of the C(2) position is of key importance
in modulating the biological properties of vitamin D.
We have synthesized a number of 2-substituted 19-nor-
vitamin D analogs and described their biological activi-
ties.19,20 We have found that introduction of
hydroxyethylidene or hydroxyethoxy groups at C(2)
causes dramatic changes in the activity profile compared
with the parent vitamin D. In the VDR-binding assay,
the 2b-hydroxyethoxy-19-norvitamin D analog 6b is 5-
fold more active than 1,25-(OH)2D3, and this to our
knowledge is the strongest affinity for the VDR among
the known 19-norvitamin D analogs; 6b also has dra-
matically enhanced transcriptional activity (30-fold
higher than that of 1a).19 In this paper, we synthesized
two new 19-norvitamin D derivatives (3b and 4b) and
performed a detailed evaluation of the biological activi-
ties of sixteen 2-substituted 19-norvitamin D analogs
with 20R- or 20S-configuration in comparison with
those of the natural hormone (1a) and the established
analog 2MD (2b) (Fig. 1).
19-norvitamin
D analog, 2-methylene-19-nor-(20S)-
1a,25-dihydroxyvitamin D3 (2MD, 2b), exhibits the
same affinity for the VDR as does 1,25-(OH)2D3 and
shows a preferential activity on bone relative to intes-
tine.9,14–16 2MD also stimulates bone formation both
in vitro and in vivo, and is now in phase II clinical tri-
als. 2b-Hydroxypropoxy-1a,25-dihydroxyvitamin D3
(ED-71) has a significant affinity for the VDR, one-
eighth that of 1,25-(OH)2D3, and its affinity for vitamin
D binding protein (DBP) is about 3-fold that of 1,25-
(OH)2D3.8,17 In an ovariectomized rat model for osteo-
porosis, ED-71 increases bone mass to a greater extent
than does 1,25-(OH)2D3. ED-71 is a promising thera-
peutic candidate for treatment of osteoporosis, and
phase III clinical trials are under way. Introduction
of substituents at the C(2) position dramatically chang-
es the biological profile of vitamin D analogs, as seen
in 2MD, ED-71, and a series of 2-substituted 19-norvi-
tamin D analogs synthesized in our laboratory.18–20
Many 2-substituted vitamin D analogs exhibit in-
creased binding affinity for the VDR and for DBP,
high HL-60 cell differentiating activity, and high calce-
mic potency.14,21–24
2. Results and discussion
2.1. Synthesis
For the synthesis of 3b and 4b, we used a Wittig-Horner
reaction of the A-ring phosphine oxide 11 with the 20-
epi-25-hydroxy Grundmann’s ketone 12 (Fig. 2). The
A-ring phosphine oxide 11 (ca. 2:1 diastereomeric mix-
ture), prepared from D-glucose as reported,18 was treat-
ed with the C/D-ring ketone 12 to afford 13 (66%, ca. 3:2
diastereomeric mixture), which was selectively deprotec-
ted to yield 14 (46%). The 2-hydroxy-19-norvitamin D
derivative 14 was oxidized to the 2-oxo derivatives 15
(86% as a single compound) under Swern’s conditions.
Cyanomethylation of 15 with diethyl (cyanometh-
yl)phosphonate gave 16 (99%) as an approximately 1:1
mixture of E and Z isomers at C(2), which was reduced
with diisobutylaluminum hydride followed by sodium
borohydride to afford the 2-hydroxyethylidene deriva-
tive 17 as a separable isomeric mixture (17a, 27%; 17b,
25%). Deprotection of 17a and 17b with camphor sul-
fonic acid yielded (E)- and (Z)-hydroxyethylidene-19-
norvitamin D analogs 3b and 4b, respectively. The
1,25-(OH)2D3 induces osteoclast formation.25–27 Bone-
resorbing, multinucleated active osteoclasts are induced
by cell-to-cell contact of osteoblasts with osteoclast pre-
cursors. Receptor activator of nuclear factor-j B ligand
(RANKL) is an essential factor for osteoclastogenesis.
RANKL is expressed on the surface of osteoblasts and
binds to RANK (the receptor for RANKL) present on
the surface of osteoclast precursors, stimulating the dif-
ferentiation, fusion, and activation of osteoclasts. 1,25-
(OH)2D3 activates the expression of RANKL in the
osteoblast and thus induces osteoclast formation in a
dose-dependent fashion.

M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4647
R
R
R
R
S
a:
b:
OH
OH
OH
OH
HO
HO
R2
1α,25-(OH)2D3 (1a)
20S-1α,25-(OH)2D3 (1b)
19 ND (2a; R2 = H2)
2MD (2b; R2 = CH2)
R
R
R
R
OH
OH
HO
HO
HO
OH
HO
OH
OH
HO
OH
O
O
OH
6
3 (E-isomer)
4 (Z-isomer)
5
R
R
R
R
OH
8 (2α-epoxide)
HO
OH
OH
HO
HO
OH
HO
O
O
HO Me
Me OH
7 (2β-epoxide)
9
10
Figure 1. Structures of 1,25-(OH)2D3 and its analogs.
stereochemistries of the 2-hydroxyethylidene analogs 3b,
4b, 17a, and 17b at C(2) were determined by their phase-
sensitive 2D NOESY spectra. In 3b and 17a, an NOE
was observed between H-1 and vinyl proton (d 5.80
for 3b, d 5.72 for 17a). A correlation cross peak was ob-
served between H-3 and vinyl proton (d 5.83 for 4b, d
5.72 for 17b) in 4b and 17b.
nal hydroxyl group of substituents at C(2). The binding
of vitamin D analogs to the VDR is markedly affected
by their configuration at C(20): 20S-isomers have a
higher affinity than do 20R-isomers.7 Comparison of
the receptor binding of the C(2) isomeric pairs of the
19-norvitamin D analogs 3–10 was consistent with the
reported findings.7 Interestingly, the (2E)-hydroxyethy-
lidene-19-norvitamin D derivative 3b with 20S-configu-
ration showed an even weaker affinity for the VDR
than that of the 20R-counterpart 3a, whereas 20-epimer-
ization markedly increased the binding potency for the
Z-isomers (4a vs 4b). Similar trends were also observed
for (2E)- and (2Z)-ethylidene-19-norvitamin D analogs,
which were first developed by DeLuca’s group.22 19-
Norvitamin D analogs 5, 8, and 10, whose hydrophilic
oxygen atom or hydroxyl group occupies the a-configu-
ration at the C(2) position, bound less well to the VDR
than did the corresponding counterparts 6, 7, and 9.
Within the VDR ligand-binding pocket (LBP), above
the A-ring, a narrow space is open for hydrophobic
interactions with the adjacent LBP amino acid residues
Leu233, Phe150, and the phenyl ring of Tyr236. Com-
pounds 5, 8, and 10 can make unfavorable interactions
within the LBP, explaining their decreased binding affin-
ity for the VDR.
2.2. Biological activity
2.2.1. VDR affinity and transcriptional activity. The 2-
substituted 19-norvitamin D analogs were examined
for their affinity for the bovine thymus VDR; the results
are summarized in Table 1.19,20 The VDR-binding affin-
ities of the analogs 3a and 6a relative to the natural
hormone 1a were 2-fold greater and equivalent, respec-
tively. In the crystal structures of the human or rat
VDR ligand-binding domain (LBD) in complex with
1,25-(OH)2D3 (1a) and its derivatives, C(19)H2 of the li-
gands interacts with Leu233.31–35 19-Norvitamin D ana-
logs with no 19-exomethylene group exhibited lower
affinity for the VDR relative to 1a. Introduction of
2E-hydroxyethylidene or 2b-hydroxyethoxyl moieties
at the C(2) position compensates for the loss of the
van der Waals contact at C(19)H2 with Leu233. Dock-
ing studies of analogs 3a and 6a with the structural mod-
ifications described above show that the backbone
carbonyl of Asp144 is hydrogen-bonded with the termi-
The VDR-mediated transcriptional activity of the 19-
norvitamin D analogs was tested by luciferase assay

4648
M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4
A
OTES
OR
3
1,25-D3
3a
1) n-BuLi
2) aq. AcOH
O
2
4a
12
+
7a
P(O)Ph2
OTBS
8a
1
OTBS
TBSO
OR'
TBSO
0
13: R = TES; R' = TMS
(ca. 3:2; 66%)
14: R = R' = H (46%)
OTMS
11
0.001
0.01
0.1
1
10
100
1000
Conc. (nM)
1.6
1.2
0.8
0.4
0
B
OH
1,25-D3
3b
1) (EtO2)2P
O
CH2CN
DMSO
4b
2) DIBAL-H
3) NaBH4
(COCl)2
7b
8b
OTBS
15 (86%)
TBSO
O
0.0001 0.001 0.01
0.1
1
10
100 1000
Conc. (nM)
OH
2.4
C
CSA
1.8
1.2
0.6
0
3b, 4b
1,25-D3
5a
6a
OTBS
TBSO
9a
10a
R
16: R = CN (ca. 1:1; 99%)
17: R = CH2OH (52%)
0.001
0.01
0.1
1
10
100
1000
Figure 2. Synthetic scheme of the (20S)-2-hydroxyethylidene-19-nor-
vitamin D analogs.
Conc. (nM)
Figure 3. Dose–response effects of 1,25-(OH)2D3 and 19-norvitamin D
analogs on transcriptional activity. (A) d, 1,25-(OH)2D3
(ED50 = 0.3 nM); n, 3a (ED50 = 0.15 nM); m, 4a (ED50 = 1 nM); j,
7a (ED50 = 6 nM); h, 8a (ED50 = 7 nM). (B) d, 1,25-(OH)2D3
(ED50 = 0.25 nM); j, 3b (ED50 = 0.02 nM); h, 4b (ED50 = 0.06 nM);
m, 7b (ED50 = 0.28 nM); n, 8b (ED50 = 1.4 nM). (C) d, 1,25-(OH)2D3
(ED50 = 1.4 nM); h, 5a (ED50 = 0.4 nM); j, 6a (ED50 = 0.2 nM); n,
9a (ED50 = 0.5 nM); m, 10a (ED50 = 24 nM).
Table 1. Relative VDR affinity and transcriptional activity of 2-
substituted 19-norvitamin D analogsa
Compound
a:
b:
OH
OH
VDR
affinityb
Trans.
activityc
VDR
affinityb
Trans.
activityc
1
3
1.0
2.0d
1.0
5.0d
1.6
40d
12.5
4.0
10d
30d
0.2d
0.9d
32d
NT
values calculated from their dose–response curves
(Fig. 3). The two isomeric pairs 3 versus 4 or 9 versus
10 with 20R-configuration exhibited large differences in
transcriptional activity. The four analogs 5 and 6 with
the 2-hydroxyethoxy moiety proved to be much more
active than 1a in spite of their C(2)- and C(20)-configu-
rations, although the 2a-hydroxyethoxy analogs 5 have
an unfavorable hydrophilic substituent at C(2). In a pre-
vious paper,19 to clarify the interaction between the
VDR and the 2-substituted 19-norvitamin D analogs
we performed two-dimensional alanine scanning muta-
tional analysis with the 2a- and 2b-hydroxyethoxy-19-
norvitamin D derivatives; the 2a-isomer 5b showed
striking differences from the native ligand 1a and the
2b-isomer 6b. From the alanine mutational analysis in
combination with a docking model of 5b, we proposed
that 5b docks in the VDR LBP with the A-ring a-con-
formation, which is different from the native hormone
2.0d
0.3d
3.5d
7.0d
0.05d
0.04d
2.8d
0.06d
4
0.007d
0.1d
0.02
1.0d
5.0d
0.5d
0.2d
1.0d
NT
5
6
1.0d
7
0.04d
0.003d
0.3d
8
9
10
0.001d
NT, not tested.
a Potencies of 1,25-(OH)2D3 (1a) are normalized to 1.
b Bovine vitamin D receptor.
c Activity was assessed in terms of ED50
d Refs. 18–20.
.
with mouse osteopontin VDRE in COS-7 cells. The re-
sults are shown in Table 1 and Figure 3.19,20 The relative
activities of test compounds were assessed by the ED50

M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4649
Table 2. Relative potency of 1,25-(OH)2D3 and its analogs in
osteoclast formation and inhibition of CD86 expressiona
Compound
a:
b:
OH
OH
Osteoclast CD86
Osteoclast CD86
formationb inhibitionb formationb inhibitionb
1
2
1.0
1.0
NT
5.0
1.3
1.5
6.0
0.2
0.13
2.0
NT
NT
87
NT
60
NT
1.2
3
77
66
42
4
0.8
1.0
6
50
5
66
87
6
1.0
100
2.5
0.4
25
7
NT
NT
0.95
NT
NT
NT
66
8
9
10
NT
0.43
NT, not tested.
a Potencies of 1,25-(OH)2D3 (1a) are normalized to 1.
b Activity was assessed in terms of ED50
.
in our laboratory, the 2a-methyl-2b-hydroxy analog 9b
is the most potent in transcriptional activity.
2.2.2. Effect of 2-substituted 19-norvitamin D analogs on
osteoclast formation. To define VDR-mediated osteo-
clastogenesis, the effects of 19-norvitamin D analogs
on osteoclast formation were examined in a mouse co-
culture system; the results are summarized in Figure 4
and Table 2. 1,25-(OH)2D3 and 2MD induced multinu-
cleated cells that were positive for tartrate-resistant acid
phosphatase (TRAP, a marker enzyme of osteoclasts),
features typical of osteoclasts. 2MD was approximately
100 times more potent than 1,25-(OH)2D3, as reported
by DeLuca and co-workers15 Osteoclast formation by
the 19-norvitamin D derivatives with 20R-configuration
was dose-dependent with activity similar to that of 1,25-
(OH)2D3, whereas the corresponding 20S-epimers
showed almost the same potency as 2MD. Regardless
of the type of substituent at the C(2) position, 19-norvi-
tamin D analogs with a 20S-configuration were much
more active than their corresponding 20R-counterparts
in eliciting osteoclast formation. These results suggest
that the 20S-side chain structure of 19-norvitamin D
plays a crucial role for VDR-mediated osteoclast differ-
entiation. RANKL produced by osteoblasts is an essen-
tial factor for osteoclastogenesis. Osteoclast precursors
express RANK, which is the receptor for RANKL, rec-
ognize RANKL through a cell–cell contact, and differ-
entiate into osteoclasts. 1,25-(OH)2D3 up-regulates the
expression of RANKL in osteoblasts and 2MD elicits
an approximately 100-fold induction of RANKL com-
pared with 1,25-(OH)2D3.15 In our preliminary experi-
ments, we found that 3b, 6b, and 9b strongly
stimulated RANKL mRNA production in osteoblasts,
showing activity similar to that of 2MD.
Figure 4. Effects of 1,25-(OH)2D3, 2MD, and 19-norvitamin
analogs on osteoclast formation.
D
1a with the A-ring b-conformation.36 In the a-confor-
mation, the 2a-substituent of 5b takes an equatorial con-
formation and the docking model of this compound
suggests that the hydroxyl group at C(2) can form a
hydrogen bond with Asp144.
The analogs 7 and 8 with an oxirane at C(2) showed an
extremely weak transcriptional activity and the differ-
ences between VDR binding activity and transcriptional
activity were found to be small. The 2-methyl-2-hydroxy
analogs 9 and 10 were more potent in transcriptional
activity than were the corresponding epoxy analogs.
Fujishima et al. compared 2-cyclopropyl-1a,25-
(OH)2D3 and 2,2-dimethyl-1a,25-(OH)2D3, demonstrat-
ing that the VDR affinity and cell differentiation-induc-
ing activity of analogs with cyclopropane at C(2) were
similar to those of the natural hormone 1a, whereas
the 2,2-dimethyl analogs showed 3–30% of the activity
of 1a.37,38 These results are different from those for
our analogs with 2-epoxy or 2-methyl-2-hydroxy groups
and suggest that an oxygen atom attached directly to the
C(2) position plays a critical role in VDR binding.
Among the 19-norvitamin D analogs prepared to date
2.2.3. Bone calcium mobilization. 1,25-(OH)2D3 (1a)
induces calcium resorption from bone in vivo and
enhances the expression of RANKL in osteoblasts.
RANKL is a key regulator of osteoclast formation.

4650
M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
Vitamin D-mediated formation of osteoclasts is related
to bone resorption activity.
2.2.4. Effects on differentiation and maturation of den-
dritic cells. We studied the effect of 19-norvitamin D
analogs on differentiation and maturation of dendritic
cells (DCs) differentiated from mouse bone marrow
macrophages obtained from bone marrow cells cultured
with M-CSF for 4 days. First, the bone marrow macro-
phages were cultured for 3 days in the presence of GM-
CSF without or with 19-norvitamin D analogs. On day
7 of culture, cells were harvested and analyzed by fluo-
rescence-activated cell sorting (FACS) for surface
expression of CD11c, which is a marker for immature
DCs. Figure 5A shows a representative experiment
studying surface phenotype. Expression of CD11c was
not affected by the 19-norvitamin D analogs at concen-
trations of 10ꢀ10–10ꢀ7 M. Similar results were obtained
with 1,25-(OH)2D3-treated macrophages. This result
suggests that the synthetic 19-norvitamin D analogs do
not affect differentiation from bone marrow macro-
phages to immature DCs.
To investigate their support of calcium mobilization
from bone (BCM), the calcemic activities of 19-norvita-
min D analogs were tested in vitamin D-deficient rats on
a low-calcium diet (Table 3). Our experimental model
studied the time course of BCM at 6, 12, and 24 h after
a single dose of 1 lg of 1,25-(OH)2D3 or the 2-substitut-
ed 19-norvitamin D analogs. In this model, administra-
tion of vitamin D-related compounds elevated the serum
level of calcium, and different compounds showed differ-
ent time courses with respect to peak calcium level. The
natural hormone 1a exhibited peak activity around 12 h
after administration. 2MD also showed peak activity at
12 h and had a stronger effect than 1a on serum calcium,
concomitant with its greater potency for osteoclast for-
mation. Comparing the times of maximal effect, the
2E-hydroxyethylidene derivatives 3a and 3b were more
effective than 1a, whereas the 2Z-isomers 4a and 4b were
slightly less effective than and equal to 1a, respectively.
The four 2-hydroxyethoxy analogs, except for 5a, were
slightly more potent than 1a on calcemic activity. In
contrast with the 66-fold difference of relative potency
of 9b to 1a in osteoclast formation, the 2a-methyl-2b-hy-
droxy analog 9b exhibited only weak calcemic activity
compared with 1a. Our present data demonstrate that
substitution of 19-norvitamin D at the C(2) position
has variable effects on calcemic activity. These results
suggest that alteration of C(20) stereochemistry is not
essential for calcium-regulatory potency.
Immature DCs obtained by 2-day culture with GM-CSF
can be induced to form mature DCs with potent anti-
gen-presenting properties by incubation with lipopoly-
saccharide (LPS). DC maturation is accompanied by
enhanced production of the maturation marker CD86.
Immature DCs were cultured with LPS in the presence
or absence of 19-norvitamin D analogs. The effects of
the vitamin D compounds on DC maturation were com-
pared with those of 1,25-(OH)2D3 (1a) and 2MD (2b) as
positive controls. Figure 5B shows representative flow
cytometric histograms of control cells and of cells cul-
Table 3. Bone calcium mobilization by 1,25-(OH)2D3, 2MD, and 19-norvitamin D3 analogs in vitamin D-deficient rats on a low-calcium dieta
Compound
Serum Ca (6 h)/(mg/dL)
(means SEM)
Serum Ca (12 h)/(mg/dL)
(means SEM)
Serum Ca (24 h)/(mg/dL)
(means SEM)
None (control)
1,25-(OH)2D3 1a
2MD 2b
4.68 0.56b
6.73 0.56c
7.19 0.74d
4.84 0.45b
8.20 0.71c
9.57 0.01d
4.90 0.38b
7.72 0.11c
9.34 0.43d
None (control)
4.23 0.54b
5.16 0.55c
5.95 0.35d
5.98 0.49e
6.18 0.18f
5.58 0.37g
4.51 0.65b
6.70 0.18c
6.47 0.39d
6.14 0.56e
7.58 0.51f
6.68 0.29g
5.16 0.44b
6.55 0.69c
7.73 0.89d
5.53 0.28e
8.42 0.39f
6.71 0.31g
1a
3a
4a
3b
4b
None (control)
5.12 0.57b
8.02 0.62c
7.92 0.16d
8.54 0.24e
8.73 0.61f
8.15 0.67g
5.17 0.25b
7.19 0.18c
7.20 0.46d
7.04 0.47e
8.32 0.25f
8.79 0.97g
4.63 0.31b
7.10 0.24c
6.10 0.60d
7.99 0.48e
7.54 0.49f
8.00 0.56g
1a
5a
6a
5b
6b
None (control)
3.30 0.42b
5.25 0.38c
5.06 0.33d
4.71 0.52e
4.59 0.52b
7.63 0.69c
6.47 0.15d
7.33 0.50e
3.33 0.37b
6.77 0.43c
5.94 0.09d
7.28 0.74e
1a
9a
9b
Statistical analysis was done by Student’s t-test. Serum Ca at 6 h, panel 1: b from c and d, P < 0.01; panel 2: b from c, d, f, and g, P < 0.001, b from e,
P < 0.01; panel 3: b from c, P < 0.05, b from d and f, P < 0.001, b from e and g, P < 0.01; panel 4: b from c and e, P < 0.001, b from d, P < 0.01.
Serum Ca at 12 h, panel 1: b from c and d, P < 0.01; panel 2: b from c, P < 0.001, b from d, e, and f, P < 0.01, b from g, P < 0.05; panel 3: b from c, d,
f, and g, P < 0.001, b from e, P < 0.01; panel 4: b from c, d, and e, P < 0.001. Serum Ca at 24 h, panel 1: b from c and d, P < 0.01; panel 2: b from c, e,
f, and g, P < 0.001, b from d, P < 0.01, b from d and f, P < 0.01; panel 3: b from c, d, f, and g, P < 0.001, b from d, P < 0.01; panel 4: b from c and d,
P < 0.001, b from e, P < 0.01.
a Weanling male rats were maintained on the vitamin D-deficient diet containing 0.03% calcium and 0.6% phosphorus for 3 weeks.

M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4651
B
CD86
A
CD11c
Control
1,25-(OH)2D3
6b
Control
70
1,25-(OH)2D3
6b
84
M1
86
86
61
56
M1
M1
M1
M1
M1
10-10
M
10-10
M
10-12
M
10-14
M
52
84
84
51
M1
M1
M1
M1
10-8 M
10-8 M
10-12 M
10-10
M
82
83
M1
6
9
M1
M1
M1
10-7 M
10-7 M
0
-10 M
1
10-8 M
Figure 5. Flow cytometric histograms of CD11c and CD86 expressed by DCs differentiated in the absence or presence of 1,25-(OH)2D3 and 6b. (A)
Open or filled histograms: CD11c expression in the GM-CSF-untreated or treated bone marrow macrophages. (B) Open or filled histograms: CD86
expression in the LPS-untreated or treated immature DCs.
tured with 1,25-(OH)2D3 or with the 2-hydroxyethoxy
derivative 6b. Reduction of CD86 was observed in the
90
A
presence of 1,25-(OH)2D3, with maximal effect occurring
at 10ꢀ8 M. With 6b, however, the maximal inhibitory ef-
fect was observed at 10ꢀ10 M. Dose-dependent inhibi-
tion of DC maturation by 1,25-(OH)2D3 and 19-
norvitamin D analogs is illustrated in Figure 6, and
the experiments are summarized in Table 2. 19-Norvita-
min D analogs prevented the LPS-induced maturation
of immature DCs, maintaining DCs at the immature
stage characterized by low CD86 expression. The fifteen
19-norvitamin D analogs tested, excluding 7a, 8a, 8b,
and 10b, as well as 2MD were more effective than
1,25-(OH)2D3 in inhibiting CD86 induction. The
(20S)-2E-hydroxyethylidene and (20S)-2-hydroxyethoxy
derivatives 3b, 5b, and 6b exhibited 42-, 50-, and 100-
fold higher potency than the natural hormone 1a,
respectively, having potency similar to that of 2MD.
The rank order of potency for osteoclast formation
among the eight test compounds almost paralleled that
for inhibition of CD86 expression. Treatment with 19-
norvitamin D analogs prevented the LPS-induced matu-
ration of immature DCs, maintaining DCs at the imma-
ture stage.
1,25-D3
2MD
5a
60
30
0
6a
5b
6b
1E-07 1E-06 1E-05 1E-04 0.001 0.01 0.1
1
10
100 1000
Conc. (nM)
90
60
30
0
B
3b
4b
9b
10b
IL-12 is a cytokine derived from antigen-presenting cells
that is known to play an important role in T-cell devel-
opment. To investigate the effects of 1,25-(OH)2D3 and
19-norvitamin D analogs on IL-12 mRNA expression
in DCs, we performed reverse-transcription PCR on
RNA extracted from DC cultures, as described in Sec-
tion 4. As shown in Figure 7, 1,25-(OH)2D3 inhibited
IL-12 mRNA expression in DCs in a dose-dependent
manner. Addition of 10ꢀ9 and 10ꢀ10 M 2-hydroxyeth-
1E-07 1E-06 1E-05 1E-04 0.001 0.01
0.1
1
10
100 1000
Conc. (nM)
Figure 6. Dose-dependent inhibition of CD86 expression by 1,25-
(OH)2D3, 2MD, and 19-norvitamin D analogs (A) d: 1,25-(OH)2D3
(IC50 = 0.3 nM); s: 2MD (IC50 = 0.005 nM); n: 5a (IC50 = 0.2 nM); m:
6a (IC50 = 0.05 nM); j: 5b (IC50 = 0.006 nM); h: 6b (IC50 = 0.003 nM).
(B) n: 3b (IC50 = 0.007 nM); m: 4b (IC50 = 0.05 nM); j: 9b
(IC50 = 0.012 nM); h: 10b (IC50 = 0.7 nM).

4652
M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
they display a broad spectrum of biological activity.
LPS
1,25-(OH)2D3
A
20-epi-19-Norvitamin D analogs with these C(2) substit-
uents are characterized by an extremely high potency in
the induction of differentiation of osteoclasts and the
inhibition of differentiation and maturation of dendritic
cells, exhibiting an activity similar to that of 2MD.
These 20S-19-norvitamin D analogs may be useful in
the treatment of bone and autoimmune diseases.
-
+
10-10 10-9 10-8 10-7 10-6 (M)
IL-12
GAPDH
3b
4b
2MD
B
4. Experimental
10-10 10-9
10-10 10-9
10-10 10-9 (M)
1H NMR spectra were obtained on a Bruker ARX-400
spectrometer, operating at 400 MHz. Chemical shifts
are reported in parts per million (ppm, d) downfield
from tetramethylsilane as an internal standard (d
0 ppm). Abbreviations used are: singlet (s), doublet
(d), triplet (t), multiplet (m), aromatic (arom), and
broad signal (br). Low- and high-resolution mass spec-
tra (LR-MS and HR-MS) were obtained with electronic
ionization (EI) on a JEOL JMS-AX505HA spectrome-
ter run at 70 eV for EI; m/z values are given with relative
intensities in parentheses. UV spectra were obtained on
a Beckmann DU-7500 spectrophotometer. Column
chromatography was carried out on silica gel (Wako
Pure Chem. Ind. Ltd. Wakogel C-200), unless otherwise
indicated. All reactions, unless specifically mentioned,
were conducted under an atmosphere of argon gas.
Yields are not optimized. Tetrahydrofuran (THF) was
distilled from Na benzophenone ketyl prior to use. Tol-
uene was distilled from Na. Dimethylsulfoxide (DMSO),
methylene chloride (CH2Cl2), and triethylamine were
distilled from calcium hydride. N,N-Dimethylformam-
IL-12
GAPDH
5b
6b
5a
6a
10-10 10-9
10-10 10-9 10-10 10-9 (M)
10-10 10-9
IL-12
GAPDH
Figure 7. Effects of 1,25-(OH)2D3, 2MD, and 19-norvitamin
analogs on the expression of IL-12 mRNA in DCs.
D
oxy-19-norvitamin D analogs 5 and 6 to DC culture
resulted in a dose-dependent reduction in IL-12 mRNA
expression. Of the four 2-hydroxyethoxy derivatives, the
(20S)-2b-hydroxyethoxy derivative 6b markedly de-
creased IL-12 mRNA expression at 10ꢀ9 M. 2MD also
exhibited activity higher than that of 1a in suppression
of IL-12 mRNA expression. The (20S)-2b-hydroxyeth-
oxy analog 6b inhibited both CD86 expression and
IL-12 mRNA expression in DCs. DCs play a key role
in initiation of the immune response.
˚
ide (DMF) was distilled from 4 A molecular sieves.
4.1. (20S)-1a-[(tert-Butyl-dimethylsilyl)oxy]-2-[(trimeth-
ylsilyl)oxy]-25-[(triethylsilyl)oxy]-19-norvitamin D3 tert-
butyl-dimethylsilyl ether (13)
Immature DCs are positive for CD11c and CD86,
whereas mature DCs express up-regulated levels of
CD86. Surface expression levels of CD11c were not
inhibited by 1,25-(OH)2D3 or by 19-norvitamin D ana-
logs, whereas those compounds markedly prevented
up-regulation of CD86. Our data suggest that 19-norvi-
tamin D analogs prevent differentiation from immature
DCs to mature DCs. In addition, the effect of 19-norvi-
tamin D analogs on mature DCs, leading to inhibition
of IL-12 production, may be, in part, responsible for
the induction of DCs with tolerogenic properties. Vita-
min D analogs able to regulate DC differentiation may
be useful in the treatment of autoimmune diseases and
the prevention of transplant rejection.
To stirred solution of 11 (435.2 mg, 0.660 mmol, ca. 2:1
isomeric mixture) in dry THF (5 mL) at ꢀ78 ꢁC was
added n-BuLi (412 lL, 0.660 mmol, 1.6 M solution in
hexane) and the resulting dark-orange solution was stir-
red for 15 min. To this colored solution was added a
solution of 12 (173.8 mg, 0.440 mmol) in dry THF
(3 mL) and the whole mixture was stirred for 2 h at
ꢀ78 ꢁC. The mixture was quenched with saturated
NH4Cl and extracted with AcOEt. The organic phase
was rinsed with brine, dried over MgSO4, and evaporat-
ed to dryness. The residue was purified by chromatogra-
phy on silica gel (20 g) using 2% AcOEt in hexane to
give 13a, b (243.4 mg, 66% based on 12) as a mixture
of 13a:13b = ca. 3:2 ratio, and 5% AcOEt in hexane to
afford the unreacted starting material 12 (30.0 mg,
17%) and 11 (157.6 mg).
3. Conclusion
We have synthesized a total of sixteen 2-substituted
19-norvitamin D analogs. Their profiles of biological
activity were assessed in terms of affinity for the VDR,
VDR-mediated transcriptional activity, and effects on
osteoclast and dendritic cell differentiation in compari-
son with the natural hormone 1a. The 2-hydroxyethylid-
ene, 2-hydroxyethoxy, and 2-methyl-2-hydroxy analogs
of 19-nor-1,25-(OH)2D3 are extremely interesting as
LR-MS m/z (%): 834 (no M+), 702 (10), 645 (2), 616 (7),
570 (20), 513 (5), 484 (25), 75 (100). Compound 13a (ma-
jor): 1H NMR (CDCl3) d: 0.04, 0.055, 0.058, 0.063 (each
3H, s, 4· Si–Me), 0.12 (9H, 3· Si–Me), 0.54 (3H, s, H-
18), 0.56 (6H, q, J = 7.9 Hz, 3· Si–CH2), 0.85 (3H, d,
J = 6.5 Hz, H-21), 0.87, 0.88 (each 9H, s, 2· Si–t-Bu),
0.94 (9H, t, J = 7.9 Hz, 3· Si–CH2CH3), 1.19 (6H, s,

M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4653
H-26, 27), 2.30 (1H, m), 2.50 (2H, m), 2.79 (1H, m, H-9),
3.54 (1H, m, H-2), 3.80 (1H, m, H-3), 3.88 (1H, m, H-1),
5.81 (1H, d, J = 11.1 Hz, H-7), 6.10 (1H, d, J = 11.1 Hz,
H-6). Compound 13b (minor): 1H NMR (CDCl3) d:
0.04, 0.06 (each 3H, s, 2· Si–Me), 0.07 (6H, s, 2· Si–
Me), 0.12 (9H, 3· Si–Me), 0.53 (3H, s, H-18), 0.56
(6H, q, J = 7.8 Hz, 3· Si–CH2), 0.84 (3H, d,
J = 6.6 Hz, H-21), 0.86, 0.89 (each 9H, s, 2· Si–t-Bu),
0.94 (9H, t, J = 7.8 Hz, 3· Si–CH2CH3), 1.19 (6H, s,
H-26, 27), 2.10 (1H, m), 2.44 (2H, m), 2.79 (1H, m, H-
9), 3.60 (1H, m, H-2), 3.80 (1H, dd, J = 8.7, 4.5 Hz,
H-1), 3.94 (1H, m, H-3), 5.79 (1H, d, J = 11.2 Hz, H-
7), 6.13 (1H, d, J = 11.2 Hz, H-6).
Compound 15: 1H NMR (CDCl3) d : 0.055, 0.065,
0.069, 0.10 (each 3H, s, 4· Si–Me), 0.55 (3H, s, H-18),
0.87, 0.89 (each 9H, s, 2· Si–t-Bu, overlapped with H-
21), 1.22 (6H, s, H-26, 27), 2.45 (1H, dd, J = 13.5,
8.7 Hz), 2.52 (1H, dd, J = 14.2, 4.1 Hz), 2.66 (1H, dd,
J = 13.5, 5.5 Hz), 2.72 (1H, dd, J = 14.2, 6.3 Hz), 2.83
(1 H, m, H-9), 4.35 (1H, dd, J = 6.3, 4.1 Hz), 4.55
(1H, dd, J = 8.7, 5.5 Hz), 5.81 (1H, d, J = 11.2 Hz, H-
7), 6.35 (1H, d, J = 11.2 Hz, H-6). LR-MS m/z (%):
646 (M+, 1), 598 (11), 571 (100), 439 (29). HR-MS m/
z: 571.4009 (M+ꢀt-Bu–H2O) (Calcd for C34H59O3Si2:
571.4003).
4.4. (E)- and (Z)-(20S)-1a-[(tert-Butyl-dimethylsilyl)oxy]-
2-cyanomethylene-25-hydroxy-19-norvitamin D3
tert-butyl-dimethylsilyl ether (16)
4.2. (20S)-1a-[(tert-Butyl-dimethylsilyl)oxy]-2,25-dihy-
droxy-19-norvitamin D3 tert-butyl-dimethylsilyl ether (14)
A solution of 13 (182.5 mg, 0.218 mmol, 13a:13b = ca.
3:2) in THF, AcOH, and water (v/v/v, 8:8:1, 9.5 mL)
was stirred for 2 h at 0 ꢁC and for 20 h at ambient temper-
ature and the mixture was diluted with AcOEt. The
organic layer was successively washed with 5% NaHCO3
and brine, and dried over Na2SO4. The solvent was evap-
orated in vacuo, and the residue was purified by chroma-
tography on silica gel (10 g) using 2% AcOEt in hexane to
give 14a (39.1 mg, 28%) and 14b (26.0 mg, 18%).
To a stirred solution of diethyl (cyanomethyl)phospho-
nate (24 lL, 0.148 mmol) in dry THF (1 mL) at
ꢀ40 ꢁC was added n-BuLi (95 lL, 0.151 mmol, 1.58 M
solution in hexane). The mixture was stirred for
15 min after which time a solution of 15 (48.7 mg,
0.075 mmol) in dry THF (1.2 mL) was added dropwise.
Stirring was continued for 1.5 h at ꢀ40 ꢁC, the mixture
was quenched with saturated NH4Cl, and extracted with
AcOEt. The AcOEt layer was washed with brine dried
over MgSO4, and evaporated in vacuo. The residue
was purified by chromatography on silica gel (5 g) using
10% AcOEt in hexane to afford 16a (E-isomer) and 16b
(Z-isomer) (50.0 mg, 99%) as a mixture of two isomers
in a ratio of ca. 1:1.
LR-MS m/z (%): 648 (M+, 6), 630 (7), 498 (5), 441 (42),
75 (100). HR-MS m/z: 648.4965 (Calcd for C38H72O4Si2:
648.4969). Compound 14a (major): 1H NMR (CDCl3) d:
0.067, 0.077, 0.083, 0.10 (each 3H, s, 4· Si–Me), 0.54
(3H, s, H-18), 0.86 (3H, d, J = 6.6 Hz, H-21), 0.87,
0.88 (each 9H, s, 2· Si–t-Bu), 1.22 (6 H, s, H-26, 27),
2.27 (1H, d, J = 3.2 Hz, OH), 2.31 (1H, dd, J = 12.6,
3.7 Hz), 2.48 (2H, m), 2.79 (1H, m, H-9), 3.51(1H, m,
H-2), 3.91, (1H, m, H-3), 4.00 (1H, m, H-1), 5.80 (1H,
d, J = 11.1 Hz, H-7), 6.15 (1H, d, J = 11.1 Hz, H-6).
LR-MS m/z(%): 669 (no M+), 651 (16), 594 (89), 567
(100), 519 (28), 462 (14), 408 (8). HR-MS m/z:
651.4841 (M+ꢀH2O) (Calcd for C40H69O2NSi2:
651.4867). Compound 16a (E-isomer): 1H NMR
(CDCl3) d : 0.054, 0.067, 0.099, 0.121 (each 3H, s, 4·
Si–Me), 0.55 (3H, s, H-18), 0.83, 0.92 (each 9H, s, 2·
Si–t-Bu), 0.86 (3H, d, J = 6.5 Hz, H-21), 1.22 (6H, s,
H-26, 27), 2.80 (1H, m, H-9), 3.12 (1H, m, H-10), 4.46
(1H, m, H-1), 4.99 (1H, t, J = 2.8 Hz, H-3), 5.47 (1H,
d, J = 1.8 Hz, C@CHCN), 5.82 (1H, d, J = 11.1 Hz,
H-7), 6.19 (1 H, d, J = 11.1 Hz, H-6). Compound 16b
1
Compound 14b (minor): H NMR (CDCl3) d : 0.06,
0.07, 0.08, 0.10 (each 3H, s, 4· Si–Me), 0.53 (3H, s, H-
18), 0.86, 0.90 (each 9H, s, 2· Si–t-Bu, overlapped with
H-21), 1.21 (6H, s, H-26, 27), 2.18 (1H, dd, J = 13.0,
4.5 Hz), 2.39 (3H, m), 2.80 (1H, m, H-9), 3.59 (1H, m,
H-2), 4.00 (2H, m, H-1, 3), 5.80 (1H, d, J = 11.2 Hz,
H-7), 6.18 (1H, d, J = 11.2 Hz, H-6).
1
(Z-isomer): H NMR (CDCl3) d: 0.063, 0.075, 0.112,
0.132 (each 3H, s, 4· Si–Me), 0.54 (3H, s, H-18), 0.83,
0.92 (each 9H, s, 2· Si–t-Bu), 0.86 (3H, d, J = 6.5 Hz,
H-21), 1.22 (6H, s, H-26, 27), 2.80 (1H, m, H-9), 2.99
(1H, m, H-10), 4.57 (1H, m, H-3), 5.04 (1H, t,
J = 2.8 Hz, H-1), 5.47 (1H, d, J = 1.8 Hz, C@CHCN),
5.79, (1H, d, J = 11.1 Hz, H-7), 6.32 (1H, d,
J = 11.2 Hz, H-6, 7).
4.3. (20S)-1a-[(tert-Butyl-dimethylsilyl)oxy]-2-oxo-25-
hydroxy-19-norvitamin D3 tert-butyl-dimethylsilyl
ether (15)
To a stirred solution of oxalyl chloride (18 lL,
0.206 mmol) in dry CH2Cl2 (1 mL) at ꢀ78 ꢁC was added
a solution of DMSO (29 lL, 0.414 mmol) in dry CH2Cl2
(0.2 mL). After 5 min of stirring, a solution of 14
(61.0 mg, 0.094 mmol, 14a:14b = ca. 3:2) in dry CH2Cl2
(1.2 mL) was added dropwise. The reaction mixture was
stirred for 15 min at ꢀ78 ꢁC, and Et3N (131 lL,
0.940 mmol) was added. The whole mixture was stirred
for 30 min at ꢀ78 ꢁC and for 10 min at 0 ꢁC, quenched
with ice water, and extracted with CH2Cl2. The CH2Cl2
extract was washed with brine, dried over MgSO4, and
evaporated to dryness. The residue was purified by chro-
matography on silica gel (5 g) using 20% AcOEt in hex-
ane to afford 15 (52.0 mg, 86%) as a single compound.
4.5. (E)- and (Z)-(20S)-1a-[(tert-Butyl-dimethylsilyl)oxy]-
2-(2-hydroxy-ethylidene)-25-hydroxy-19-norvitamin D3
tert-butyl-dimethylsilyl ether (17)
To a stirred solution of 16 (20.0 mg, 0.030 mmol,
16a:16b = ca. 1:1) in dry toluene (1 mL) at ꢀ78 ꢁC was
added dropwise diisobutylaluminum hydride (60 lL,
0.060 mmol, 1.0 M solution in hexane). The mixture
was stirred for 3 h at ꢀ78 ꢁC and for 1 h at ꢀ20 ꢁC.
Additional diisobutylaluminum hydride (30 lL,
0.030 mmol) was added, and stirring was continued for

4654
M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
5.5 h at ꢀ20 ꢁC. After addition of saturated potassium
sodium tartrate, the mixture was poured into ice water
and extracted with AcOEt. The AcOEt phase was
washed with brine, dried over MgSO4, and concentrated
to dryness. The crude product was dissolved in ethanol
(EtOH, 1 mL) and sodium borohydride (NaBH4,
1.1 mg, 0.030 mmol) was added portionwise at 0 ꢁC.
After being stirred for 1 h, the mixture was poured into
ice water and extracted with AcOEt. The organic layer
was washed with brine and dried over MgSO4. Solvent
was removed in vacuo, and the residue was purified by
chromatography on silica gel (4 g) using 15% AcOEt
in hexane to give 17a (5.5 mg, 27%) and 17b (5.0 mg,
25%). The unreacted starting material 16 was recovered
(7.5 mg, 38%).
acid (11.4 mg, 0.049 mmol) in dry MeOH (0.5 mL) gave
4b (4.8 mg, 66%).
Compound 4b: 1H NMR (CDCl3) d: 0.55 (3H, s, H-18),
0.85 (3H, d, J = 6.5 Hz, H-21), 1.21 (6H, s, H-26, 27),
2.21 (1H, br t, J = ꢁ 13 Hz, H-4), 2.33 (1H, dm, H-
10), 2.70 (1H, d, J = 12.8, 4.7 Hz, H-4), 2.82 (2H, m,
H-9, 10), 4.24 (1H, dd, J = 12.6, 6.4 Hz, CH2OH), 4.38
(1H, dd, J = 12.6, 7.4 Hz, CH2OH), 4.46 (1 H, m, H-
3), 4.87 (1H, t, J = 4.2 Hz, H-1), 5.83 (2H, m, H-7,
C@CH), 6.40 (1H, d, J = 11.1 Hz, H-6). LR-MS m/z
(%): 446 (M+, 9), 428 (10), 410 (31), 392 (100), 374
(79), 263 (84). HR-MS m/z: 446.3385 (Calcd for
C28H46O4: 446.3396). UV kmax (EtOH): 246, 254,
263 nm.
1
Compound 17a (E-isomer): H NMR (CDCl3) d: 0.02,
4.6.1. Reagents and animals. 1,25-(OH)2D3 was a gift
from Mercian Corp. (Tokyo, Japan). 19-Norvitamin D
analogs (3–10) and 2MD were synthesized in our labora-
tory18–20 and these structures are shown in Figure 1.
0.07, 0.08 (3H, 3H, 6H, s, 4· Si–Me), 0.55 (3H, s, H-
18), 0.85, 0.92 (each 9H, s, 2· Si–t-Bu, overlapped with
H-21), 1.22 (6H, s, H-26, 27), 2.29 (2H, m, H-4), 2.79
(1H, m, H-9), 2.88 (1H, dd, J = 12.7, 4.3 Hz, H-10),
4.19 (1H, dd, J = 12.7, 6.8 Hz, CH2OH), 4.31 (1H, dd,
J = 12.7, 6.7 Hz, CH2OH), 4.37 (1H, dd, J = 9.7,
4.3 Hz, H-1), 4.81 (1H, t, J = 3.8 Hz, H-3), 5.72 (1H, t,
J = 6.8 Hz, C@CH), 5.85 (1H, d, J = 11.2 Hz, H-7),
6.15 (1H, d, J = 11.2 Hz, H-6). Compound 17b (Z-iso-
[26,27-Methyl-3H]-1,25-(OH)2D3
(specific
activity
6.62 TBq/mmol) was purchased from Amersham (Buck-
inghamshire, UK). All vitamin D samples were dis-
solved in 95% EtOH. Concentrations of vitamin D
samples were determined by ultraviolet (UV) spectros-
copy using the molar absorptivity e = 34,800 (kmax
254 nm) for 2-ethylidene analogs (3–4), e = 40,500 (kmax
252 nm) for 2-hydroxyethoxy analogs (5–6), e = 32,600
(kmax 251 nm) for 2-epoxy analogs (7–8), and
e = 37,500 (kmax 252 nm) for 2-methyl-2-hydroxy ana-
logs (9–10). Vitamin D receptor (VDR) binding assays
were carried out using a bovine thymus VDR kit (Ya-
masa Shoyu, Co., Ltd, Chiba, Japan). Radioactivity
was determined with a TRI-CARB 1900TR Analyzer
(PACKARD) liquid scintillation counter using a liquid
scintillation cocktail ACS-II obtained from Amersham
(Buckinghamshire, UK). GM-CSF was purchased from
PeproTech EC Ltd, (London, UK). LPS was purified
from Escherichia coil strain K235 as described.39 Five-
to 8-week-old male ddY mice were obtained from Japan
SLC, Inc. (Shizuoka, Japan) and male weanling rats
(Sprague–Dawley strain) were maintained for 3 weeks
on vitamin D-deficient diet containing 0.03% Ca and
0.06% phosphorus (Teklad, Madison, USA).
1
mer): H NMR (CDCl3) d: 0.01, 0.07, 0.08, 0.09 (each
3H, s, 4· Si–Me), 0.54 (3H, s, H-18), 0.83, 0.93 (each
9H, s, 2· Si–t-Bu), 0.85 (3H, d, J = 6.5 Hz, H-21), 1.22
(6H, s, H-26, 27), 2.14 (1H, br t, J = ꢁ11.5 Hz, H-4),
2.55 (1H, dd, J = 12.3, 5.0 Hz, H-4), 2.82 (2H, m, H-9,
10), 4.22 (1H, dd, J = 12.3, 7.1 Hz, CH2OH), 4.30 (each
1H, dd, J = 12.7, 7.0 Hz, CH2OH), 4.47 (1H, m, H-3),
4.86 (1H, t, J = 3.1 Hz, H-1), 5.72 (1H, m, C@CH),
5.81 (1H, d, J = 11.1 Hz, H-7), 6.25 (1H, d,
J = 11.1 Hz, H-6).
4.6. (E)- and (Z)-(20R)-1a,25-Dihydroxy-2-(2-hydroxy-
ethylidene)-19-norvitamin D3 (3b and 4b)
A mixture of 17a (11.0 mg, 0.016 mmol) and (ꢀ)-10-
camphor sulfonic acid (11.4 mg, 0.049 mmol) in dry
MeOH (0.5 mL) was stirred for 2 h at ambient tempera-
ture. NaHCO3 (5%) was added, and the solution was
extracted with AcOEt. The organic phase was washed
with brine, dried over MgSO4, and evaporated in vacuo.
The residue was chromatographed on silica gel (3 g)
using 3% MeOH in AcOEt to afford 3b (6.4 mg, 88%).
4.6.2. Vitamin D receptor binding assay. Bovine thymus
VDR receptors were dissolved in 0.05 M phosphate
buffer (pH 7.4) containing 0.3 M KCl (45 mL) and
5 mM dithiothreitol just before use. The receptor solu-
tions (500 lL) were pre-incubated with an increasing
amount of 1,25-(OH)2D3 (10ꢀ11–10ꢀ6 M) or 19-norvita-
min D analogs (3–10) in 50 lL of EtOH for 60 min at
25 ꢁC. [3H]-1,25-(OH)2D3 (ꢁ5000 cpm) in 50 lL of
EtOH was added, and the receptor mixture was left to
stand overnight at 4 ꢁC. Two hundred microliters of
dextran-coated charcoal suspension (Yamasa Shoyu
Co., Ltd.) was added, and the mixture was vortexed.
After 30 min at 4 ꢁC, the bound-free (B-F) separation
(separation of vitamin bound to VDR [B] from vitamin
D unbound to VDR [F]) was carried out by centrifugation
at 3000 rpm for 10 min. Then, 500 lL aliquot of the
supernatant was mixed with ACS-II (9.5 mL) in each vial,
and the radioactivity was measured with a liquid scintilla-
Compound 3b: 1H NMR (CDCl3) d: 0.54 (3H, s, H-18),
0.86 (3H, d, J = 6.5 Hz, H-21), 1.21 (6H, s, H-26, 27),
2.42 (2H, m, H-4), 2.81 (1H, m, H-9), 3.15 (1H, d,
J = 12.8, 4.9 Hz, H-10), 4.15 (1H, dd, J = 12.4, 5.9 Hz,
CH2OH), 4.39 (2H, m, H-1, CH2OH), 4.84 (1H, m, H-
3), 5.80 (1H, m, C@CH), 5.88 (1H, d, J = 11.1 Hz, H-
7), 6.29 (1H, d, J = 11.1 Hz, H-6). LR-MS m/z (%):
446 (M+, 9), 428 (9), 410 (21), 392 (82), 374 (100), 263
(84). HR-MS m/z: 446.3407 (Calcd for C28H46O4:
446.3396). UV kmax (EtOH): 246 (e 30,700), 254 (e
34,800), 263 (e 23,000) nm.
The same procedure as described above, but using 17b
(11.0 mg, 0.016 mmol) and (ꢀ)-10-camphor sulfonic

M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
4655
tion counter. Each assay was performed at least twice in
duplicate. The relative VDR-binding affinity of the 19-
norvitamin D analogs was calculated by comparison of
the compound concentrations required for 50% displace-
ment of [3H]-1a,25-(OH)2D3 from the receptor protein.
EtOH by jugular injection. After 6, 12, and 24 h, blood
was taken to determine serum calcium and rats were
euthanized. Serum calcium concentration was deter-
mined by colorimetric assay using o-cresolphthalein
complexone (Calcium C-test Wako, Wako Pure chemi-
cal, Osaka, Japan) according to the manufacturer’s
instructions. Serum calcium was measured with a spec-
trometer model BECKMAN DU-600. Statistical analy-
sis was done by Student’s t-test.
4.6.3. Transfection and transactivation assay. COS-7
cells were cultured in Dulbecco’s modified Eagle’s medi-
um (DMEM) supplemented with 5% fetal bovine serum
(FBS, JRH Bioscience, Lenexa, USA). Cells were seed-
ed in 24-well plates at a density of 2 · 104 per well 24 h
before transfection. The next day, the medium was re-
placed with 250 lL of fresh serum-free medium (Opti-
MEM). Then a DNA/Trans IT-LT1 reagent (Mirus,
Madison, USA) mixture containing 0.28 lg of a recep-
tor plasmid (SPP · 3-TK-Luc), 0.2 lg of wild-type
hVDR expression plasmid (pCMX-hVDR), and
0.02 lg of the internal control plasmid containing sea
pansy luciferase expression constructs (pRL-CMV)
was prepared according to the manufacturer’s proce-
dures and added to each well. The SPP · 3-TK-Luc
reporter plasmid contains three copies of the mouse
osteopontin vitamin D response element. The cells were
further incubated for 4 h and the medium was replaced
with fresh DMEM containing 5% FBS pretreated with
dextran-coated charcoal. The next day, transfected cells
were treated with either 1,25-(OH)2D3 (10ꢀ7–10ꢀ11 M),
19-norvitamin D analogs (10ꢀ6–10ꢀ14 M) or EtOH
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 (Toyo
Ink, Inc., Tokyo, Japan) according to the manufactur-
er’s instruction. Transactivation measured by the lucif-
erase activity was normalized with the luciferase
activity of the same cells determined by the sea pansy
luciferase assay kit (Toyo, Ink). All experiments were
done in triplicate.
4.6.6. Preparation of mouse bone marrow macrophage.
Bone marrow cells obtained from 4- to 6-week-old ddY
male mice were suspended in a-MEM supplemented
with 10% FBS in 100-mm diameter dishes (107 cells/
10 mL/dish) in the presence of M-CSF (100 ng/mL).
After the cells were cultured for 4 days, the adherent
cells were harvested by treatment with 0.05% trypsin–
EDTA (Life Technology, Inc.) for 5 min. The harvested
cells were resuspended in RPMI 1640 medium (Sigma)
supplemented with 5% FBS. At day 0, cells were seeded
in 24-well plates (2 · 105 cell /2 mL/well) with GM-CSF
(5 ng/mL) in the presence of increasing concentrations
of 1,25-(OH)2D3 or 19-norvitamin D analogs. On day
2, half of the cells was stained with biotin-conjugated
anti-CD11c antibody followed by streptavidin–fluores-
cein isothiocyanate (BD PharMingen, San Diego,
USA). On the other hand, the other half of the cells
was replaced with fresh medium containing the same
concentration of GM-CSF, 1,25-(OH)2D3, and 19-nor-
vitamin D analogs. LPS (1 lg/mL final concentration)
was added to the cultures to stimulate the maturation
of dendritic cells. After further culturing for 1 day, the
cells were stained with phycoerythrin-conjugated CD86
antibody (from BD PharMingen, San Diego, USA).
The stained cells were assessed on a FACS can flow
cytometer (Becton Dickinson, Mountain View, USA),
and data were analyzed with Cell Quest software (Bec-
ton Dickinson, Mountain View, USA). All data with
standard deviation (SD) are mean values for at least
three independent experiments.
4.6.4. Osteoclast differentiation assay. Bone marrow
cells were obtained from tibiae of 5- to 8-week-old
male mice of the ddY strain. Primary osteoblastic cells
were prepared from the calvariae of newborn ddY
mice as previously described.39,40 Briefly, mouse bone
marrow cells (1.5 · 105 cells/well) and primary osteo-
clasts (3 · 103 cells/well) were cocultured for 7 days
in the presence of 1,25-(OH)2D3, 2MD or 19-norvita-
min D analogs (10ꢀ8–10ꢀ12 M) in 0.3 mL of a-MEM
(Sigma, St. Louis, USA) supplemented with 10%
FBS in 48-well plates. Cells were replenished on day
3 with fresh medium. Cells were then fixed with 10%
formaldehyde in PBS and stained for tartrate-resistant
acid phosphatase (TRAP) as described.40,41 TRAP-
positive multinucleated cells containing three or more
nuclei were counted as osteoclasts, under microscopic
examination. The results were expressed as means
SEM of three cultures.
4.6.7. Total RNA extraction and RT-PCR analysis. For
reverse-transcribed PCR analysis, bone marrow cells
prepared from ddY mice were cultured as described
above. At day 7, total RNA was extracted from mouse
bone marrow cells by the Trizol method (Invitrogen,
CA, USA) according to the manufacturer’s instruction
manual.
Total RNA (1 lg) was reverse-transcribed using Super-
script II (Invitrogen, USA) according to the manufac-
turer’s protocols. Aliquots of the obtained cDNA pool
were subjected to PCR amplification with Go Taq
DNA polymerase (Promega Co., USA). Primers for
mouse IL-12 and GAPDH used in this study are as fol-
lows: IL-12, 50-cagatagcccatcaccctgt-30 (forward), 50-acg
gccagagaaaaactgaa-30 (reverse); and GAPDH, 50-gaaggt
cggtgtgtaacggatttggc-30 (forward), 50-catgtaggccatgaggt
ccaccac-30 (reverse). The PCR program was as follows:
an initial denaturation at 94 ꢁC for 2 min was followed
by 36 cycles of denaturation at 94 ꢁC for 30 s, annealing
at 58 ꢁC for 30 s, and extension at 72 ꢁC for 45 s, and
then a final extension step at 72 ꢁC for 5 min. PCR prod-
4.6.5. Measurement of bone calcium mobilization. Twen-
ty-day-old weanling male rats from the low vitamin D
colony were purchased from SLC, Inc. and fed the vita-
min D-deficient diet containing 0.03% calcium and 0.6%
phosphorus diet for 3 weeks. The rats were given 1 lg of
19-norvitamin D analogs or 1,25-(OH)2D3 in 50 lL of

4656
M. Shimazaki et al. / Bioorg. Med. Chem. 14 (2006) 4645–4656
20. Shimizu, M.; Iwasaki, Y.; Shimazaki, M.; Amano, Y.;
Yamamoto, K.; Reischl, W.; Yamada, S. Bioorg. Med.
Chem. Lett. 2005, 15, 1451.
ucts were separated by 2% agarose gel electrophoresis
and were stained with ethidium bromide.
21. Sicinski, R. R.; Perlman, K. L.; DeLuca, H. F. J. Med.
Chem. 1994, 37, 3730.
Acknowledgments
22. Sicinski, R. R.; Rotkiewicz, P.; Kolinski, A.; Sicinska, W.;
Prahl, J. M.; Smith, C. M.; DeLuca, H. F. J. Med. Chem.
2002, 45, 3366.
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24. Takayama, H.; Kittaka, A.; Fujishima, T.; Suhara, Y.
Recent Results in Cancer Research; Springer: Berlin,
Heidelberg, 2003, Vol. 164.
We thank Dr. Ryutaro Kamijo, Professor, Showa Uni-
versity. We also thank Mr. Masahiro Sato for his advice
and technical assistance.
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