24,24-Dimethylvitamin D3-26,23-lactones and their 2α-functionalized analogues as highly potent VDR antagonists

Article abstract of DOI:10.1016/j.tet.2004.05.113

Novel vitamin D receptor (VDR) antagonists, 24,24-dimethyl-1α- hydroxyvitamin D₃-26,23-lactones (8 and 9) and their C2α functionalized analogues (8a-c and 9a-c) were efficiently synthesized and their biological activities were evaluated. The co

Full text of DOI:10.1016/j.tet.2004.05.113

Tetrahedron 60 (2004) 7951–7961
24,24-Dimethylvitamin D3-26,23-lactones and their
2a-functionalized analogues as highly potent VDR antagonistsq
Nozomi Saito,a Manami Masuda,a Toshihiro Matsunaga,a Hiroshi Saito,b Miyuki Anzai,b
Kazuya Takenouchi,b Daishiro Miura,b Seiichi Ishizuka,b Midori Takimoto-Kamimurab
and Atsushi Kittakaa
,
*
aFaculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan
bTeijin Institute for Bio-medical Research, Tokyo 191-8512, Japan
Received 6 May 2004; accepted 28 May 2004
Available online 7 July 2004
Abstract—Novel vitamin D receptor (VDR) antagonists, 24,24-dimethyl-1a-hydroxyvitamin D3-26,23-lactones (8 and 9) and their C2a
functionalized analogues (8a–c and 9a–c) were efficiently synthesized and their biological activities were evaluated. The construction of
vitamin D3 triene skeleton was achieved by palladium-catalyzed alkenylative cyclization of A-ring precursor enyne (22 and 22a–c) with
CD-ring bromoolefin having a 24,24-dimethyl-a-methylene-g-lactone unit on the side chain (13 and 14). The CD-ring precursors 13 and 14
were prepared by using chromium-mediated allylation of the aldehyde 10 derived from vitamin D2. On the other hand, the A-ring enyne
having 2a-(3-hydroxypropyl) group (22b) was newly synthesized from epoxide 15 using regio- and stereoselective alkylation methodology.
The potency of the antagonistic activity of the newly designed analogues (8 and 9) increased up to 12 times that of TEI-9647 (2).
Furthermore, introduction of the three motifs, that is, a methyl (8a and 9a), an v-hydroxypropyl (8b and 9b) or an v-hydroxypropoxyl group
(8c and 9c) into the C2a position of 8 and 9, respectively, resulted in remarkable enhancement, up to 89 times, of the antagonistic activity
on VDR.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
1a,25-Dihydroxyvitamin D3 (1), which is a hormonally
active form of vitamin D, exerts various biological profiles
including calcium and phosphorous homeostasis, cell
proliferation and differentiation of various types of tumor
cells, and immune reaction.1,2 Most of the biological
responses of 1 are mediated by its specific receptor, vitamin
D receptor (VDR), which is a member of the nuclear
receptor superfamily and acts as a ligand-dependent gene
transcription factor with coactivators.3,4 Recently, we have
Figure 1. Structures of 1a,25-dihydroxyvitamin D3 (1) and its
C2a-modified analogues 1a–c.
synthesized several 1a,25-dihydroxyvitamin D3 analogues,
which systematically introduced an alkyl, v-hydroxyalkyl,
and v-hydroxyalkoxyl group into the C2a position of 1.5
Some of these 2a-modified vitamin D3 analogues exhibited
groups showed 2- to 4-fold higher binding affinity to the
bovine thymus VDR relative to 1 (Fig. 1).
unique biological activities with potent agonistic activity.6,7
In particular, introduction of the 2a-methyl (1a),5a,b 2a-(3-
hydroxypropyl) (1b),5c,d and 2a-(3-hydroxypropoxy) (1c)5e
In 1999, the first VDR antagonists, 25-dehydro-1a-hydroxy-
vitamin D3-26,23-lactones, TEI-9647 (2) and TEI-9648 (3)
were discovered during the course of studies on the
side-chain modification of the 1a,25-dihydroxyvitamin
q Supplementary data associated with this article can be found in the online
version, at doi: 10.1016/j.tet.2004.05.113
D3-26,23-lactone metabolite8 derived from 1 (Fig. 2).9,10
Both vitamin D3 analogues 2 and 3 specifically antagonize
the VDR-mediated genomic action of 1.11 That is, 2 and 3
inhibit differentiation of human leukemia cells (HL-60
cells)9a as well as 25-hydroxyvitamin D3-24-hydroxylase
Keywords: Active vitamin D3; VDR antagonist; a-Methylene-g-lactone.
*
Corresponding author. Tel./fax: þ81-426-85-3713;
e-mail address: akittaka@pharm.teikyo-u.ac.jp
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.05.113

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N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
double functionalization of the C2a and C24 positions of 2
(4a and 5a) remarkably increased the antagonistic activity
of 2 to 62-fold stronger than that of the original 2.
On the basis of our previous results, we newly designed
novel vitamin D3 lactone analogues 8 and 9, which have the
dimethyl groups at the C24 position, to investigate further
structure–activity relationships on the lactone core structure
(Fig. 3). From the point of manufacturing new drug
candidates, reduction of the number of chiral centers is
favorable. Moreover, we expected that biological activity of
the vitamin D3 lactone analogues would be enhanced by
introduction of the above three motifs, that is, the methyl
(8a and 9a), the 3-hydroxypropyl (8b and 9b) and the
3-hydroxypropoxyl group (8c and 9c) as in our previous
studies.6,7,12 Here, we report the synthesis and biological
evaluation of the novel 24,24-dimethylvitamin D3-26,23-
lactones and their 2a-modified analogues.
Figure 2. Structures of 1a-hydroxyvitamin D3-26,23-lactones (TEI-9647: 2
and TEI-9648: 3), their 2a-modified (2a–c and 3a–c), 24-modified (4–7),
and 2,24-double modified analogues (4a and 5a).
2. Results
gene expression in human osteosarcoma cells9b and in
HL-60 cells9d induced by 1. Furthermore, TEI-9647 (2)
antagonizes the genomic-mediated calcium metabolism
regulated by 1 in vivo in rat.9e The interesting biological
profiles of the vitamin D3 analogues 2 and 3 prompted us to
investigate structure–activity relationship of the vitamin D3
lactones from the standpoint of developing more potent
anti-D molecules.
2.1. Synthesis and biological evaluation of 24,24-
dimethylvitamin D3-26,23-lactones
For the synthesis of the 24,24-dimethylvitamin D3 lactone
analogues, we utilized the Pd-catalyzed A-ring/CD-ring
connective strategy.13 First of all, we prepared the CD-ring
precursors having the 24,24-dimethyl-a-methylene-g-lac-
tone side chain via the low-valent Cr-mediated allylation–
lactonization process14 (Scheme 1). The aldehyde 10 was
synthesized from vitamin D2,12 and the alcohol 1115 was
treated with PBr3 to give allylic bromide 12 in 83% yield.
When aldehyde 10 reacted with 12 in the presence of the
Cr(II) complex generated from CrCl3 and LiAlH4, two
hydrindan derivatives 13 and 14, which were diastereomers
with respect to the C23 position on the lactone ring (based
on steroidal numbering), were obtained in 80% yield in the
ratio of 1 to 2. The absolute streochemistries at C23 position
Quite recently, we found that some pertinent modifications
of 2 and 3 enhanced their biological activities.12 Namely,
introduction of the above three motifs, that is, the methyl,
the 3-hydroxypropyl or the 3-hydroxypropoxy group, into
the C2a position of 2 and 3 (2a–c and 3a–c) raised the
potential of the antagonistic activity up to 30-fold.12a On the
other hand, it was also found that the VDR binding affinity
and antagonistic activity of 2 and 3 were affected by the
structure including stereochemistry of the lactone part
(4–7).12b Especially, introducing the methyl group into the
C24 position on the lactone ring improved the antagonistic
activity to be up to 2.5-fold more potent than that of
TEI-9647 (2). Furthermore, we disclosed that simultaneous
Figure 3. 24,24-Dimethylvitamin D3 lactones (8 and 9) and their
2a-modified analogues (8a–c and 9a–c).
Scheme 1. Preparation of CD-ring precursors.

N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
7953
Figure 4. Stereoscopic views of the crystal structure of compounds 13 (upper) and 14 (lower). The thermal displacement parameters are drawn at both 50%
probability (13 and 14).
Scheme 2. Improved synthesis of A-ring precursor having 3-hydroxypropyl side chain 22b.
of 13 and 14 were confirmed by X-ray structual determi-
nation, respectively (Fig. 4).16
89% yield. The alcohol 20 reacted with TsCl, then the
resulting sulfonate derivative was treated with TBAF to
afford epoxide 21. Introduction of the TMS-ethynyl group
into 21 followed by deprotection under basic conditions
gave a triol. The resulting triol was protected by TBS groups
to provide the desired A-ring precursor 22b in totally 12
steps from epoxide 15.20
Next, the A-ring precursor having the v-siloxypropyl side-
chain 22b was synthesized in an improved manner from
known epoxide 1517 by using our regio- and stereoselective
alkylation methodology reported previously5e,18 (Scheme
2). The epoxide 15 was treated with allyl magnesium
chloride19 followed by hydroboration–oxidation to give
diol 16 in 3 steps in 91% yield. Protection of two hydroxyl
groups with Piv and TBS provided 18, which was then
converted into bromide 19 via radical benzylidene acetal
cleavage by NBS. Pyranose ring-opening reaction of 19
with activated-zinc in the presence of NaBH3CN gave 20 in
Construction of the vitamin D3 triene unit was achieved by
Pd-catalyzed alkenylative cyclization of 225a with 13 or 14;
Table 1. Biological activities of 24,24-dimethylvitamin D3 lactones 8 and 9
Compounds
VDR binding affinitya Antagonistic activityb (IC50, nM)
1
100
12
29
22
—
8.3
3.7
3.2
0.71
TEI-9647 (2)
4c
5c
8
37
TEI-9648 (3)
7
12
5
111.6
160.0
51.0
6c
7c
9
18
51.5
a
The potency of 1 is normalized to 100.
b
c
Antagonistic activity was assessed in terms of IC50 for differentiation of
HL-60 cells induced by 10 nM of 1.
See: Ref. 12b.
Scheme 3. Synthesis of 24,24-dimethylvitamin D3-26,23-lactones 8 and 9.

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N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
and then deprotection of the silyl groups by HF gave
the desired 24,24-dimethylvitamin D3 lactones 8 and 9
(Scheme 3).
Table 2. Biological activities of 2a-modified 24,24-dimethylvitamin D3
lactones 8a–c and 9a–c
Compounds
VDR binding affinitya Antagonistic activityb (IC50, nM)
First, the receptor binding affinity and antagonistic activity
of 24,24-dimethylvitamin D3 lactones 8 and 9 were
evaluated to see the biological effects of the 24,24-dimethyl
groups (Table 1). We also show the data of 24-methyl-
vitamin D3 lactones (4–7) for comparison. Binding
affinities of 8 and 9 to the chick intestinal VDR were
examined as described previously.21 The affinity of
(23S)-24,24-dimethylactone derivative 8 increased to be
3.1-fold more potent than that of 2. In the case of TEI-9648
type analogue 9, introducing the dimethyl unit into the C24
position raised the binding affinity to 2.6 times higher
compared with that of 3. The antagonistic activities of 8 and
9 were assessed by the NBT-reduction method22 in terms of
inhibition of HL-60 cell differentiation induced by 1
(10 nM). Surprisingly, introduction of the dimethyl groups
into the original antagonist 2 resulted in marked enhance-
ment of the antagonistic activity to be ca. 12 times stronger
than that of 2. Although TEI-9648 (3) type analogue 9
showed weaker antagonistic activity than that of 2, the
dimethyl analogue 9 exhibited 2.2-fold higher activity
compared to the original compound 3. These results
indicated that the vitamin D3 lactone derivatives having
the two-methyl groups on the C24 position (8 and 9) altered
binding affinity for the VDR and worked on antagonism on
the VDR more effectively than the mono methyl analogues
(4–7).
TEI-9647 (2)
12
67
71
36
8.3
0.093
0.7
8a
8b
8c
0.3
TEI-9648 (3)
7
111.6
5.8
7.7
9a
9b
9c
48
53
12
28.0
a
The potency of 1 is normalized to 100.
Antagonistic activity was assessed in terms of IC50 for differentiation of
HL-60 cells induced by 10 nM of 1.
b
than that of 2 by introduction of the three motifs into the
C2a position of 8 (8a–c). Such C2a modification exhibited
remarkable effect on antagonistic activity. Especially, VDR
antagonistic activity of 2a-methyl analogue 8a increased to
be ca. 89-fold more potent than that of the original 2 (7.6
times stronger than 8). The other lactone derivatives 8b and
8c also showed 12- and 28-fold stronger antagonistic
activity than that of 2, respectively. In the case of TEI-
9648 type derivatives (9a–c), receptor binding affinity was
raised to be 1.7–7.6 fold more potent than TEI-9648 (3).
Although 9a–c generally showed weaker antagonistic
activities than 8a–c, C2a functionalization of 9 signifi-
cantly increased the activities in comparison with the
original compound 3 (4–19 more potent than 3) and 24,24-
dimtehylvitamin D3 lactone derivative 9 (1.8–8.9 times
stronger than 9).
2.2. Effect of C2a modifications of 24,24-
dimethylvitamin D3 lactones
Next, we turned our attention to C2a-functionalization
of 24,24-dimethylvitamin D3-lactone analogues 8 and 9.
According to our previous results, C2a-functionalization of
24-methylvitamin D3 lactones effectively enhanced both
binding affinity to the VDR and antagonistic activity.12b
Therefore, we expected a high increase in VDR binding
affinity and marked improvement of the VDR antagonistic
activity through such functionalizations of the new 24,24-
dimethylvitamin D3 lactones. The 2a-modified vitamin D3
analogues (8a–c and 9a–c) were similarly synthesized
from the corresponding CD-ring unit 13 and 14 with the
A-ring precursor 22a,17a 22b and 22c,5e respectively
(Scheme 4).
3. Discussion
In the generally accepted mechanism of transactivation
mediated by VDR, a ligand first binds to the ligand binding-
domain (LBD) of an apo form of VDR. Next, the VDR–
ligand complex changes the conformation into a transcrip-
tionally active holo form, which binds to the coactivators to
activate transcription of the target gene.23 In this confor-
mational change process, the appropriate positioning of
helix 12 of VDR, which is the most C-terminal a-helix and
presents an interaction site with the other proteins such as
coactivators in the active holo form, is essential, and
regulates whether the function of the ligand on the VDR
exhibits agonism or antagonism.24
The antagonist 2 binds to the LBD of a VDR, and the
binding of 2 changes the conformation of the VDR into an
unusual transcriptionally inactive form.25 We at present
speculate that some amino acid residues in the LBD
participate in the unusual conformational change of the
VDR through the interaction with the exo-methylene
lactone moiety of 2. Namely, there are two cysteines, that
is, Cys403 on helix 11 and Cys410 on the hinge region
between helix 11 and helix 12, in the LBD of the hVDR.26
The nucleophilic thiol groups of the cysteines could attack
on the a-methylene-g-lactone of 2 via 1,4-addition to give
the corresponding cysteine adduct.27 Such interaction
between the LBD and the ligand might prevent the usual
positioning of helix 12. As the result, the VDR-2 complex
Scheme 4. Synthesis of 2a-modified 24,24-dimethylvitamin D3 lactones
8a–c and 9a–c.
The evaluation of biological activities of 8a–c and 9a–c
disclosed that C2a modifications were also effective to
enhance the biological potency of 24,24-dimethylvitamin
D3 lactones (Table 2). That is, VDR binding affinity of TEI-
9647 (2) type analogues increased to 3.0–5.9 times stronger

N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
7955
could not form transcriptionally active conformation.
Therefore, it is thought that the antagonist, whose exo-
methylene unit is located on more favorable position to
interact with Cys403 and/or Cys410, would show more
potent antagonistic activity.
5.1.2. (S)-5-{(R)-2-[(1R,4E,3aR,7aR)-4-Bromomethyl-
ene-7a-methylperhydroinden-1-yl]propyl}-4,4-di-
methyl-3-methylenedihydrofuran-2-one (13) and (R)-5-
{(R)-2-[(1R,4E,3aR,7aR)-4-bromomethylene-7a-methyl-
perhydroinden-1-yl]propyl}-4,4-dimethyl-3-methylene-
dihydrofuran-2-one (14). To a suspension of CrCl3
(739 mg, 4.7 mmol) in THF (23 mL) was added LiAlH4
(94 mg, 2.3 mmol) at 0 8C, and the mixture was stirred at
room temperature for 30 min. To the mixture were added a
solution of 10 (486 mg, 2.3 mmol) in THF (8 mL) and a
solution of 12 (350 mg, 1.2 mmol) at room temperature, and
the resulting mixture was stirred at the same temperature for
1 h. To the mixture was added H2O at 0 8C, and the aqueous
layer was extracted with Et2O. The organic layer was
washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼10/
1) to give a mixture of 13 and 14 (390 mg, 80% in the ratio
of 2 to 1). Further separation was performed by recycle-
HPLC (column: SHIMADZU Shim-pack PREP-SIL(H)-
KIT, eluent: hexane/AcOEt¼8/1, flow rate: 10 mL/min,
detector: UV (235 nm)). 13: mp. 130 8C (recrystallized from
Et2O–hexane); [a]2D5¼þ31.4 (c 0.85, CHCl3); IR (film,
Although it is yet unclear why the newly synthesized 24,24-
dimethylvitamin D3 lactones (8 and 9) and their C2a
modified analogues (8a–c and 9a–c) exhibited more potent
antagonistic activity than the original 2 and 3, they might be
situated in the above mentioned preferable position to
interact with the cysteine residues after the binding to the
LBD of the VDR.
4. Conclusion
We have succeeded in developing highly potent vitamin D
receptor antagonists (8, 8a–c, 9 and 9a–c), 24,24-dimethyl-
1a-hydroxyvitamin D3-26,23-lactones and their C2a-func-
tionalized analogues. Recently, the VDR antagonists are
expected to be potent therapeutic agents for some diseases
caused by hypersensitivity to 1a,25-dihydroxyvitamin D3,
such as Paget’s bone disease.28 We expect that these
analogues with potent anti-vitamin D activity would
contribute to understanding the mechanisms involved in
the expression of antagonistic activity on VDR as well as to
finding the seeds of new medicines for treating Paget’s bone
disease.
CHCl3) 1767, 1653, 1634, 1371, 1133 cm21 1H NMR
;
(400 MHz, CDCl3) d 0.58 (s, 3H), 1.05 (s, 3H), 1.08 (d,
J¼6.6 Hz, 3H), 1.21 (s, 3H), 1.25–1.70 (m, 11H), 1.95–
2.04 (m, 3H), 2.88 (dd, J¼15.9, 3.9 Hz, 1H), 4.10 (dd,
J¼9.0, 2.9 Hz, 1H), 5.46 (s, 1H), 5.65 (s, 1H), 6.14 (s, 1H);
13C NMR (100 MHz, CDCl3) d 11.8, 19.7, 22.1, 22.6, 23.0,
24.3, 27.9, 31.0, 35.3, 35.5, 39.8, 42.6, 45.6, 55.7, 55.9,
86.1, 97.5, 118.9, 114.8, 146.1, 170.5; EI-LRMS m/z 394
(Mþ), 315, 256, 227; EI-HRMS Calcd for C21H31O729Br
394.1507, found 394.1508. Anal. Calcd for C21H31O2Br: C,
63.79; H, 7.90. Found: C, 64.13; H, 8.27. 14: mp. 117 8C
(recrystallized from Et2O–hexane); [a]2D5¼þ141.2 (c 0.38,
CHCl3); IR (film, CHCl3) 1767, 1651, 1638, 1458,
5. Experimental
5.1. General
All manipulations were performed under an argon atmos-
phere unless otherwise mentioned. All solvents and reagents
were purified when necessary using standard procedures.
Column chromatography was performed on silica gel 60 N
(Kanto Chemical Co., Inc., 100–210 mm), and flash
column chromatography was performed on silica gel 60
(Merck, 40–63 mm). NMR spectra were measured on a
JEOL AL-400 magnetic resonance spectrometer. Infrared
spectra were recorded on JASCO FTIR-8000 spectrometer.
Mass spectra were measured on JEOL JMX-SX 102 mass
spectrometer. Specific optical rotations were measured on
JASCO DIP-370 digital polarimeter.
1
1190 cm21; H NMR (400 MHz, CDCl3) d 0.59 (s, 3H),
1.00 (d, J¼6.3 Hz, 3H), 1.05 (s, 3H), 1.11 (dd, J¼13.4,
11.2 Hz, 1H), 1.21 (s, 3H), 1.25–1.36 (m, 3H), 1.44–1.66
(m, 7H), 1.89 (m, 1H), 1.96–2.04 (m, 2H), 2.89 (dd,
J¼15.6, 6.8 Hz, 1H), 4.14 (d, J¼10.5 Hz, 1H), 5.47 (s, 1H),
5.65 (s, 1H), 6.15 (s, 1H); 13C NMR (100 MHz, CDCl3) d
11.9, 18.6, 22.1, 22.5, 22.8, 25.1, 27.6, 31.0, 32.9, 35.9,
39.9, 41.9, 45.6, 55.9, 56.2, 84.2, 97.6, 119.1, 144.7, 146.1,
170.3; EI-LRMS m/z 394 (Mþ), 315, 256, 227; EI-HRMS
Calcd for C21H31O729Br 394.1507, found 394.1508. Anal.
Calcd for C21H31O2Br: C, 63.79; H, 7.90. Found: C, 63.57;
H, 8.20.
5.1.1. Methyl 2-(bromomethyl)-3-methylbut-2-enoate
(12). To a solution of 11 (200 mg, 1.4 mmol) in Et2O was
added PBr3 (80 mL, 0.83 mmol) at 0 8C, and the mixture
was stirred at room temperature for 1 h. To the mixture was
added H2O at 0 8C, and the aqueous layer was extracted with
Et2O. The organic layer was washed with saturated NaCl aq.
solution, dried over Na2SO4, and concentrated. The residue
was purified by flash column chromatography on silica gel
(hexane/AcOEt¼20/1) to give 12 (240 mg, 83%) as a
5.1.3. Methyl 4,6-O-benzylidene-3-allyl-3-deoxy-a-D-
altropyranoside. To a solution of 15 (264 mg, 1.0 mmol)
in THF (1.5 mL) was added a solution of allylmagnesium
chloride in THF (2.0 M, 1.5 mL, 3.0 mmol) at room
temperature, and the mixture was stirred at 80 8C for 1 h.
To the mixture was added water at 0 8C, and the aqueous
layer was extracted with Et2O. The organic layer was
washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼2/1)
to give the desired allylated compound (314 mg, quant.) as
an amorphous solid. IR (neat) 3422, 2930, 1642, 1456,
1
colorless oil. IR (neat) 1723, 1628, 1373 cm21; H NMR
(400 MHz, CDCl3) d 1.99, (s, 3H), 2.16 (s, 3H), 3.79 (s, 3H),
4.31 (s, 2H); 13C NMR (100 MHz, CDCl3) d 23.0, 24.0,
29.4, 51.7, 124.6, 153.8, 166.9; EI-LRMS m/z 205 (Mþ),
191, 175; EI-HRMS Calcd for C7H11O729Br 205.9942, found
205.9951.
1
1381 cm21; H NMR (400 MHz, CDCl3) d 1.90 (m, 1H),
2.26 (m, 1H), 2.53 (dd, J¼7.1, 7.1 Hz, 2H), 3.39 (s, 3H),

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N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
3.78 (dd, J¼10.2, 10.2 Hz, 1H), 3.97 (m, 1H), 4.00 (ddd,
J¼10.2, 10.2, 5.1 Hz, 1H), 4.12 (dd, J¼10.2, 5.1 Hz, 1H),
4.29 (dd, J¼10.2, 5.1 Hz, 1H), 4.60 (s, 1H), 5.06 (d, J¼
10.2 Hz, 1H), 5.11 (d, J¼17.2 Hz, 1H), 5.60 (s, 1H), 5.83
(ddt, J¼17.2, 10.2, 7.1 Hz, 1H), 7.36–7.37 (m, 3H), 7.48–
7.50 (m, 2H); 13C NMR (100 MHz, CDCl3) d 29.1, 42.8,
55.6, 59.9, 69.7, 69.9, 76.4, 102.3, 102.7, 116.9, 126.4 (2C),
128.5 (2C), 129.2, 137.3, 137.9; EI-LRMS m/z 306 (Mþ);
EI-HRMS Calcd for C17H22O5 306.1467, found 306.1469.
pyranoside (18). To a solution of 17 (270 mg, 0.66 mmol)
in CH2Cl2 (6.6 mL) were added 2,6-lutidine (0.39 mL,
3.3 mmol) and TBSOTf (0.23 mL, 0.99 mmol) at 0 8C, and
the mixture was stirred at room temperature for 13 h. To the
mixture was added water at 0 8C, and the aqueous layer was
extracted with Et2O. The organic layer was washed with
saturated NaCl aq. solution, dried over Na2SO4, and
concentrated. The residue was purified by column chroma-
tography on silica gel (hexane/AcOEt¼9/1) to give 18
(332 mg, 96%) as a colorless oil. [a]1D9¼þ35.9 (c 0.62,
1
5.1.4. Methyl 4,6-O-benzylidene-3-deoxy-3-(3-hydroxy-
propyl)-a-D-altropyranoside (16). To a solution of the
above allylated compound (1.8 g, 5.9 mmol) in THF
(12 mL) was added a solution of BH3·THF in THF
(1.0 M, 14.7 mL, 14.7 mmol) at 0 8C, and the mixture was
stirred at room temperature for 23 h. To the mixture was
added 3N NaOH aq. solution (7.7 mL) and 30% H2O2 aq.
solution (7.7 mL) at 0 8C, and the mixture was stirred at
room temperature for 3 h. To the mixture was added
saturated NH4Cl aq. solution at 0 8C, and the aqueous layer
was extracted with AcOEt. The organic layer was washed
with saturated NaCl aq. solution, dried over Na2SO4, and
concentrated. The residue was purified by flash column
chromatography on silica gel (hexane/AcOEt¼1/2) to give
alcohol (1.72 g, 91%) as a colorless oil. [a]1D9¼þ84.2 (c
CHCl3); IR (neat) 2955, 1728, 1258, 1107, 1051 cm21; H
NMR (400 MHz, CDCl3) d 0.086 (s, 3H), 0.092 (s, 3H),
0.92 (s, 9H), 1.19 (s, 9H), 1.60–1.89 (m, 4H), 2.05 (m, 1H),
3.34 (s, 3H), 3.77 (dd, J¼10.1, 10.1 Hz, 1H), 3.87 (br s, 1H),
3.92 (ddd, J¼10.1, 10.1, 5.0 Hz, 1H), 4.03–4.14 (m, 3H),
4.26 (dd, J¼10.1, 5.0 Hz, 1H), 4.45 (s, 1H), 5.59 (s, 1H),
7.34–7.37 (m, 3H), 7.47–7.49 (m, 2H); 13C NMR
(100 MHz, CDCl3) d 24.9, 24.8, 18.1, 21.1, 25.8 (3C),
27.2 (3C), 27.8, 38.8, 43.4, 55.0, 59.4, 64.4, 69.7, 70.7, 76.4,
101.9, 102.6, 126.2 (2C), 128.6 (2C), 128.9, 137.8, 178.4;
EI-LRMS m/z 522 (Mþ), 491, 358, 244, 159; EI-HRMS
Calcd for C28H46O7 522.3013, found 522.3015.
5.1.7. Methyl 4-O-benzoyl-6-bromo-2-[(tert-butyldi-
methylsilyl)oxy]-3-deoxy-3-[(3-pivaloyloxy)propyl]-6-
deoxy-a-D-altropyranoside (19). To a solution of 18
(2.28 g, 4.4 mmol) in CCl4 (22 mL) were added BaCO3
(861 mg, 4.4 mmol) and NBS (932 mg, 5.2 mmol) at room
temperature, and the mixture was stirred at 80 8C for
30 min. To the mixture was added saturated Na2S2O3 aq.
solution and saturated NaHCO3 aq. solution, and the
aqueous layer was extracted with Et2O. The organic layer
was washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼9/1)
to give 19 (2.46 g, 94%) as a colorless oil. [a]1D8¼þ18.4 (c
2.92, CHCl3); IR (neat) 2957, 1727, 1267, 1116,
1
2.31, CHCl3); IR (neat) 3403, 2936, 1103, 1051 cm21; H
NMR (400 MHz, CDCl3) d 1.52–1.86 (m, 4H), 2.17–2.18
(m, 3H), 3.36 (s, 3H), 3.61 (t, J¼6.3 Hz, 2H), 3.76 (dd,
J¼10.1, 10.1 Hz, 1H), 3.92 (m, 1H), 3.97 (ddd, J¼10.1,
10.1, 5.2 Hz, 1H), 4.11 (dd, J¼10.1, 5.2 Hz, 1H), 4.27 (dd,
J¼10.1, 5.2 Hz, 1H), 4.57 (s, 1H), 5.57 (s, 1H), 7.34–7.39
(m, 3H), 7.45–7.48 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 20.8, 31.4, 42.5, 55.2, 59.5, 62.8, 69.5, 70.2, 76.4, 102.0,
102.1, 126.1 (2C), 128.2 (2C), 129.0, 137.5; EI-LRMS m/z
324 (Mþ), 292, 274, 215, 162, 143, 105, 77; EI-HRMS
Calcd for C17H24O6 324.1573, found 324.1575.
1
5.1.5. Methyl 4,6-O-benzylidene-3-deoxy-3-[(3-pivaloyl-
oxy)propyl]-a-D-altropyranoside (17). To a solution of 16
(227 mg, 0.7 mmol) in pyridine (3.5 mL) was added
pivaloyl chloride (95 mL, 0.77 mmol) at 0 8C, and the
mixture was stirred at room temperature for 12 h. To the
mixture was added water at 0 8C, and the aqueous layer was
extracted with AcOEt. The organic layer was washed with
saturated NaCl aq. solution, dried over Na2SO4, and
concentrated. The residue was purified by column chroma-
tography on silica gel (hexane/AcOEt¼4/1) to give 17
(270 mg, 94%) as a colorless oil. [a]1D9¼þ66.1 (c 2.38,
CHCl3); IR (neat) 3474, 2963, 1725, 1287, 1103,
1042 cm21; H NMR (400 MHz, CDCl3) d 0.10 (s, 3H),
0.12 (s, 3H), 0.93 (s, 9H), 1.08 (s, 9H), 1.43–1.73 (m, 3H),
1.87 (m, 1H), 2.16 (ddt, J¼6.0, 5.9, 6.3 Hz, 1H), 3.43 (s,
3H), 3.58 (dd, J¼10.7, 7.6 Hz, 1H), 3.66 (dd, J¼10.7,
2.8 Hz, 1H), 3.82 (dd, J¼6.0, 2.4 Hz, 1H), 4.00 (t, J¼
6.1 Hz, 2H), 4.10 (ddd, J¼7.8, 7.6, 2.8 Hz, 1H), 4.55
(d, J¼2.4 Hz, 1H), 5.38 (dd, J¼7.8, 5.9 Hz, 1H), 7.43–7.47
(m, 2H), 7.59 (m, 1H), 7.99–8.02 (m, 2H); 13C NMR
(100 MHz, CDCl3) d 24.8, 24.6, 18.1, 21.7, 25.8 (3C),
26.9, 27.1 (3C), 33.4, 38.7, 42.1, 55.4, 64.2, 69.0, 71.0, 71.1,
103.7, 128.5 (2C), 129.5, 129.6 (2C), 133.3, 165.3, 178.3;
EI-LRMS m/z 569 (Mþ-OMe), 423, 339, 322, 213; EI-
HRMS Calcd for C27H42O769Br 569.1934, found 569.1931.
1
1049 cm21; H NMR (400 MHz, CDCl3) d 1.18 (s, 9H),
1.63–1.90 (m, 4H), 2.18 (m, 1H), 2.32 (br s, 1H), 3.36 (s,
3H), 3.77 (dd, J¼10.2, 10.2 Hz, 1H), 3.91 (m, 1H), 3.97
(ddd, J¼10.2, 10.2, 5.0 Hz, 1H), 4.01–4.14 (m, 3H), 4.27
(dd, J¼10.2, 5.0 Hz, 1H), 4.58 (s, 1H), 5.58 (s, 1H), 7.33–
7.38 (m, 3H), 7.44–7.47 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 20.9, 27.2 (3C), 27.5, 38.8, 42.5, 55.2, 59.5, 64.3,
69.5, 70.1, 76.2, 101.9, 102.1, 126.1 (2C), 128.1 (2C),
128.9, 137.6, 178.6; EI-LRMS m/z 408 (Mþ), 390, 358, 241,
162, 105; EI-HRMS Calcd for C22H32O7 408.2140, found
408.2140.
5.1.8. (2S,3R,4R)-4-[(Benzoyl)oxy]-2-[(tert-butyldi-
methylsilyl)oxy]-3-[3-(pivaloyloxy)propyl]hexa-5-ene-1-
ol (20). To a solution of 19 (2.40 g, 4.0 mmol) in 1-pro-
panol/H2O (5/1, 24 mL) were added activated Zn dust
(45.5 g, 0.7 mol) and NaBH3CN (9.8 g, 0.14 mol) at 95 8C,
and the mixture was stirred at the same temperature for
1.5 h. To the mixture was added saturated NH4Cl aq.
solution, and the aqueous layer was extracted with AcOEt.
The organic layer was washed with saturated NaCl aq.
solution, dried over Na2SO4, and concentrated. The residue
was purified by column chromatography on silica gel
(hexane/AcOEt¼6/1) to give 20 (1.76 g, 89%) as a colorless
5.1.6. Methyl 4,6-O-benzylidene-2-(tert-butyldimethyl-
silyloxy)-3-deoxy-3-[(3-pivaloyloxy)propyl]-a-D-altro-

N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
7957
oil. [a]1D9¼þ25.4 (c 2.62, CHCl3); IR (neat) 3495, 2957,
(m, 1H), 8.03–8.06 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 25.8, 26.2, 27.2 (3C), 38.7, 45.9, 46.8, 53.1, 64.1, 75.5,
118, 2, 128.4 (2C), 129.5 (2C), 129.9, 133.1, 134.2, 165.3,
178.4; EI-LRMS m/z 360 (Mþ), 259, 136, 80; EI-HRMS
Calcd for C21H28O5 360.1937, found 360.1935.
1
1725, 1598, 1271, 1111, 1069 cm21; H NMR (400 MHz,
CDCl3) d 0.03 (s, 3H), 0.06 (s, 3H), 0.90 (s, 9H), 1.18
(s, 9H), 1.42–1.63 (m, 2H), 1.67–1.90 (m, 3H), 2.06
(ddt, J¼6.3, 5.6, 6.1 Hz, 1H), 3.62 (dd, J¼11.0, 4.9 Hz, 1H),
3.72 (dd, J¼11.0, 5.3 Hz, 1H), 3.95 (ddd, J¼5.6, 5.3,
4.9 Hz, 1H), 4.04 (t, J¼6.1 Hz, 2H), 5.28 (ddd, J¼10.5,
1.2, 1.2 Hz, 1H), 5.37 (ddd, J¼17.1, 1.2, 1.2 Hz, 1H), 5.56
(dd, J¼6.7, 6.3 Hz, 1H), 5.92 (ddd, J¼17.1, 10.5, 6.7 Hz,
1H), 7.42–7.46 (m, 2H), 7.56 (m, 1H), 8.02–8.04 (m, 2H);
13C NMR (100 MHz, CDCl3) d 24.6, 24.2, 18.2, 22.8,
25.9 (3C), 27.2 (3C), 27.9, 38.8, 44.8, 64.4, 65.0, 73.0,
75.8, 118.4, 128.3 (2C), 129.5 (2C), 130.3, 132.9,
134.6, 165.4, 178.4; EI-LRMS m/z 391 (Mþ-OPiv), 239,
105; EI-HRMS Calcd for C22H35O4Si 391.2305, found
391.2307.
5.1.11. (3R,4R,5S)-4-(3-Hydroxypropyl)oct-1-en-7-yne-
3,5-diol. To a solution of ethynyltrimethylsilane (0.32 mL,
2.2 mmol) in THF (3 mL) was added a solution of n-BuLi in
hexane (1.56 M, 1.1 mL, 1.7 mmol) at 278 8C, and the
mixture was stirred at the same temperature for 30 min. To
the mixture were added a solution of 21 (400 mg, 1.1 mmol)
in THF (8 mL) and BF3·OEt2 (0.15 mL, 1.2 mmol) at
278 8C, and the resulting mixture was stirred at the same
temperature for 3 h. To the mixture was added saturated
NH4Cl aq. solution, and the aqueous layer was extracted
with Et2O. The organic layer was washed with saturated
NaCl aq. solution, dried over Na2SO4, and concentrated.
The residue was dissolved in MeOH (11 mL). To the
mixture was added NaOMe (300 mg, 5.6 mmol) at 0 8C, and
the mixture was stirred at 40 8C for 15.5 h. To the mixture
was added saturated NH4Cl aq. solution at 0 8C, and the
aqueous layer was extracted with AcOEt. The organic layer
was washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼1/4)
to give the desired triol (217 mg, 99%) as a colorless oil.
[a]2D0¼þ9.88 (c 0.77, CHCl3); IR (neat) 3304, 2940, 2118,
1630, 1051 cm21; 1H NMR (400 MHz, CDCl3) d 1.45–1.77
(m, 5H), 2.02 (dd, J¼2.7, 2.7 Hz, 1H), 2.33 (ddd, J¼16.8,
7.1, 2.7 Hz, 1H), 2.48 (ddd, J¼16.8, 7.1, 2.7 Hz, 1H), 3.03
(br s, 3H), 3.62–3.74 (m, 2H), 4.20 (dt, J¼1.5, 7.1 Hz, 1H),
4.41 (m, 1H), 5.25 (ddd, J¼10.5, 1.6, 1.6 Hz, 1H), 5.37
(ddd, J¼17.1, 1.7, 1.7 Hz, 1H), 5.91 (ddd, J¼17.1, 10.5,
4.9 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 20.4, 24.3,
30.4, 44.8, 62.8, 70.3, 70.4, 73.6, 81.0, 115.5, 139.5;
EI-LRMS m/z 180 (Mþ-H2O), 161, 105, 79; EI-HRMS
Calcd for C11H16O2 180.1150, found 180.1149.
5.1.9. (3R,4R)-3-Benzoyloxy-4-[(1S)-1-{(tert-butyldi-
methylsilyl)oxy}-2-(p-toluenesulfonyl)ethyl]-7-(pivaloyl-
oxy)hept-1-ene. To a solution of 20 (1.72 g, 3.4 mmol) in
pyridine (7 mL) was added TsCl (3.0 g, 16 mmol) at 0 8C,
and the mixture was stirred at room temperature for 17.5 h.
To the mixture was added water at 0 8C, and the aqueous
layer was extracted with Et2O. The organic layer was
washed with saturated NaCl aq. solution, dried over
Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼9/1)
to give the desired sulfonate (2.24 g, 99%) as a colorless oil.
[a]1D9¼þ18.6 (c 0.46, CHCl3); IR (neat) 2957, 1725, 1599,
1269, 1107 cm21;1H NMR (400 MHz, CDCl3) d 20.06 (s,
3H), 20.04 (s, 3H), 0.83 (s, 9H), 1.17 (s, 9H), 1.33–1.79
(m, 4H), 1.95 (m, 1H), 2.43 (s, 3H), 3.92–4.05 (m, 4H), 4.14
(ddd, J¼6.2, 5.5, 2.8 Hz, 1H), 5.26 (ddd, J¼10.4, 1.2,
1.2 Hz, 1H), 5.35 (ddd, J¼17.3, 1.2, 1.2 Hz, 1H), 5.43 (dd,
J¼7.3, 7.1 Hz, 1H), 5.84 (ddd, J¼17.3, 10.4, 7.1 Hz, 1H),
7.30–7.34 (m, 2H), 7.42–7.48 (m, 2H), 7.59 (m, 1H), 7.78–
7.80 (m, 2H), 8.01–8.03 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 25.0, 24.3, 18.0, 21.7, 22.3, 25.8 (3C), 27.2 (3C),
27.6, 38.8, 45.0, 64.1, 70.0, 71.3, 75.5, 118.9, 127.9 (2C),
128.4 (2C), 129.5 (2C), 129.8 (2C), 130.1, 132.7, 133.0,
134.6, 144.8, 165.2, 178.3; EI-LRMS m/z 475 (Mþ-OTs),
329, 229; EI-HRMS Calcd for C27H43O5Si 475.2880, found
475.2887.
5.1.12. (3R,4R,5S)-3,5-Bis[(tert-butyldimethylsilyl)oxy]-
4-[3-{(tert-butyldimethylsilyl)oxy}propyl]oct-1-en-7-yne
(22b). To the solution of the above triol (14 mg, 71 mmol) in
CH2Cl2 (0.7 mL) were added 2,6-lutidine (49 mL,
0.42 mmol) and TBSOTf (65 mL, 0.28 mmol) at 0 8C, and
the mixture was stirred at room temperature for 1 h. To the
mixture was added water at 0 8C, and the aqueous layer was
extracted with Et2O. The organic layer was washed with
saturated NaCl aq. solution, dried over Na2SO4, and
concentrated. The residue was purified by column chroma-
tography on silica gel (hexane) to give 22b (36 mg, 94%) as
a colorless oil. [a]2D2¼þ8.32 (c 0.77, CHCl3); IR (neat)
3314, 2932, 1256, 1102 cm21; 1H NMR (400 MHz, CDCl3)
d 0.03 (s, 3H), 0.04 (s, 6H), 0.05 (s, 3H), 0.07 (s, 3H), 0.09
(s, 3H), 0.89 (s, 27H), 1.32 (m, 2H), 1.56 (m, 2H), 1.75
(ddt, J¼6.4, 4.0, 6.8 Hz, 1H), 1.95 (t, J¼2.8 Hz, 1H), 2.38
(ddd, J¼16.8, 6.2, 2.8 Hz, 1H), 2.42 (ddd, J¼16.8, 6.2,
2.8 Hz, 1H), 3.56 (t, J¼6.8 Hz, 2H), 4.03 (dt, J¼4.0, 6.2 Hz,
1H), 4.12 (dd, J¼7.6, 6.4 Hz, 1H), 5.08 (d, J¼10.0 Hz, 1H),
5.14 (d, J¼17.2 Hz, 1H), 5.84 (ddd, J¼17.2, 10.0, 7.6 Hz,
1H); 13C NMR (100 MHz, CDCl3) d 25.1 (2C), 24.4,
24.2, 23.9, 23.4, 18.25, 18.32, 18.5, 22.2, 26.0 (3C),
26.08 (3C), 26.12 (3C), 26.2, 32.6, 49.2, 63.6, 69.9, 71.5,
75.9, 82.1, 115.5, 140.4; EI-LRMS m/z 483 (Mþ-tBu);
5.1.10. (3R,4R)-3-[(Benzoyl)oxy]-4-(S)-oxiranyl-7-(piva-
loyloxy)hept-1-ene (21). To a solution of the above
sulfonate (110 mg, 0.17 mmol) in THF (1.7 mL) was
added a solution of TBAF in THF (1.0 M, 0.26 mL,
0.26 mmol) at 0 8C, and the mixture was stirred at room
temperature for 6 h. To the mixture was added water at 0 8C,
and the aqueous layer was extracted with Et2O. The organic
layer was washed with saturated NaCl aq. solution, dried
over Na2SO4, and concentrated. The residue was purified by
column chromatography on silica gel (hexane/AcOEt¼9/1)
to give 21 (64 mg, quant.) as a colorless oil. [a]2D0¼þ28.7
(c 2.31, CHCl3); IR (neat) 2973, 1725, 1601, 1269,
1
1157 cm21; H NMR (400 MHz, CDCl3) d 1.15 (s, 9H),
1.50 (m, 1H), 1.59–1.78 (m, 2H), 1.79–1.92 (m, 2H), 2.59
(dd, J¼4.9, 2.6 Hz, 1H), 2.77 (dd, J¼4.9, 3.9 Hz, 1H), 2.93
(ddd, J¼8.3, 3.9, 2.6 Hz, 1H), 4.06 (t, J¼6.3 Hz, 2H), 5.28
(ddd, J¼10.5, 1.2, 1.0 Hz, 1H), 5.37 (ddd, J¼17.1, 1.2,
1.0 Hz, 1H), 5.62 (dddd, J¼6.6, 6.6, 1.0, 1.0 Hz, 1H), 5.90
(ddd, J¼17.1, 10.5, 6.6 Hz, 1H), 7.44–7.47 (m, 2H), 7.58

7958
N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
EI-HRMS Calcd for C25H51O3Si3 483.3146, found
483.3141.
1.26 (s, 3H), 1.45–1.76 (m, 9H), 1.83–2.04 (m, 5H), 2.31
(dd, J¼13.4, 6.5 Hz, 1H), 2.60 (dd, J¼13.4, 3.4 Hz, 1H),
2.83 (dd, J¼12.0, 3.9 Hz, 1H), 4.14 (dd, J¼11.6, 1.6 Hz,
1H), 4.24 (br s, 1H), 4.43 (br s, 1H), 5.00 (s, 1H), 5.33 (s,
1H), 5.47 (s, 1H), 6.12 (d, J¼11.4 Hz, 1H), 6.15 (s, 1H),
6.37 (d, J¼11.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) d
12.1, 18.6, 22.3, 22.8, 23.6, 25.1, 27.6, 29.1, 32.9, 35.9,
40.5, 42.0, 42.9, 45.3, 46.0, 56.4, 57.0, 66.8, 70.8, 84.3,
111.7, 117.2, 119.1, 124.7, 133.1, 142.6, 146.2, 147.5,
170.4; EI-LRMS m/z 454 (Mþ), 418, 403; EI-HRMS Calcd
for C29H42O4 454.3083, found 454.3083.
5.2. General procedure for the synthesis of vitamin D3
lactones
To a solution of an A-ring precursor (1.5 equiv. to a CD-ring
precursor), and the CD-ring precursor in toluene were added
Et3N and Pd(PPh3)4 (30 mol% to the CD-ring precursor)
and the mixture was stirred at 110 8C. After the mixture was
filtered through a silica gel pad, the filtrate was concen-
trated. The crude product was dissolved in MeCN (1 mL).
To the solution was added 10% solution of conc. HF in
MeCN (1 mL) at 0 8C, the mixture was stirred at room
temperature. To the mixture was added saturated NaHCO3
aq. solution, and the aqueous layer was extract with AcOEt.
The organic layer was washed with saturated NaCl aq.
solution, dried over Na2SO4, and concentrated. The residue
was purified by flash column chromatography on silica gel
or thin-layer chromatography on silica gel to give the
vitamin D3 derivative. Further purification for biological
assays was conducted by reversed-phase recycle HPLC
(YMC-Pack ODS column, 20£150 mm, 9.9 mL/min,
eluent: CH3CN/H2O¼90/10).
5.2.3. (23S)-25-Dehydro-1a-hydroxy-2a,24,24-tri-
methylvitamin D3-26,23-lactone (8a). According to the
general procedure, a crude product, which was obtained
from 13 (25 mg, 63 mmol), 22a (41 mg, 107 mmol), Et3N
(0.6 mL) and Pd(PPh3)4 (37 mg, 32 mmol) in toluene
(0.6 mL) at 110 8C for 1.5 h, was treated with conc. HF in
MeCN for 1 h. After usual work up, the crude product was
purified by column chromatography on silica gel (hexane/
AcOEt¼1/1) to give 8a (14 mg, 47% in 2 steps) as an
amorphous solid. UV (EtOH) lmax¼266.0 nm; [a]2D6¼
þ11.6 (c 1.08, CHCl3); IR (film, CHCl3) 3441, 1752,
1671, 1636, 1051 cm21; 1H NMR (400 MHz, CDCl3) d 0.55
(s, 3H), 1.04 (s, 3H), 1.06 (d, J¼6.7 Hz, 3H), 1.07 (d, J¼
6.7 Hz, 3H), 1.21 (s, 3H), 1.25–1.70 (m, 13H), 1.88–2.04
(m, 4H), 2.23 (dd, J¼13.8, 8.1 Hz, 1H), 2.66 (dd, J¼13.8,
4.0 Hz, 1H), 2.82 (m, 1H), 3.84 (ddd, J¼7.6, 7.6, 4.2 Hz,
1H), 4.10 (dd, J¼8.8, 3.4 Hz, 1H), 4.31 (d, J¼3.2 Hz, 1H),
5.00 (d, J¼1.7 Hz, 1H), 5.27 (dd, J¼2.0, 1.0 Hz, 1H), 5.45
(s, 1H), 6.01 (d, J¼11.2 Hz, 1H), 6.13 (s, 1H), 6.38 (d,
J¼11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 12.0, 12.6,
19.7, 22.3, 23.0, 23.5, 24.3, 27.9, 29.1, 35.4, 35.6, 40.4,
42.6, 43.5, 44.2, 46.0, 56.2, 56.7, 71.7, 75.4, 86.2, 113.1,
117.0, 118.8, 124.7, 133.1, 142.8, 146.2, 146.5, 170.5;
EI-LRMS m/z 468 (Mþ), 451, 434, 419; EI-HRMS Calcd
for C30H44O4 468.3240, found 468.3248.
5.2.1. (23S)-25-Dehydro-1a-hydroxy-24,24-dimethyl-
vitamin D3-26,23-lactone (8). According to the general
procedure, a crude product, which was obtained from 13
(31 mg, 78 mmol), 22 (43 mg, 117 mmol), Et3N (0.8 mL)
and Pd(PPh3)4 (33 mg, 28 mmol) in toluene (0.8 mL) at
110 8C for 1 h, was treated with conc. HF in MeCN for 1 h.
After usual work up, the crude product was purified by
column chromatography on silica gel (hexane/AcOEt¼1/1)
to give 8 (17 mg, 48% in 2 steps) as an amorphous solid. UV
(EtOH) lmax¼264.0 nm; [a]2D4¼221.8 (c 0.85, CHCl3); IR
(film, CHCl3) 3382, 1765, 1663, 1057 cm21 1H NMR
;
(400 MHz, CDCl3) d 0.56 (s, 3H), 1.05 (s, 3H), 1.07 (d,
J¼6.6 Hz, 3H), 1.21 (s, 3H), 1.25–1.72 (m, 13H), 1.88–
2.06 (m, 5H), 2.32 (dd, J¼13.4, 6.3 Hz, 1H), 2.60 (dd,
J¼13.4, 3.5 Hz, 1H), 2.83 (dd, J¼10.4, 3.8 Hz, 1H), 4.10
(dd, J¼10.4, 3.4 Hz, 1H), 4.23 (br s, 1H), 4.43 (br s, 1H),
5.00 (dd, J¼1.6, 1.5 Hz, 1H), 5.33 (dd, J¼1.7, 1.6 Hz, 1H),
5.45 (s, 1H), 6.02 (d, J¼11.2 Hz, 1H), 6.13 (s, 1H), 6.38 (d,
J¼11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d 12.0, 19.7,
22.3, 23.0, 23.6, 24.4, 27.9, 29.1, 35.4, 35.6, 40.4, 42.6,
42.9, 45.3, 46.0, 56.2, 56.7, 66.8, 70.8, 86.3, 111.6, 117.1,
118.8, 124.8, 133.0, 142.8, 146.2, 147.6, 170.5; EI-LRMS
m/z 454 (Mþ), 418, 403; EI-HRMS Calcd for C29H42O4
454.3083, found 454.3083.
5.2.4. (23R)-25-Dehydro-1a-hydroxy-2a,24,24-tri-
methylvitamin D3-26,23-lactone (9a). According to the
general procedure, a crude product, which was obtained
from 14 (26 mg, 66 mmol), 22a (40 mg, 105 mmol), Et3N
(1 mL) and Pd(PPh3)4 (24 mg, 21 mmol) in toluene (1 mL)
at 110 8C for 2.5 h, was treated with conc. HF in MeCN for
1 h. After usual work up, the crude product was purified by
column chromatography on silica gel (hexane/AcOEt¼1/1)
to give 9a (18 mg, 58% in 2 steps) as an amorphous solid.
UV (EtOH) lmax¼266.5 nm; [a]2D6¼þ78.5 (c 1.38, CHCl3);
1
IR (film, CHCl3) 3476, 1750, 1649, 1051 cm21; H NMR
(400 MHz, CDCl3) d 0.56 (s, 3H), 1.00 (d, J¼6.7 Hz, 3H),
1.06 (s, 3H), 1.08 (d, J¼6.7 Hz, 3H), 1.12 (m, 1H), 1.21 (s,
3H), 1.26–1.34 (m, 3H), 1.46–1.72 (m, 10H), 1.90–2.04
(m, 3H), 2.23 (dd, J¼13.4, 7.9 Hz, 1H), 2.67 (dd, J¼13.4,
4.0 Hz, 1H), 2.83 (m, 1H), 3.85 (ddd, J¼7.5, 7.5, 4.2 Hz,
1H), 4.15 (dd, J¼11.6, 1.3 Hz, 1H), 4.31 (br s, 1H), 5.01 (d,
J¼2.0 Hz, 1H), 5.28 (s, 1H), 5.47 (s, 1H), 6.01 (d, J¼
11.2 Hz, 1H), 6.15 (s, 1H), 6.38 (s, J¼11.2 Hz, 1H); 13C
NMR (100 MHz, CDCl3) d 12.1, 12.6, 18.6, 22.3, 22.9,
23.5, 25.1, 27.6, 29.1, 32.9, 35.9, 40.6, 42.0, 43.4, 44.2,
46.0, 56.4, 57.0, 71.7, 75.3, 84.3, 113.1, 117.1, 119.1, 124.6,
133.2, 142.6, 146.2, 146.5, 170.4; EI-LRMS m/z 468 (Mþ),
451, 434, 419, 404; EI-HRMS Calcd for C30H44O4
468.3240, found 468.3264.
5.2.2. (23R)-25-Dehydro-1a-hydroxy-24,24-dimethyl-
vitamin D3-26,23-lactone (9). According to the general
procedure, a crude product, which was obtained from 14
(30 mg, 76 mmol), 22 (48 mg, 114 mmol), Et3N (0.8 mL)
and Pd(PPh3)4 (26 mg, 23 mmol) in toluene (0.8 mL) at
110 8C for 30 min, was treated with conc. HF in MeCN for
30 min. After usual work up, the crude product was purified
by column chromatography on silica gel (hexane/AcOEt¼1/
1) to give 9 (27 mg, 78% in 2 steps) as an amorphous solid.
UV (EtOH) lmax¼265.0 nm; [a]2D4¼þ56.2 (c 1.15, CHCl3);
1
IR (film, CHCl3) 3426, 1759, 1672, 1057 cm21; H NMR
(400 MHz, CDCl3) d 0.57 (s, 3H), 0.99 (d, J¼6.6 Hz, 3H),
1.05 (s, 3H), 1.11 (dd, J¼10.5, 1.47 Hz, 1H), 1.21 (s, 3H),

N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
7959
5.2.5. (23S)-25-Dehydro-1a-hydroxy-2a-(3-hydroxypro-
pyl)-24,24-dimethylvitamin D3-26,23-lactone (8b).
steps) as an amorphous solid. UV (EtOH) lmax¼267.0 nm;
[a]2D3¼þ13.3 (c 0.69, CHCl3); IR (neat) 3330, 1763, 1649,
1
According to the general procedure, a crude product,
which was obtained from 13 (18 mg, 46 mmol), 22b
(37 mg, 68 mmol), Et3N (0.4 mL) and Pd(PPh3)4 (30 mg,
26 mmol) in toluene (0.4 mL) at 110 8C for 4 h, was treated
with conc. HF in MeCN for 4 h. After usual work up, the
crude product was purified by column chromatography on
silica gel (hexane/AcOEt¼1/4) to give 8b (9 mg, 39% in 2
steps) as an amorphous solid. UV (EtOH) lmax¼267.5 nm;
[a]2D6¼þ13.7 (c 0.85, CHCl3); IR (film, CHCl3) 3380, 1763,
1624, 1096 cm21; H NMR (400 MHz, CDCl3) d 0.70 (s,
3H), 1.05 (s, 3H), 1.06 (d, J¼6.6 Hz, 3H), 1.21 (s, 3H),
1.23–1.72 (m, 11H), 1.84–2.04 (m, 5H), 2.24 (dd, J¼13.6,
9.2 Hz, 1H), 2.53 (br s, 3H), 2.68 (dd, J¼13.6, 4.6 Hz, 1H),
2.82 (m, 1H), 3.38 (dd, J¼7.4, 3.3 Hz, 1H), 3.83 (m, 4H),
4.05 (m, 1H), 4.10 (dd, J¼8.9, 3.3 Hz, 1H), 4.45 (d,
J¼2.9 Hz, 1H), 5.10 (d, J¼1.5 Hz, 1H), 5.39 (d, J¼1.5 Hz,
1H), 5.46 (s, 1H), 6.02 (d, J¼11.2 Hz, 1H), 6.13 (s, 1H),
6.42 (d, J¼11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d
12.1, 19.7, 22.3, 23.0, 23.5, 24.3, 27.9, 29.1, 32.0, 35.6,
40.4, 41.0, 42.6, 46.0, 52.5, 56.2, 56.7, 61.2, 68.4, 68.5,
71.9, 84.5, 86.2, 116.1, 117.2, 118.8, 125.4, 131.6, 143.1,
144.2, 146.2, 170.5; EI-LRMS m/z 528 (Mþ), 511, 494, 477,
435; EI-HRMS Calcd for C32H48O6 528.3451, found
528.3451.
1
1653, 1057 cm21; H NMR (400 MHz, CDCl3) d 0.55 (s,
3H), 1.05 (s, 3H), 1.07 (d, J¼6.6 Hz, 3H), 1.21 (s, 3H),
1.24–1.54 (m, 10H), 1.58–1.77 (m, 7H), 1.92–2.02 (m,
5H), 2.25 (dd, J¼13.5, 8.9 Hz, 1H), 2.66 (dd, J¼13.6,
4.3 Hz, 1H), 2.83 (m, 1H), 3.69–3.71 (m, 2H), 3.89 (ddd,
J¼8.3, 8.3, 4.4 Hz, 1H), 4.11 (dd, J¼9.0, 3.2 Hz, 1H), 4.38
(d, J¼2.9 Hz, 1H), 5.00 (d, J¼1.6 Hz, 1H), 5.28 (d, J¼
1.6 Hz, 1H), 5.46 (s, 1H), 6.00 (d, J¼11.4 Hz, 1H), 6.14 (s,
1H), 6.40 (d, J¼11.4 Hz, 1H); 13C NMR (100 MHz, CDCl3)
d 12.0, 12.6, 19.7, 22.3, 23.0, 23.5, 24.3, 27.9, 29.1,
35.4, 35.6, 40.4, 42.6, 43.5, 44.2, 46.0, 56.2 (2C), 56.7
(2C), 71.7, 75.4, 86.2, 113.1, 117.0, 118.8, 124.7, 133.1,
142.8, 146.2, 146.5, 170.5; EI-LRMS m/z 512 (Mþ), 495,
478, 461; EI-HRMS Calcd for C32H48O5 512.3502, found
512.3490.
5.2.8. (23R)-25-Dehydro-1a-hydroxy-2a-(3-hydroxypro-
poxy)-24,24-dimethylvitamin D3-26,23-lactone (9c).
According to the general procedure, a crude product,
which was obtained from 14 (30 mg, 76 mmol), 22c
(71 mg, 128 mmol), Et3N (0.8 mL) and Pd(PPh3)4 (27 mg,
23 mmol) in toluene (0.8 mL) at 110 8C for 3 h, was treated
with conc. HF in MeCN for 1 h. After usual work up, the
crude product was purified by column chromatography on
silica gel (hexane/AcOEt¼1/4) to give 9c (23 mg, 57% in 2
steps) as an amorphous solid. UV (EtOH) lmax¼267.0 nm;
[a]2D3¼þ64.9 (c 1.77, CHCl3); IR (film, CHCl3) 3397, 1763,
5.2.6. (23R)-25-Dehydro-1a-hydroxy-2a-(3-hydroxypro-
pyl)-24,24-dimethylvitamin
D3-26,23-lactone
(9b).
1
According to the general procedure, a crude product,
which was obtained from 14 (39 mg, 76 mmol), 22b
(68 mg, 126 mmol), Et3N (0.8 mL) and Pd(PPh3)4 (25 mg,
22 mmol) in toluene (0.8 mL) at 110 8C for 4 h, was treated
with conc. HF in MeCN for 1.5 h. After usual work up, the
crude product was purified by column chromatography on
silica gel (hexane/AcOEt¼1/4) to give 9b (18 mg, 46% in 2
steps) as an amorphous solid. UV (EtOH) lmax¼268.0 nm;
[a]2D2¼þ69.4 (c 1.38, CHCl3); IR (film, CHCl3) 3393, 1757,
1649, 1638 1076 cm21; 1H NMR (400 MHz, CDCl3) d 0.56
(s, 3H), 0.99 (d, J¼6.3 Hz, 3H), 1.06 (s, 3H), 1.11 (m, 1H),
1.21 (s, 3H), 1.26–1.35 (m, 5H), 1.48–1.86 (m, 11H),
1.97–2.05 (m, 3H), 2.25 (dd, J¼13.3, 8.7 Hz, 2H), 2.28 (br
s, 1H), 2.66 (dd, J¼13.3, 4.2 Hz, 1H), 2.83 (m, 1H), 3.69–
3.70 (m, 2H), 3.90 (ddd, J¼8.2, 8.2, 4.3 Hz, 1H), 4.15 (dd,
J¼11.4, 1.1 Hz, 1H), 4.38 (d, J¼2.9 Hz, 1H), 4.99 (d,
J¼1.7 Hz, 1H), 5.28 (d, J¼1.7 Hz, 1H), 5.47 (s, 1H), 6.00
(d, J¼11.2 Hz, 1H), 6.15 (s, 1H), 6.39 (d, J¼11.2 Hz, 1H);
13C NMR (100 MHz, CDCl3) d 12.2, 14.2, 18.6, 22.3, 22.9,
23.5, 25.1, 27.6, 29.0, 30.1, 33.0, 35.9, 40.6, 42.0, 44.3,
46.0, 49.0, 56.4, 56.9, 62.7, 70.3, 73.5, 84.3, 113.6, 117.1,
119.1, 124.5, 133.0, 142.6, 146.2, 146.4, 170.4; EI-LRMS
m/z 512 (Mþ), 495, 478, 461; EI-HRMS Calcd for C32H48O5
512.3502, found 512.3502.
1638, 1076 cm21; H NMR (400 MHz, CDCl3) d 0.56 (s,
3H), 0.99 (d, J¼6.6 Hz, 3H), 1.05 (s, 3H), 1.11 (m, 1H),
1.21 (s, 3H), 1.23–1.35 (m, 4H), 1.47–1.56 (m, 3H), 1.66–
1.88 (m, 6H), 1.96–2.05 (m, 2H), 2.24 (dd, J¼13.6, 8.8 Hz,
1H), 2.68 (dd, J¼13.6, 4.4 Hz, 1H), 2.73 (br s, 3H), 2.83 (m,
1H), 3.37 (dd, J¼7.6, 3.2 Hz, 1H), 3.74–3.91(m, 4H), 4.06
(ddd, J¼8.2, 8.2, 4.4 Hz, 1H), 4.15 (dd, J¼11.5, 1.2 Hz,
1H), 4.45 (d, J¼2.9 Hz, 1H), 5.09 (d, J¼1.7 Hz, 1H), 5.39
(s, 1H), 5.47 (s, 1H), 6.12 (d, J¼11.2 Hz, 1H), 6.15 (s, 1H),
6.41 (d, J¼11.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) d
12.2, 18.6, 22.3, 22.9, 23.5, 25.1, 27.6, 29.1, 31.9, 32.9,
35.9, 40.6, 41.0, 42.0, 46.0, 56.4, 56.9, 61.1, 68.3, 68.4,
71.8, 84.3, 84.4, 116.0, 117.3, 119.1, 125.2, 131.8, 142.9,
144.2, 146.2, 170.4; EI-LRMS m/z 528 (Mþ), 511, 494, 477,
435; EI-HRMS Calcd for C32H48O6 528.3451, found
528.3449.
5.3. Vitamin D receptor (VDR) binding assay
[26,27-Methyl-3H]-1a,25-dihydroxyvitamin D3 (specific
activity 6.623 TBq/mmol, 15,000 dpm, 15.7 pg) and various
amounts of 1a,25-dihydroxyvitamin D3 and an analogue to
be tested were dissolved in 50 mL of absolute ethanol in
12£75-mm polypropylene tubes. The chick intestinal
VDR (0.2 mg) and 1 mg of gelatin in 1 mL of phosphate
buffer solution (25 nM KH2PO4, 0.1 M KCl, 1 mM
dithiothreitol, pH 7.4) were added to each tube in an ice
bath. The assay tubes were incubated in shaking water bath
for 1 h at 25 8C and then chilled in an ice bath. 1 mL of 40%
polypropylene glycol 6000 in distilled water was added to
each tube, which was the mixed vigorously and centrifuged
at 2,260£g for 60 min at 4 8C. After the supernatant
was decanted, the bottom of the tube containing the pellet
was cut off into a scintillation vial containing 10 mL of
5.2.7. (23S)-25-Dehydro-1a-hydroxy-2a-(3-hydroxypro-
poxy)-24,24-dimethylvitamin D3-26,23-lactone (8c).
According to the general procedure, a crude product,
which was obtained from 13 (14 mg, 35 mmol), 22c
(35 mg, 63 mmol), Et3N (0.4 mL) and Pd(PPh3)4 (13 mg,
11 mmol) in toluene (0.4 mL) at 110 8C for 2 h, was treated
with conc. HF in MeCN for 1.5 h. After usual work up, the
crude product was purified by column chromatography on
silica gel (hexane/AcOEt¼1/4) to give 8c (13 mg, 69% in 2

7960
N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
2. Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr. Rev.
1995, 16, 200.
dioxane-based scintillation fluid and the radioactivity was
counted with a Beckman liquid scintillation counter (Model
LS6500). The relative potency of the analogues were
calculated from their concentration needed to displace 50%
of [26,27-methyl-3H]-1a,25-dihydroxyvitamin D3 from
the receptor compared with the activity of 1a,25-dihydroxy-
vitamin D3 (assigned a 100% value).
3. (a) Evans, R. M. Science 1988, 240, 889. (b) Umezono, K.;
Murakami, K. K.; Thompson, C. C.; Evans, R. M. Cell 1991,
65, 1255.
4. Takeyama, K.; Masuhiro, Y.; Fuse, H.; Endoh, H.; Murayama,
A.; Kitanaka, S.; Suzawa, M.; Yanagisawa, J.; Kato, S. Mol.
Cell Biol. 1999, 19, 1049.
5. (a) Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.; Miura, D.;
Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8,
151. (b) Konno, K.; Fujishima, T.; Maki, S.; Liu, Z.; Miura,
D.; Chokki, M.; Ishizuka, S.; Yamaguchi, K.; Kan, Y.;
Kurihara, M.; Miyata, N.; Smith, C.; DeLuca, H. F.;
Takayama, H. J. Med. Chem. 2000, 43, 4247. (c) Suhara, Y.;
Nihei, K.-i.; Tanigawa, H.; Fujishima, T.; Konno, K.;
Nakagawa, K.; Okano, T.; Takayama, H. Bioorg. Med.
Chem. Lett. 2000, 10, 1129. (d) Suhara, Y.; Nihei, K.-i.;
Kurihara, M.; Kittaka, A.; Yamaguchi, K.; Fujishima, T.;
Konno, K.; Miyata, N.; Takayama, H. J. Org. Chem. 2001, 66,
8760. (e) Kittaka, A.; Suhara, Y.; Takayanagi, H.; Fujishima,
T.; Kurihara, M.; Takayama, H. Org. Lett. 2000, 2, 2619.
6. For our account, see: Takayama, H.; Kittaka, A.; Fujishima,
T.; Suhara, Y. In Vitamin D Analogs in Cancer Prevention and
Therapy. Recent Results in Cancer Research 164; Reichrath,
J., Friedrich, M., Tilgen, W., Eds.; Springer: Berlin, 2003; pp
289–317.
5.4. Assay for HL-60 cell differentiation
Nitro blue tetrazolium (NBT)-reducing activity was used as
a cell differentiation marker. HL-60 cells were cultured in
RPMI-1640 medium supplemented with 10% heat-inacti-
vated FCS. Exponentially proliferating cells were collected,
suspended in fresh medium and seeded in culture plates
(Falcon, Becton Dickinson and Company, Franklin Lakes,
NJ). Cell concentration at seeding was adjusted to 2£104
cells/mL and the seeding volume was 1 mL/well. An
ethanol solution of 1a,25-dihydroxyvitamin D3 (final
concentration: 1028 M) and an analogue (final concen-
tration: 10211 to 1026 M) was added to the culture medium
at 0.1% volume and culture was continued for 96 h at 37 8C
in a humidified atmosphere of 5% CO2/air without medium
change. The same amount of vehicle was added to the
control culture. NBT-reducing assay was performed accord-
ing to the method of Collins.22 Briefly, cells were collected,
washed with PBS, and suspended in serum-free medium.
NBT/TPA solution (dissolved in PBS) was added. Final
concentrations of NBT and TPA were 0.1% and 100 ng/mL,
respectively. Then, the cell suspensions were incubated at
37 8C for 25 min. After incubation, cells were collected by
centrifugation and resuspended in FCS. Cytospin smears
were prepared, and the counter-staining of nuclei was done
with Kemechrot solution. At least 500 cells per preparation
were observed.
7. This concept was also good for 19-nor 1, see: (a) 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. (b) Yoshida, A.;
Ono, K.; Suhara, Y.; Saito, N.; Takayama, H.; Kittaka, A.
Synlett 2003, 1175. (c) For the alternative synthesis of ED-71,
see: Hatakeyama, S.; Ikeda, T.; Maeyama, J.; Esumi, T.;
Iwabuchi, Y.; Irie, H.; Kawase, A.; Kubodera, N. Bioorg. Med.
Chem. Lett. 1997, 7, 2871.
8. (a) Ishizuka, S.; Ishimoto, S.; Norman, A. W. Biochemistry
1984, 23, 1473. (b) Ishizuka, S.; Ohba, T.; Norman, A. W. In
Vitamin D: Molecular, Cellular and Chemical Endocrinology;
Norman, A. W., Schaefer, K., Grigoleit, H. G., von Herrath,
D., Eds.; Walter de Gruyter: Berlin, 1988; pp 143–144.
9. (a) Miura, D.; Manabe, K.; Ozono, K.; Saito, M.; Gao, Q.;
Norman, A. W.; Ishizuka, S. J. Biol. Chem. 1999, 274, 16392.
(b) Ozono, K.; Saito, M.; Miura, D.; Michigami, T.; Nakajima,
S.; Ishizuka, S. J. Biol. Chem. 1999, 274, 32376. (c) Miura, D.;
Manabe, K.; Gao, Q.; Norman, A. W.; Ishizuka, S. FEBS Lett.
1999, 460, 297. (d) Ishizuka, S.; Miura, D.; Eguchi, H.; Ozono,
K.; Chokki, M.; Kamimura, T.; Norman, A. W. Arch.
Biochem. Biophys. 2000, 380, 92. (e) Ishizuka, S.; Miura,
D.; Ozono, K.; Chokki, M.; Mimura, H.; Norman, A. W.
Endocrinology 2001, 142, 59. (f) Ishizuka, S.; Miura, D.;
Ozono, K.; Saito, M.; Eguchi, H.; Chokki, M.; Norman, A. W.
Steroids 2001, 66, 227.
6. Supporting Information Available
Charts of vitamin D receptor binding assay of compounds 8,
8a–c, 9, and 9a–c, and assay for HL-60 cell differentiation
to test antagonistic activity of compounds 8, 8a–c, 9, and
9a–c. This material is available online with the paper in
Science Direct.
Acknowledgements
The authors thank Miss J. Shimode and Miss A. Tonoki
(Teikyo University) for spectroscopic measurements. We
also thank emeritus professor Hiroaki Takayama for helpful
discussions. This study was supported by Grants-in-Aid
from the Ministry of Education, Culture, Sports, Science
and Technology, Japan. N.S. acknowledges TAKEDA
SCIENCE FOUNDATION for support.
10. The other type of VDR antagonist of 25-carboxylic esters
ZK159222 and ZK168281, see: (a) Herdick, M.; Steinmeyer,
A.; Carlberg, C. J. Biol. Chem. 2000, 275, 16506. (b) Bury, Y.;
Steinmeyer, A.; Carlberg, C. Mol. Pharmacol. 2000, 58,
1067. (c) Herdick, M.; Steinmeyer, A.; Carlberg, C.
Chem. Biol. 2000, 7, 885. (d) Toell, A.; Gonzalez, M. M.;
Ruf, D.; Steinmeyer, A.; Ishizuka, S.; Carlberg, C. Mol.
References and notes
¨ ¨ ¨
Pharmacol. 2001, 59, 1478. (e) Vaisanen, S.; Perakyla, M.;
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Karkkainen, J. I.; Steinmeyer, A.; Carlberg, C. J. Mol. Biol.
2002, 315, 229.
11. Carlberg, C. J. Cell. Biochem. 2003, 88, 274.
1. (a) In Vitamin D; Feldman, D., Glorieux, F. H., Pike, J. W.,
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H. F. Adv. Drug Res. 1996, 28, 269.

N. Saito et al. / Tetrahedron 60 (2004) 7951–7961
7961
12. (a) 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. (b) 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.
Arch. Biochem. Biophys. 1976, 176, 235. (b) Inaba, M.;
DeLuca, H. F. Biochim. Biophys. Acta 1989, 1010, 20.
22. Collins, S. J.; Ruscetti, F. W.; Gallagher, R. E.; Gallo, R. C.
J. Exp. Med. 1979, 149, 969.
23. For recent description of VDR regulated transactivation
mechanism, see: Masuno, H.; Yamamoto, K.; Wang, X.;
Choi, M.; Ooizumi, H.; Shinki, T.; Yamada, S. J. Med. Chem.
2002, 45, 1825.
13. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992,
114, 9836.
14. Okuda, Y.; Nakatsukasa, S.; Oshima, K.; Nozaki, H. Chem.
Lett. 1985, 481.
24. Recent review on the function of VDR in response to 1 and the
other synthetic ligands, see: Carlberg, C. In Vitamin D Analogs
in Cancer Prevention and Therapy. Recent Results in Cancer
Research 164; Reichrath, J., Friedrich, M., Tilgen, W., Eds.;
Springer: Berlin, 2003; pp 29–42; See also, Ref. 11.
25. Bula, C. M.; Bishop, J. E.; Ishizuka, S.; Norman, A. W. Mol.
Endocrinol. 2000, 14, 1788.
15. Xu, L.-H.; Ku¨ndig, E. P. Helv. Chim. Acta 1994, 77, 1480.
16. Crystal data of 13 are as follows: space group P21, Z¼2,
˚
a¼9.394(2), b¼7.571(1), c¼14.713(2) A, b¼106.10(1)8,
3
3
˚
V¼1005.3(3) A , Dc¼1.445 g/cm . Crystal data of 14 are as
follows: space group P21, Z¼2, a¼11.137(2), b¼7.609(2),
3
˚
˚
c¼12.433(2) A, b¼100.02(1)8, V¼1037.5(2) A , Dc¼1.400 g/
cm3. Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication numbers CCDC 239774 for 13 and CCDC 239775
for 14. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ,
UK (fax: þ44-1223-336033 or e-mail: dposit@ccdc.cam.a-
c.uk).
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