Synthesis of a 1α-C-methyl analogue of 25-hydroxyvitamin D3: interaction with a mutant vitamin D receptor Arg274Leu

Article abstract of DOI:10.1016/j.tet.2009.06.029

Vitamin D₃ analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1α-OH of 1α,25-dihydroxyvitamin D₃ to anchor the ligand t

Full text of DOI:10.1016/j.tet.2009.06.029

Tetrahedron 65 (2009) 7135–7145
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of a 1a-C-methyl analogue of 25-hydroxyvitamin D3: interaction
with a mutant vitamin D receptor Arg274Leu
,
Shinobu Honzawa a y, Naoyuki Takahashi a, Atsushi Yamashita b, Takayuki Sugiura b, Masaaki Kurihara c,
,
,
*
Midori A. Arai a z, Shigeaki Kato d, Atsushi Kittaka a
a Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 229-0195, Japan
b Department of Hygienic Chemistry and Nutrition, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 229-0195, Japan
c National Institute of Health Sciences, Tokyo 158-8501, Japan
d Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 May 2009
Received in revised form 5 June 2009
Accepted 5 June 2009
Available online 13 June 2009
Vitamin D3 analogues have been developed for a mutant vitamin D receptor (VDR), Arg274Leu. The
mutant VDR has a mutation at Arg274, which forms an important hydrogen bond with 1
dihydroxyvitamin D3 to anchor the ligand tightly in the VDR ligand binding pocket. Stereoselective
synthesis of the A-ring part of the novel vitamin D analogue, 2 -(3-hydroxypropyl)-1 -methyl-25-hy-
droxyvitamin D3 (4), from -galactose was accomplished with the key steps of the introduction of the
methyl and allyl groups to the chiral building blocks. The new analogue 4 is ca. 7.3-fold more active than
the natural hormone 1 ,25-dihydroxyvitamin D3 (1).
a-OH of 1a,25-
a
a
D
Keywords:
Vitamin D analogue
Vitamin D receptor
a
Ó 2009 Elsevier Ltd. All rights reserved.
Mutant vitamin D receptor
Structure–function relationships
1. Introduction
residues in the ligand binding pocket (LBP) of VDR through hydrogen
bonds. However, hydrophobic interactions between the hydrophobic
moieties oftheligand andhydrophobicaminoacidresidues intheLBP
of VDR seem to be important. In the case of the human VDR, the triene
moiety connecting the C-ring and A-ring of 1 is sandwiched between
Ser-275 and Trp-286 on one side, and Leu-233 on the other side, and
the D-ring side chain is surrounded by hydrophobic residues, for
example, Leu-227, Val-234, Val-300, Leu-309, Leu-404, and Val-418.
Recently, adamantyl vitamin D analogues have been reported that
showed VDR partial agonism and antagonism, in which hydrophobic
interactionsof therigid and bulkyadamantyl sidechain to helixes and
loops of the VDR ligand binding domain affected stability of the VDR
active conformation.6 Alanine scanning mutational analysis of VDR
afforded important information of ligand recognition by the VDR and
transactivation potency of the ligand. These were also related to
hydrophobic interactions (Fig. 1).7
Hydrogen bonding is one of the most important interactions
between biomacromolecules such as proteins and nucleic acids,
and biologically active small molecules such as hormones and drug
molecules.1 Also very important is the hydrophobic interactions
between such molecules.2 The latter interactions are weaker than
the former, but cannot be neglected when they account for much of
the biological system.
Vitamin D3, a lipophilic hormonal molecule of low molecular
weight, has long been known to play a significant role in the sus-
tainment of life through calcium and phosphate homeostasis.3 Re-
cently, it has been clarified that this molecule is also involved in
controlling cell differentiation and proliferation through binding with
a specific nuclear hormone receptor, vitamin D receptor (VDR).4 The
identity of the vitamin D3 action is the dihydroxylated metabolite,
1
a
,25-dihydroxyvitamin D3 (1
experiments5 have shown that the three hydroxy groups of 1, that is,
-OH, 3 -OH and 25-OH, interact with hydrophilic amino acid
a,25(OH)2D3, 1). X-ray crystallographic
OH
H
1a
b
C
D
H
1: R1 = OH, R2 = H
* Corresponding author. Tel./fax: þ81 42 685 3713.
2: R1 = OH, R2 = CH3
A
2
E-mail address: akittaka@pharm.teikyo-u.ac.jp (A. Kittaka).
3
1
3: R1 = OH, R2 = (CH2)3OH
4: R1 = CH3, R2 = (CH2)3OH
y
Present address: Faculty of Pharmaceutical Sciences, Niigata University of
HO
R1
R2
Pharmacy and Applied Life Sciences (NUPALS), Niigata 956-8603, Japan.
z
Present address: Graduate School of Pharmaceutical Sciences, Chiba University,
Figure 1. Vitamin D analogues.
Chiba 263-8522, Japan.
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.06.029

7136
S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
C
D
OH
OH
H
H
Vitamin D3
Br
5
+
A
R3O
R3O
HO
CH3
OH
OH
CH3
OR2
CH3
R4O
CH3
OR4
HO
4
O
8
7
6
Pd-catalyzed
coupling reaction
Epoxide ring opening
by Grignard reagent
Ph
OH
OH
O
O
O
O
D-Galactose
Ph
O
COOR1
O
Ph
O
COOR1
HO
OH
CH3
OH
10
11
9
Stereoselective 1,4-addition
Scheme 1. Retrosynthetic analysis of the analogue.
Mutations of hydrophilic amino acid residues would diminish
affinity, and have been found to cause hereditary vitamin D re-
sistant rickets (HVDRR).8 We focused on the mutation of Arg-274,
2. Results and discussion
The analogue 4 was synthesized by a convergent method
(Scheme 1). A 2 -(3-hydroxypropyl) group was introduced to en-
hance affinity for the VDR.13–16 2
-(3-Hydroxypropyl) group is one
which forms a hydrogen bond with the 1
a
-OH group of 1. The
a
replacement of Arg-274 with Leu diminished affinity to ca. 1/
a
1000.9
of the active motifs that we have been developing, and we have al-
ready shown that the moiety itself recovers affinity for the mutant
VDR (Arg274Ala).17The CD-ring moiety 5 was prepared fromvitamin
D3 as reported,18 and the A-ring enyne 6 was synthesized from
We anticipated that the loss of affinity would be recovered
through hydrophobic interactions between the hydrophobic pocket
formed by the mutation and the hydrophobic substituent of the
modified ligand. Such analogues, which possess a hydrophobic 1
benzyloxy group, have been reported by Koh’s group.10 Other
analogues have been reported by us,11 which have the 1
-hydroxy-
methyl group reported by Posner’s group to be hypocalcemic.12 So
we decided to design and synthesize the 1 -methylated analogue 4
a
-
D
-galactose in order to introduce efficiently all of the chiral carbon
centers in 6. The key steps are (1) regioselective introduction of the
allyl group that corresponds to the 2 -(3-hydroxypropyl) group for
step 8 to 7, (2) stereoselective 1,4-addition using an organocopper
reagent in order to introduce the 1
-methyl group for step 10 to 9.19
a
a
a
a
as a candidate for an alternative ligand for the mutant VDR
(Arg274Leu) (Fig. 1).
Coupling of the CD-ring 5 and the A-ring enyne 6 was carried out
with a palladium-catalyzed Trost coupling reaction.18
OH
Ph
O
OH
OH
O
Ph
O
CHO
O
O
benzaldehyde
NaIO4, NaOH
H2O
HO
O
OH
ZnCl2
OH
O
OH
HO
OH
OH
O
Ph
D-Galactose
O
11
O
Ph
O
O
HO
12
OH
OTBS
(EtO)2P(O)CH2COOEt
NaH, THF
O
TBSCl
O
DMF, imidazole
97%
Ph
O
COOEt
Ph
O
COOEt
3 steps 12%
13
14
OTBS
(R)
OTBS
O
O
CuI, MeMgBr
(S)
+
Ph
O
COOEt
TMSCl, THF
-78 °C ~ rt
Ph
O
COOEt
CH3
CH3
15
15'
60%
16%
Scheme 2. Synthesis of A-ring enyne [part 1].

S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
7137
The A-ring enyne 6 was synthesized as follows (Scheme 2); the
aldehyde 1220was subjected to Horner–Wadsworth–Emmons ole-
The benzylidene acetal of 15 was deprotected by hydro-
genolysis, followed by lactonization to give the 5-membered lac-
tone 17. The primary hydroxy group was protected as a trityl ether,
18, and a 2-step reduction of lactone gave the diol 19, whose pri-
fination to give an
droxy group was protected by a TBS group. A methyl group was
introduced into the -unsaturated ester 14 with good stereo-
a,b-unsaturated ester, 13, whose secondary hy-
a
,b
mary hydroxy group was protected as
(Scheme 4).
a pivaloyl ester, 20
selectivity to give the (R)-configured ester 15 as the major isomer.
The configuration of the methyl group of 15 was confirmed by NMR
experiments (COSY, differential NOE) with the lactones 16 and 160
(Scheme 3 and Fig. 2). The stereoselectivity could be explained by
a ‘modified’ Felkin–Ahn model (Fig. 3).21
The set up was now ready for the introduction of the allyl group.
The secondary hydroxy group of 20 was mesylated, and treatment
with TBAF in the presence of zeolite gave the epoxide 22 in good
yield, which was treated with excess allyl Grignard reagent in THF
Ph
O
O
OTBS
O
O
O
O
TBAF
R1
O
O
COOEt
Ph
O
R1
73%
Ph
R2
R1
R2
O
R2
15: R1 = CH3, R2 = H
2
16: R1 = CH3, R2 = H
16': R1 = H, R2 = CH
15': R1 = H, R = CH
3
3
Scheme 3. Formation of lactones 16 and 160.
NOE experiments (600 MHz, CDCI )
Coupling constants
3
Ph
Ph
O
H
O
H
H
H
H
O
O
O
H
H
H
H
H3
H3
O
O
H1
CH3
2.9%
H
H
H
O
H3C
H3C
1.5%
H1
1.9%
12.0 Hz
H1
H4
16'
H4
6.8 Hz
O
O
H2
H2
2.3%
16
16
Figure 2. Determination of the configuration of the methyl group.
"Modified" Felkin-Ahn model
OTBS
H
H
OTBS
COOEt
COOEt
COOEt
O
H
O
H
Ph
O
O
H
TBSO
H
O
14
Ph
"Cu"
H
H
"Cu"
OTBS
O
O
(R)
Ph
COOEt
CH3
15
Figure 3. Modified Felkin–Ahn model to explain the diastereoselective methylation of 14.
O
O
OTBS
O
O
O
Pd(OH)2/C, H2
EtOH
TrCl, pyridine
DMAP
Ph
O
COOEt
TrO
TBSO
HO
TBSO
CH3
CH3
CH3
90% (2 steps)
15
17
18
OH
OH
1) DIBAL-H, toluene
2) NaBH4, THF, EtOH
75%
PivCl
OH
OPiv
TrO
TrO
pyridine
CH3
TBSO
CH3
TBSO
95%
19
Scheme 4. Synthesis of A-ring enyne [part 2].
20

7138
S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
to give 23. The allyl group was introduced regioselectively into
a site far from the trityloxy group by this method.22 The position of
the allyl group could be assigned by 2D-NMR (COSY and HMBC). At
the same time, the pivaloyl group was also deprotected during the
allylation, so reprotection as a TBS ether needed to be carried out to
afford 24 (Scheme 5).
with luciferase as reported previously.11 The analogue 3211 without
substitution at the 1-position was also assayed as a reference. For
the wild-type hVDR, the analogue synthesized this time showed
only 6% affinity as compared with 1a,25(OH)2D3 (1) (Table 1). This
result is reasonable because the 1 -methyl group of 4 is not able to
a
form a hydrogen bond with the wild-type receptor.
MsCl
OH
OMs
O
pyridine
99%
TBAF, zeolite (A-4)
OPiv
OPiv
OPiv
TrO
TBSO
TrO
TBSO
TrO
THF
CH3
CH3
86%
H3C
22
20
21
TBSCl
allylMgCl
THF
imidazole
OH
OTBS
DMF
TrO
TrO
OH CH3
23
OH CH3
24
95%
90%
Scheme 5. Synthesis of A-ring enyne [part 3].
Transformation to the A-ring enyne could be carried out in
a relatively conventional manner (Scheme 6). Hydroboration at the
terminal olefin of 24 led to the diol 25, whose primary hydroxy
Next, an assay was carried out using the mutant hVDR
(Arg274Leu) (Table 2). As shown previously, the 2 -hydroxypropyl
group itself could recover the loss of affinity for the mutant (the
a
OH
OPiv
1) BH3·THF
THF
1) PivCl
pyridine
OTBS
OTBS
OH
TrO
TrO
TrO
2) H2O2, H2O
2) TBAF
OH CH3
OH CH3
OH CH3
26
81%
81% (2 steps)
25
24
OPiv
SeCN
OPiv
1)
TsCl, CH2Cl2
, PBu3, THF
NO2
LiHMDS
THF
91%
2) H2O2, THF
cat. Bu2SnO, Et3N
TsO
HO
3) TsOH, CH3OH
OH CH3
28
OH CH3
54% (3 steps)
85%
27
OPiv
1)
TMS
BF3·OEt2
, n-BuLi
TBSOTf
2,6-lutidine
HO
CH3
OH
TBSO
CH3
OTBS
2) NaOCH3, CH3OH
92% (2 steps)
O
CH2Cl2
62%
CH3
29
31
30
Scheme 6. Synthesis of A-ring enyne [part 4].
group was protected as a pivaloyl ester. The TBS group was
deprotected, and then dehydration according to Greeco’s pro-
cedure23 afforded the terminal alkene, whose trityl group was re-
moved by TsOH to give 27. The primary hydroxy group could be
selectively esterified as the tosylate 28,24 and treatment with base
gave the epoxide 29. Alkynylation25 followed by persilylation gave
the target enyne 31.
OH
OH
H
H
1) (Ph3P)4Pd, toluene
Et3N
Br
5
+
2) TBAF, THF
29% (2 steps)
Trost coupling between 5 and 31 followed by deprotection
afforded the target molecule 4 in 29% yield. This compound could
be purified by reverse phase HPLC for biological assays
(Scheme 7).
HO
CH3
TBSO
CH3
OTBS
OH
4
31
The analogues were assayed for the wild-type human VDR
(hVDR) and the mutant hVDR(Arg274Leu). Assays were carried out
Scheme 7. Construction of the analogue 4.

S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
7139
Table 1
analogue 32 is 5.6-fold more potent than the natural hormone
1).17 4 was found to be more potent than 32, and the most potent
of the analogues tested this time (7.3-fold more potent than the
natural hormone 1). This is consistent with our expectation that
Transcriptional activity for wild-type hVDR
the 1a-methyl group of 4 would fit the hydrophobic pocket in
OH
the LBP of the mutant VDR, thus enhancing affinity for the
mutant VDR.
H
A hydrophobic interaction is one of the most important ones
between proteins such as receptors and ligands and between
3
32
4
:
:
:
R = OH
R = H
R = CH3
enzymes and their substrates. We have examined 1
analogues which possess 2 -alkyl, 2 -alkoxy, 2 -(
alkyl) or 2 -( -hydroxyalkoxy) groups in order to develop much
a
,25(OH)2D3
a
a
a u-hydroxy-
a
u
HO
R
more effective vitamin D agonists and to consider the effects of
the A-ring conformation on biological activity.13–16 In the ligand
binding domain (LBD) of VDR, there is a hydrophobic cavity
OH
Compounds
Relative luciferase activity
around the A-ring of 1
ceptor and a water channel near the C-2 position of the ligand.5,26
Among the analogues synthesized, the 2 -methyl analogue has
a,25(OH)2D3 in a complex with the re-
1
a
,25(OH)2D3 (1)
100
210
3
3
32
4
a
been shown to have increased affinity for the VDR, and more
potent HL-60 cell differentiation activity and transcriptional ac-
tivity in reporter assays. Recently, an X-ray crystallographic
analysis26 showed the structure of the complex of the hVDR LBD
6
The potency of 1a,25(OH)2D3 is normalized to 100.
Table 2
a-methyl 1a
,25(OH)2D3 analogue 2.27 From the results,
Transcriptional activity for mutant hVDR (Arg274Leu)
with the 2
the 2 -methyl group seemed to interact with Phe-150, Leu-233,
a
and Ser-237 to make additional van der Waals contacts. These
contacts should increase the affinity for the VDR up to 4-fold. The
OH
role of the 2a
-(3-hydroxypropyl) group14b,c for compound 4 was
also clarified in the same study. Interestingly, this moiety comes
into the water channel, and the terminal OH group replaces one
of the water molecules constituting the water channel to main-
tain hydrogen bond networks, thus stabilizing the complex up
to 3-fold.
H
3
:
:
:
R = OH
R = H
R = CH3
32
4
Similar effects appear to work for the complex of the mutant
hVDR (Arg274Leu) with 4. Molecular modeling demonstrated
R
HO
OH
that around the 1
hydrophobic than that of the wild-type hVDR because of the
mutation. The distance from the 1 -methyl group to the side
a-methyl group there appears a cavity, more
Compounds
Relative luciferase activity
a
1
a
,25(OH)2D3 (1)
0.8a (100)b
3.9a (570)b
3.8a (560)b
6.2a (730)b
chain of Leu-274 was calculated to be 3.2 Å, which is reasonable
for a van der Waals contact (Fig. 4). The terminal hydroxyl group
of the 2a-substitution of 4 would work as one of the water
3
32
4
molecules which binds to the Asp-144 peptide backbone in
the LBP.
a
The potency of 1
a
,25(OH)2D3 for wild type VDR is normalized to 100.
b
The potency of 1
a
,25(OH)2D3 for mutant VDR (Arg274Leu) is normalized to 100.
Figure 4. Molecular modeling of the complex of the analogue 4 and the mutant hVDR (Arg274Leu).

7140
S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
3. Conclusion
the ester 13 (2.3 g,16% for 3 steps) as a white solid. Analytically pure
13 was obtained by recrystallization from EtOH.
1H NMR (400 MHz, CDCl3)
We have developed the agonist
4
for
a
mutant hVDR
d
1.30 (3H, t, J¼7.2 Hz), 2.64 (1H, d,
(Arg274Leu), in which the A-ring part was synthesized from
-galactose in a stereoselective manner. The analogue has a
-methyl group that is anticipated to interact with the hydro-
phobic pocket formed by the mutation. 4 is ca. 7.3-fold and 1.6-fold
more active than the natural hormone 1 and 1 -hydroxy analogue
J¼11.2 Hz), 3.70 (1H, dddd, J¼1.2, 1.6, 1.6, 11.2 Hz), 4.15 (1H, dd,
J¼1.2, 12.0 Hz), 4.21 (2H, q, J¼7.2 Hz), 4.28 (1H, dd, J¼1.6, 12.0 Hz),
4.67 (1H, ddd, J¼1.6, 1.6, 4.0 Hz), 5.67 (1H, s), 6.20 (1H, dd, J¼1.6,
15.6 Hz), 6.96 (1H, dd, J¼4.0, 15.6 Hz), 7.39–7.41 (3H, m), 7.52–7.54
D
1
a
a
(2H, m). 13C NMR (100 MHz, CDCl3)
d 14.6, 60.9, 65.8, 72.7, 78.9,
3, respectively, and the latter enhancement might be caused by
hydrophobic interactions.
101.6, 123.2, 126.2, 128.6, 129.5, 137.5, 143.2, 166.1. IR (KBr): 698,
750, 808, 1028, 1150, 1186, 1244, 1302, 1402, 1659, 1699, 2975,
3507 cmꢁ1. MS m/z: 278 (Mþ), 172. HRMS calcd for [C15H18O5]
19
278.1155, found 278.1151. [
a
]
ꢁ46.7 (c 1.12, CHCl3). Mp 107.0–
D
4. Experimental section
4.1. General
108.0 ꢀC (EtOH). Anal. Calcd for C15H18O5: C, 64.74; H, 6.52. Found:
C, 64.69; H, 6.66.
4.3. Ethyl (E)-3-[(2S,4R,5R)-5-(tert-butyldimethylsilyloxy)-2-
phenyl-1,3-dioxan-4-yl]acrylate (14)
1H and 13C NMR spectra were recorded on JEOL AL-400 NMR
(400 MHz) and ECP-600 NMR (600 MHz) spectrometers. 1H NMR
spectra were referenced with (CH3)4Si (
stantard. 13C NMR spectra were referenced with deuterated solvent
77.0 ppm for CDCl3). IR spectra were recorded on JASCO FT-IR-
d 0.00 ppm) as an internal
Under an Ar atmosphere, to an ice cold solution of 13 (5.7 g,
20.5 mmol) in DMF (50 mL) was added imidazole (2.8 g, 41.0 mmol)
and TBSCl (4.6 g, 30.8 mmol), and the mixture was stirred at room
temperature overnight. The reaction was quenched by adding sat-
urated aqueous NaHCO3 (50 mL), and the mixture was extracted
with Et2O (500 mL). The organic layer was washed with brine
(50 mL), dried (MgSO4) and concentrated. Purification by silica gel
flash column chromatography (hexane/AcOEt (15:1 to 9:1)) gave
the TBS ether 14 (7.8 g, 97%) as a colorless syrupy oil.
(d
800 Fourier Transform Infrared Spectrophotometer. Low- and high
resolution mass spectra were recorded on a JEOL JMX-SX 102A
spectrometer. FAB mass spectra were measured using m-nitro-
benzyl alcohol matrix. Elemental analyses were conducted with
a Perkin Elmer PE 2400II CHNS/O analyzer. Melting points were
determined with a Yanagimoto micromelting point apparatus
without correction. Optical rotations were measured on a JASCO
DIP-370 digital polarimeter. Column chromatography was per-
1H NMR (400 MHz, CDCl3)
d 0.04 (3H, s), 0.08 (3H, s), 0.91 (9H, s),
1.27 (3H, t, J¼7.2 Hz), 3.70 (1H, ddd, J¼1.6, 1.6, 1.6 Hz), 4.07 (1H, dd,
J¼1.6, 12.0 Hz), 4.15 (1H, dd, J¼1.6, 12.0 Hz), 4.20 (2H, q, J¼7.2 Hz),
4.56 (1H, ddd, J¼1.6, 1.6, 4.0 Hz), 5.60 (1H, s), 6.13 (1H, dd, J¼1.6,
15.6 Hz), 6.90 (1H, dd, J¼4.0, 15.6 Hz), 7.35–7.40 (3H, m), 7.52–7.54
formed on silica gel 60 N (Kanto Chemical Co., Inc., 100–210 mm) or
silica gel 60 (Merck, 0.040–0.063 mm). Preparative thin layer
chromatography was performed on silica gel 60 F254 (Merck,
0.5 mm).
(2H, m). 13C NMR (100 MHz, CDCl3)
d
ꢁ4.8, ꢁ4.4, 14.3, 18.2, 25.8,
60.3, 66.1, 72.3, 78.8, 101.1, 122.4, 126.3, 128.2, 129.0, 138.0, 144.3,
166.0. IR (neat): 777, 1026, 1097, 1163, 1267, 1364, 1400, 1460, 1666,
4.2. Ethyl (E)-3-[(2S,4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-
yl]acrylate (13)
1720, 2859, 2932 cmꢁ1. MS m/z: 392 (Mþ), 335, 305, 259. HRMS
19
calcd for [C21H32O5Si] 392.2025, found 392.2022. [
CHCl3).
a]
ꢁ35.8 (c 1.17,
D
Under an Ar atmosphere, a mixture of
D-galactose (10.0 g,
55.5 mmol), ZnCl2 (3.8 g, 27.8 mmol) and benzaldehyde (8.4 mL,
83.3 mmol) was stirred at room temperature overnight. Benzalde-
hyde (8.4 mL, 83.3 mmol) was added further, and stirring was
continued at the same temperature overnight. The reaction was
quenched by adding ice-cold water (200 mL). Layers were sepa-
rated, and the organic layer was extracted with water (500 mL).
Combined aqueous layers were basified by 10% Na2CO3, and pre-
cipitate was filtered off through Celite. The filtrate was washed with
hexane (700 mL), and concentrated under reduced pressure to
a volume of 1/10. The resulting benzylidene acetal solution was
basified (pH>10) by 1 M aqueous NaOH, and the solution was
cooled with an ice bath. NaIO4 (47.5 g, 0.22 mol) was added, and the
mixture was stirred at room temperature for 6 h. Water (300 mL)
and NaCl were added, and precipitates were removed by filtration
through Celite. The filtrate was extracted with AcOEt (2.5 L), and
the organic layer was washed with brine (200 mL) and dried
(Na2SO4). The solvent was removed in vacuo to give pale yellow oil
of 12.
Under an Ar atmosphere, to a suspension of NaH (60% in oil,
823 mg, 34.3 mmol) in THF (50 mL) was added ethyl
diethylphosphonoacetate (6.8 mL, 34.3 mmol), and stirred at 0 ꢀC
for 15 min. A solution of crude aldehyde 12 above prepared in THF
(30 mL) was added via cannula, and further stirred at the same
temperature for 10 min. The reaction was quenched by adding
saturated aqueous NH4Cl solution (50 mL), and the mixture was
extracted with AcOEt (400 mL). The organic layer was washed with
brine (30 mL), dried (MgSO4) and concentrated. Purification by
silica gel flash column chromatography (hexane/AcOEt (2:1)) gave
4.4. Ethyl (R)-3-[(2S,4R,5R)-5-(tert-butyldimethylsilyloxy)-2-
phenyl-1,3-dioxan-4-yl]butylate (15) and ethyl (S)-3-
[(2S,4R,5R)-5-(tert-butyldimethylsilyloxy)-2-phenyl-
1,3-dioxan-4-yl]butylate (150)
Under an Ar atmosphere,
a suspension of CuI (97 mg,
0.50 mmol) in THF (2 mL) was stirred at ꢁ78 ꢀC, and CH3MgBr
(0.93 M in THF, 3.2 mL, 2.98 mmol) was added. After stirred at
ꢁ40 ꢀC for 30 min, the mixture was cooled to ꢁ78 ꢀC, and
(CH3)3SiCl (65 mL, 0.50 mmol) was added, followed by a solution of
14 (100 mg, 0.25 mmol) in THF (4 mL). Stirring was continued at the
same temperature for 1.5 h, and the temperature was gradually
raised up to room temperature. The reaction was quenched by
adding saturated aqueous NH4Cl solution buffered to pH 9 (5 mL),
and the mixture was extracted with Et2O (40 mL). The organic layer
was washed with brine (5 mL), dried (MgSO4), and concentrated.
Purification by preparative TLC (hexane/AcOEt (20:1)) gave 15
(61 mg, 60%) and 150 (17 mg, 16%) as colorless syrupy oils.
4.4.1. Compound 15
1H NMR (400 MHz, CDCl3)
d 0.11 (3H, s), 0.14 (3H, s), 0.95 (9H, s),
0.97 (3H, d, J¼6.8 Hz), 1.16 (3H, t, J¼7.2 Hz), 2.19 (1H, dd, J¼8.0,
15.2 Hz), 2.46–2.56 (1H, m), 2.65 (1H, dd, J¼4.8, 15.2 Hz), 3.50 (1H,
dd, J¼0.8, 9.6 Hz), 3.71 (1H, ddd, J¼0.8, 1.2, 1.6 Hz), 3.95 (1H, dd,
J¼1.2, 12.0 Hz), 4.00 (2H, q, J¼7.2 Hz), 4.19 (1H, dd, J¼1.6, 12.0 Hz),
5.47 (1H, s), 7.30–7.37 (3H, m), 7.47–7.49 (2H, m). 13C NMR
(100 MHz, CDCl3)
d
ꢁ4.5, ꢁ3.8, 14.2, 15.2, 18.3, 25.9, 30.8, 37.7, 59.9,

S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
7141
64.4, 72.3, 83.4, 101.1, 126.3, 128.0, 128.7, 138.6, 173.1. IR (neat): 775,
1026, 1074, 1252, 1304, 1364, 1460, 1734, 2857, 2932, 2957 cmꢁ1. MS
catalyst was removed by filtration, and the filtrate was concen-
trated to give the crude lactone 17 (301 mg) as colorless syrup. 17
was dissolved in pyridine (10 mL), and TrCl (460 mg, 1.65 mmol)
and DMAP (134 mg, 1.10 mmol) were added. After stirred at 75 ꢀC
overnight, the mixture was cooled to room temperature, and di-
luted with water (30 mL). The mixture was extracted with Et2O
(170 mL), and the organic layer was washed with brine (20 mL),
dried (MgSO4) and concentrated. Purification by silica gel flash
column chromatography (hexane/AcOEt (15:1)) gave 18 (514 mg,
90% for 2 steps) as a white wax.
m/z: 408 (Mþ). HRMS calcd for [C22H36O5Si] 408.2332, found
19
408.2323. [
a
]
þ4.1 (c 1.02, CHCl3).
D
4.4.2. Compound 150
1H NMR (400 MHz, CDCl3)
d 0.12 (3H, s), 0.15 (3H, s), 0.95 (9H, s),
1.07 (3H, d, J¼6.8 Hz), 1.26 (3H, t, J¼6.8 Hz), 2.17 (1H, dd, J¼8.6,
14.2 Hz), 2.42–2.46 (1H, m), 2.49 (1H, dd, J¼4.2, 14.2 Hz), 3.57 (1H,
dd, J¼1.2, 8.6 Hz), 4.13 (1H, ddd, J¼1.2, 1.2, 1.5 Hz), 3.96 (1H, dd,
J¼1.2, 12.5 Hz), 4.20 (2H, q, J¼6.8 Hz), 4.20 (1H, dd, J¼1.5, 12.5 Hz),
5.51 (1H, s), 7.33–7.38 (3H, m), 7.49–7.51 (2H, m). 13C NMR
1H NMR (400 MHz, CDCl3)
d
ꢁ0.18 (3H, s), ꢁ0.06 (3H, s), 0.78
(9H, s), 1.14 (3H, d, J¼6.8 Hz), 2.10 (1H, dd, J¼6.0, 17.6 Hz), 2.38–2.46
(1H, m), 2.70 (1H, dd, J¼8.8, 17.6 Hz), 3.09 (1H, dd, J¼4.8, 9.2 Hz),
3.40 (1H, dd, J¼8.4, 9.2 Hz), 3.61 (1H, ddd, J¼2.4, 4.8, 8.4 Hz), 4.41
(1H, dd, J¼2.4, 5.2 Hz), 7.22–7.32 (10H, m), 7.42–7.45 (5H, m). 13C
(100 MHz, CDCl3)
d
ꢁ4.4, ꢁ3.7, 14.3, 15.7, 18.3, 25.9, 31.3, 36.9, 60.3,
64.8, 72.2, 83.0, 101.5, 126.2, 128.1, 128.7, 138.7, 172.5. IR (neat): 775,
835, 1028, 1101, 1179, 1254, 1364, 1462, 1734, 2930 cmꢁ1. MS m/z:
408 (Mþ). HRMS calcd for [C22H36O5Si] 408.2332, found 408.2342.
NMR (100 MHz, CDCl3)
d
ꢁ4.9, ꢁ4.4, 18.0, 19.6, 25.7, 31.0, 37.0, 64.0,
21
[
a]
ꢁ2.9 (c 0.62, CHCl3).
71.9, 86.5, 87.1, 127.1, 127.8, 128.5, 143.8, 176.8. IR (neat): 1076, 1175,
D
1213, 1255, 1451, 1491, 1781, 2930, 2955, 3061 cmꢁ1. MS m/z: 516
4.5. (2R,4R,8R,9R)-8-Methyl-2-phenyltetrahydropyrano[3,2-
d]-1,3-dioxin-6-one (16)
(Mþ), 459 (Mþꢁt-Bu), 439 (MþꢁC6H5). HRMS calcd for [C32H40O4Si]
23
516.2683, found 516.2711. [
a]
ꢁ19.7 (c 1.11, CHCl3).
D
Under an Ar atmosphere, to a cold (0 ꢀC) solution of 15 (290 mg,
0.71 mmol) in THF (5 mL) was added TBAF (1 M in THF, 1.1 mL,
1.1 mmol), and the mixture was stirred at room temperature for
21 h. The mixture was diluted with Et2O (20 mL), and washed with
brine (5 mL). The organic layer was dried (MgSO4), and concen-
trated. Purification by silica gel column chromatography (hexane/
AcOEt (3:2)) gave the lactone 16 (129 mg, 73%) as a colorless syrupy
oil.
4.8. (3R,4R,5R)-5-(tert-Butyldimethylsilyloxy)-3-methyl-6-
trityloxyhexane-1,4-diol (19)
Under an Ar atmosphere, to a cold (ꢁ78 ꢀC) solution of 18
(514 mg, 0.99 mmol) in toluene (10 mL) was added DIBAL-H (1 M in
toluene, 3.0 mL, 3.0 mmol), and stirred at the same temperature for
30 min. The reaction was quenched by adding 10% aqueous
Rochelle salt solution (20 mL) at ꢁ78 ꢀC, and the mixture was
extracted with AcOEt (180 mL). The organic layer was washed with
brine (10 mL), dried (MgSO4) and concentrated. The residue was
dissolved in THF (5 mL) and EtOH (5 mL), and cooled to 0 ꢀC. NaBH4
(112 mg, 2.97 mmol) was added, and the mixture was stirred at the
same temperature for 30 min, and room temperature for 40 min.
The mixture was cooled in an ice bath, and water was added. The
mixture was extracted with AcOEt (170 mL), and the organic layer
was washed with brine (10 mL), dried (MgSO4) and concentrated.
Purification by silica gel flash column chromatography (hexane/
AcOEt (5:1)) gave the diol 19 (363 mg, 75%) as a colorless syrupy oil.
1H NMR (400 MHz, CDCl3)
d
1.15 (3H, d, J¼6.8 Hz), 2.22 (1H, m),
2.52 (1H, dd, J¼6.8, 18.0 Hz), 2.58 (1H, dd, J¼12.0,18.0 Hz), 4.06 (1H,
dd, J¼1.2, 1.2 Hz), 4.11 (1H, dd, J¼1.2, 12.8 Hz), 4.22 (1H, ddd, J¼1.2,
1.2, 1.2 Hz), 4.42 (1H, dd, J¼1.2, 12.8 Hz), 5.61 (1H, s), 7.37–7.39 (3H,
m), 7.46–7.48 (2H, m). 13C NMR (100 MHz, CDCl3)
d 16.3, 31.2, 32.7,
69.5, 72.8, 73.7, 100.6, 126.0, 128.2, 129.1, 137.5, 170.1. IR (neat): 702,
882, 1013, 1094, 1237, 1456, 1399, 1732, 2926 cmꢁ1. MS m/z: 248
(Mþ), 190, 171, 142. HRMS calcd for [C14H16O4] 248.1048, found
19
248.1052. [
a
]
þ4.4 (c 0.59, CHCl3).
D
4.6. (2R,4R,8S,9R)-8-Methyl-2-phenyltetrahydropyrano[3,2-
d]-1,3-dioxin-6-one (160)
1H NMR (400 MHz, CDCl3)
d
ꢁ0.13 (3H, s), ꢁ0.02 (3H, s), 0.81
(9H, s), 0.91 (3H, d, J¼6.4 Hz), 1.55–1.63 (1H, m), 1.65–1.73 (1H, m),
1.78–1.88 (1H, m), 2.11 (1H, br s), 2.37 (1H, br s), 3.04 (1H, dd, J¼4.8,
9.6 Hz), 3.33 (1H, dd, J¼7.2, 9.6 Hz), 3.51 (1H, dd, J¼2.4, 8.4 Hz), 3.61
(1H, ddd, J¼5.1, 7.8, 10.0 Hz), 3.74 (1H, ddd, J¼5.6, 5.6, 10.0 Hz), 3.79
(1H, ddd, J¼2.4, 4.8, 7.2 Hz), 7.21–7.32 (10H, m), 7.42–7.45 (5H, m).
Under an Ar atmosphere, to a cold (0 ꢀC) solution of 150 (18 mg,
44 mmol) in THF (0.5 mL) was added TBAF (1 M in THF, 0.1 mL,
0.1 mmol), and the mixture was stirred at room temperature for
1 h. The mixture was diluted with Et2O (20 mL), and washed with
water (5 mL) and brine (5 mL). The organic layer was dried
(MgSO4), and concentrated. Purification by preparative TLC (hex-
ane-AcOEt (1: 1)) gave the lactone 160 (7 mg, 54%) as a colorless
syrupy oil.
13C NMR (100 MHz, CDCl3)
d
ꢁ4.9, ꢁ4.1, 17.4, 18.1, 25.8, 33.7, 36.9,
61.2, 64.7, 71.2, 75.7, 87.0, 127.0, 127.8, 128.6, 143.9. IR (neat): 706,
837, 1073, 1256, 1449, 2361, 2886, 2955, 3384 cmꢁ1. FABMS m/z:
543 (MþNa)þ. HRFABMS calcd for [C32H44O4SiNa] 543.2906, found
22
543.2913. [
a
]
ꢁ4.1 (c 0.96, CHCl3).
D
1H NMR (600 MHz, CDCl3)
d
1.15 (3H, d, J¼7.2 Hz), 2.29 (1H, dd,
J¼3.6, 17.4 Hz), 2.35–2.41 (1H, m), 2.94 (1H, dd, J¼6.0, 17.4 Hz), 4.01
(1H, dd, J¼1.8, 3.6 Hz), 4.11 (1H, dd, J¼1.8, 12.6 Hz), 4.28 (1H, ddd,
J¼1.8, 1.8, 1.8 Hz), 4.45 (1H, dd, J¼1.8, 12.6 Hz), 5.60 (1H, s), 7.36–
4.9. (3R,4R,5R)-5-(tert-Butyldimethylsilyloxy)-4-hydroxy-3-
methyl-6-trityloxyhexyl pivalate (20)
7.40 (3H, m), 7.47–7.49 (2H, m). 13C NMR (100 MHz, CDCl3)
d
17.1,
To a cold (0 ꢀC) solution of 19 (344 mg, 0.66 mmol) in pyridine
30.8, 32.9, 69.4, 70.2, 74.4, 100.9, 126.2, 128.3, 129.3, 137.3, 170.2. IR
(7 mL) was added PivCl (97 mL, 0.79 mmol), and the mixture was
(neat): 808, 932, 1030, 1229, 1366, 1450, 1715, 2928 cmꢁ1. MS m/z:
stirred at room temperature for 3 h. The reaction was quenched by
adding saturated aqueous NH4Cl solution (10 mL) and extracted
with AcOEt (100 mL). The organic layer was washed with brine
(5 mL), dried (MgSO4) and concentrated. Purification by silica gel
flash column chromatography (hexane/AcOEt (20:1)) gave the ester
20 (378 mg, 95%) as a colorless syrupy oil.
248 (Mþ). HRMS calcd for [C14H16O4] 248.1048, found 248.1050.
19
[
a]
ꢁ9.3 (c 0.46, CHCl3).
D
4.7. (4R,5R)-5-[(R)-1-(tert-Butyldimethylsilyloxy)-2-
trityloxyethyl]-4-methyldihydrofuran-2-one (18)
1H NMR (400 MHz, CDCl3)
d
ꢁ0.17 (3H, s), ꢁ0.05 (3H, s), 0.79
A mixture of 15 (449 mg, 1.10 mmol), Pd(OH)2 (20 wt % on
charcoal, 154 mg, 0.22 mmol) in EtOH (5 mL) was stirred at room
temperature under a H2 atmosphere. After stirred for 1 h, the
(9H, s), 0.90 (3H, d, J¼6.8 Hz), 1.18 (9H, s), 1.42–1.52 (1H, m), 1.60–
1.64 (1H, m), 2.14 (1H, d, J¼9.2 Hz), 2.11–2.19 (1H, m), 3.03 (1H, dd,
J¼4.8, 9.2 Hz), 3.34 (1H, dd, J¼8.8, 9.2 Hz), 3.46 (1H, apparent t,

7142
S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
J¼8.8 Hz), 3.76 (1H, ddd, J¼4.8, 8.8, 9.2 Hz), 4.07–4.20 (2H, m), 7.21–
7.30 (10H, m), 7.32–7.44 (5H, m). 13C NMR (100 MHz, CDCl3)
brine (5 mL), dried (Na2SO4) and concentrated. Purification by silica
gel flash column chromatography (hexane/AcOEt (5:1)) gave 23
(150 mg, 90%) as a colorless syrupy oil.
d
ꢁ4.9,
ꢁ4.1, 16.3, 18.0, 25.8, 27.3, 31.7, 33.0, 38.7, 63.2, 64.5, 70.8, 75.3, 87.0,
127.0, 127.7, 128.6, 143.9, 178.5. IR (neat): 775, 837, 1076, 1161, 1254,
1285, 1366, 1395, 1449, 1475, 1723, 2932, 2959, 3555 cmꢁ1. FABMS
1H NMR (400 MHz, CDCl3)
d
0.91 (3H, d, J¼6.8 Hz),1.24–1.37 (1H,
m), 1.40–1.44 (1H, m), 1.45–1.56 (1H, m), 1.64–1.69 (1H, m), 2.03–
2.18 (2H, m), 3.14 (1H, dd, J¼4.0, 9.2 Hz), 3.17 (1H, dd, J¼8.0, 9.2 Hz),
3.49 (1H, ddd, J¼5.6, 7.6, 10.4 Hz), 3.60 (1H, ddd, J¼5.6, 7.6, 10.4 Hz),
3.91 (1H, ddd, J¼4.0, 4.0, 8.0 Hz), 4.89 (1H, dd, J¼1.6, 10.0 Hz), 4.93
(1H, dd, J¼1.6, 17.2 Hz), 5.72 (1H, ddt, J¼7.2, 10.0, 17.2 Hz), 7.23–7.33
m/z: 627 (MþNa)þ. HRFABMS calcd for [C37H52O5SiNa] 627.3482,
22
found 627.3471. [
a]
þ0.3 (c 1.02, CHCl3).
D
4.10. (3R,4R,5R)-5-(tert-Butyldimethylsilyloxy)-4-methane-
sulfonyloxy-3-methyl-6-trityloxyhexyl pivalate (21)
(10H, m), 7.42–7.44 (5H, m). 13C NMR (100 MHz, CDCl3)
d 17.1, 18.1,
30.6, 37.2, 44.8, 61.3, 66.7, 71.3, 86.8, 115.4, 127.0, 127.8, 128.6, 138.8,
143.8. IR (neat): 908, 1069, 1248, 1373, 1449, 1493, 2878, 2932,
Under an Ar atmosphere, to a cold (0 ꢀC) solution of 20 (330 mg,
0.55 mmol) in pyridine (5 mL) was added MsCl (105
mL, 1.38 mmol),
3427 cmꢁ1. MS m/z: 430 (Mþ). HRMS calcd for [C29H34O3] 430.2508,
24
and the mixture was stirred at the same temperature for 4 h, and at
room temperature for 50 min. The reaction was quenched by
adding water (10 mL) and the mixture was extracted with AcOEt
(100 mL). The organic layer was washed with brine (5 mL), dried
(MgSO4) and concentrated. Purification by silica gel column chro-
matography (hexane/AcOEt (9:1 to 5:1)) gave the mesylate 21
(372 mg, 99%) as a colorless syrupy oil.
found 430.2488. [
a
]
þ5.4 (c 0.95, CHCl3).
D
4.13. (2S,3R)-3-[(R)-3-(tert-Butyldimethylsilyloxy)-1-
methylpropyl]-1-trityloxyhex-5-en-2-ol (24)
Under an Ar atmosphere, to a cold (0 ꢀC) solution of diol 23
(688 mg, 1.60 mmol) in DMF were added imidazole (327 mg,
4.80 mmol) and TBSCl (289 mg, 1.92 mmol), and stirred at room
temperature for 1 h. The reaction mixture was cooled in an ice bath,
and saturated aqueous NaHCO3 solution (10 mL) was added. The
mixture was extracted with Et2O (150 mL), and the organic layer
was washed with brine (10 mL), dried (MgSO4) and concentrated.
Purification by silica gel column chromatography (hexane/AcOEt
(15:1)) gave the TBS ether 24 (832 mg, 95%) as a colorless syrupy oil.
1H NMR (400 MHz, CDCl3)
d
ꢁ0.04 (3H, s), 0.02 (3H, s), 0.89 (9H,
s), 0.97 (3H, d, J¼6.8 Hz), 1.14 (9H, s), 1.37–1.46 (1H, m), 1.89–2.01
(2H, m), 2.87 (3H, s), 3.23 (1H, dd, J¼6.0, 10.0 Hz), 3.29 (1H, dd,
J¼6.4, 10.0 Hz), 3.96 (1H, ddd, J¼3.2, 6.0, 6.4 Hz), 3.99–4.10 (2H, m),
4.66 (1H, dd, J¼3.2, 6.8 Hz), 7.22–7.30 (10H, m), 7.32–7.45 (5H, m).
13C NMR (100 MHz, CDCl3)
d
ꢁ4.7, ꢁ4.2, 16.6, 18.1, 25.8, 27.2, 30.8,
31.2, 38.5, 38.6, 62.2, 64.4, 71.3, 86.1, 87.4, 127.1, 127.8, 128.7, 143.6,
178.3. IR (neat): 777, 837, 918, 1174, 1342, 1360, 1450, 1479, 1728,
1H NMR (400 MHz, CDCl3)
d
ꢁ0.05 (6H, s), 0.81 (9H, s), 0.84 (3H,
2905, 2959 cmꢁ1. FABMS m/z:706 (MþNa)þ. HRFABMS calcd for
d, J¼6.8 Hz), 1.24–1.28 (1H, m), 1.32–1.37 (1H, m), 1.39–1.47 (1H, m),
1.57–1.64 (1H, m), 1.99 (1H, ddd, J¼7.2, 7.2, 14.4 Hz), 2.08 (1H, ddd,
J¼7.2, 7.2, 14.4 Hz), 2.16 (1H, br s), 3.05 (1H, dd, J¼4.0, 9.2 Hz), 3.12
(1H, dd, J¼8.4, 9.2 Hz), 3.42 (1H, dt, J¼7.2, 10.0 Hz), 3.53 (1H, dt,
J¼7.2, 10.0 Hz), 3.85 (1H, ddd, J¼4.0, 4.0, 8.4 Hz), 4.82 (1H, d,
J¼10.0 Hz), 4.86 (1H, d, J¼17.2 Hz), 5.64 (1H, ddt, J¼7.2, 10.0,
17.2 Hz), 7.16–7.27 (10H, m), 7.37–7.39 (5H, m). 13C NMR (100 MHz,
22
[C38H54O7SiSNa] 705.3258, found 705.3239. [
CHCl3).
a]
þ11.0 (c 0.70,
D
4.11. (R)-3-[(1S,2R)-2-(Trityloxymethyl)oxiranyl]butyl
pivalate (22)
Under an Ar atmosphere, to a mixture of zeolite A-4 (pre-dried in
vacuo at 130 ꢀC for 2 h, 5 mg), TBAF (1 M in THF, 0.1 mL, 0.1 mmol),
CDCl3)
d
ꢁ5.2, ꢁ5.2, 16.8, 18.4, 26.0, 30.6, 31.2, 37.5, 44.8, 61.7, 66.8,
71.0, 86.8, 115.3, 127.0, 127.8, 128.6, 138.9, 143.8. IR (neat): 706, 775,
835, 1091, 1254, 1448, 1491, 1597, 1640, 2930, 3061, 3505 cmꢁ1. MS
inTHF (0.5 mL) was added at 0 ꢀC a solution of 21 (37 mg, 54
mmol) in
THF (0.6 mL) via cannula, and the mixture was stirred at the same
temperature for 30 min, and at room temperature for 2.5 h. The
mixture was diluted with Et2O (10 mL) and filtered through Celite.
The filtrate was diluted with water (5 mL), and the mixture was
extracted with Et2O (50 mL). The organic layer was washed with
brine (5 mL), dried (MgSO4), and concentrated. Purification by silica
gel column chromatography (hexane/AcOEt (15:1 to 10:1)) gave the
epoxide 22 (22 mg, 86%) as a colorless syrupy oil.
m/z: 487 (Mþꢁt-Bu). HRMS calcd for [C31H39O3Si] [(Mꢁt-Bu)þ]
25
487.2668, found 487.2693. [
a
]
þ7.2 (c 1.40, CHCl3).
D
4.14. (4R,5S)-4-[(R)-3-(tert-Butyldimethylsilyloxy)-1-
methylpropyl]-6-trityloxyhexane-1,5-diol (25)
Under an Ar atmosphere, to a cold (0 ꢀC) solution of the terminal
alkene 24 (458 mg, 0.84 mmol) in THF (5 mL) was added BH3$THF
complex (1 M in THF,1.7 mL,1.7 mmol), and the mixture was stirred
at room temperature for 1.25 h. The reaction mixture was cooled in
an ice bath, and to this solution were added successively water
(1 mL),15% aqueous NaOH (1 mL) and 30% aqueous H2O2 (1 mL). The
mixture was stirred at room temperature for 1 h. After cooled in an
ice bath, 10% aqueous Na2S2O3 solution (20 mL) was added, and the
mixture was extracted with AcOEt (60 mL). The organic layer was
washed with brine (10 mL), dried (MgSO4) and concentrated. Puri-
fication by silica gel flash column chromatography (hexane/AcOEt
(5:1)) gave the diol 25 (382 mg, 81%) as a colorless syrupy oil.
1H NMR (400 MHz, CDCl3)
d
1.05 (3H, d, J¼6.4 Hz), 1.10 (9H, s),
1.46–1.54 (1H, m), 1.60–1.70 (2H, m), 2.70 (1H, dd, J¼4.0, 9.6 Hz),
3.12 (1H, dd, J¼4.8, 9.6 Hz), 3.20 (1H, ddd, J¼4.0, 4.8, 10.0 Hz), 3.38
(1H, dd, J¼6.0, 10.0 Hz), 3.39 (2H, t, J¼6.8 Hz), 7.21–7.32 (10H, m),
7.44–7.47 (5H, m). 13C NMR (100 MHz, CDCl3)
d 17.5, 27.2, 29.8, 32.3,
38.6, 56.1, 61.4, 62.0, 62.1, 87.0, 127.0, 127.8, 128.5, 143.6, 178.1. IR
(neat): 756, 767, 1071, 1156, 1283, 1451, 1728, 2964 cmꢁ1. MS m/z:
472 (Mþ), 395 (MþꢁC6H5). HRMS calcd for [C31H36O4] 472.2610,
24
found 472.2626. [
a
]
ꢁ8.0 (c 0.92, CHCl3).
D
4.12. (3R,4R,5S)-4-Allyl-3-methyl-6-trityloxyhexane-
1H NMR (400 MHz, CDCl3)
d 0.00 (6H, s), 0.86 (9H, s), 0.87 (3H, d,
1,5-diol (23)
J¼6.8 Hz), 1.21–1.29 (1H, m), 1.31–1.47 (3H, m), 1.53–1.64 (4H, m),
3.13 (1H, dd, J¼8.4, 9.2 Hz), 3.18 (1H, dd, J¼4.4, 9.2 Hz), 3.45 (1H, dt,
J¼6.8, 10.0 Hz), 3.55 (2H, t, J¼6.0 Hz), 3.57 (1H, dt, J¼6.2, 10.0 Hz),
3.81 (1H, ddd, J¼4.4, 4.4, 8.4 Hz), 7.22–7.32 (10H, m), 7.41–7.44 (5H,
Under an Ar atmosphere, to a cold (0 ꢀC) solution of the epoxide
22 (184 mg, 0.39 mmol) in THF (4 mL) was added dropwise allyl
magnesium chloride (2 M in THF, 1.0 mL, 2.0 mmol), and the mix-
ture was stirred at the same temperature for 15 min, and at room
temperature for 1 h. The reaction was quenched by adding satu-
rated aqueous NH4Cl solution (5 mL), and the mixture was
extracted with Et2O (40 mL). The organic layer was washed with
m).13C NMR (100 MHz, CDCl3)
d
ꢁ5.3, ꢁ5.2,17.0,18.3, 22.5, 26.0, 30.6,
32.3, 36.7, 44.7, 61.6, 62.8, 66.8, 71.5, 86.9,127.1,127.8,128.5,143.8. IR
(neat): 706, 733, 775, 835, 907, 1094, 1254, 1377, 1449, 2858, 2930,
3389 cmꢁ1
[C35H50O4SiNa] 585.3376, found 585.3375. [
.
FABMS m/z: 585 (MþNa)þ. HRFABMS calcd for
23
a
]
þ2.2 (c 0.99, CHCl3).
D

S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
7143
4.15. (4R,5R)-7-(tert-Butyldimethylsilyloxy)-4-[(S)-1-hydroxy-
2-(trityloxy)ethyl]-5-methylheptyl pivalate
in MeOH (4 mL), and cooled in an ice bath. TsOH$H2O (270 mg,
1.35 mmol) was added, and the mixture was stirred at room tem-
perature for 3.5 h. The reaction mixture was cooled in an ice bath,
diluted with AcOEt (20 mL) and saturated aqueous NaHCO3 solution
(10 mL) was added. The mixture was extracted with AcOEt (80 mL),
and the organic layer was washed with brine (10 mL), dried (MgSO4)
and concentrated. Purification by silica gel flash column chroma-
tography (toluene/AcOEt (1:0 to 4:1)) gave the terminal alkene 27
(67 mg, 54% for 3 steps) as a pale yellow syrupy oil.
Under an Ar atmosphere, to a cold (0 ꢀC) solution of diol 25
(653 mg, 1.16 mmol) in pyridine (10 mL) was added PivCl (0.16 mL,
1.39 mmol), and stirred at the same temperature for 1.25 h, and
then at room temperature for 1.5 h. The reaction was quenched by
adding water (20 mL), and the mixture was extracted with AcOEt
(150 mL). The organic layer was washed with brine (10 mL), dried
(MgSO4), and concentrated. Purification by silica gel column chro-
matography (hexane/AcOEt (15:1)) gave the ester (734 mg, 98%) as
a colorless syrupy oil.
1H NMR (400 MHz, CDCl3)
d
1.05 (3H, d, J¼6.8 Hz), 1.93 (9H, s),
1.34–1.40 (1H, m), 1.42–1.53 (2H, m), 1.63–1.74 (2H, m), 1.90 (2H, br
s), 2.34–2.42 (1H, m), 3.56 (1H, dd, J¼8.0, 10.8 Hz), 3.60 (1H, dd,
J¼3.6, 10.8 Hz), 3.79 (1H, ddd, J¼3.6, 3.6, 8.0 Hz), 4.04 (2H, t,
J¼6.4 Hz), 5.02 (1H, ddd, J¼0.8, 1.6, 10.4 Hz), 5.04 (1H, ddd, J¼1.6,
2.4, 18.0 Hz), 5.77 (1H, ddd, J¼7.6, 10.4, 18.0 Hz). 13C NMR (100 MHz,
1H NMR (400 MHz, CDCl3)
d 0.00 (6H, s), 0.86 (9H, s), 0.88 (3H, d,
J¼7.2 Hz),1.15 (9H, s),1.29–1.63 (8H, m), 2.25 (1H, br s), 3.09 (1H, dd,
J¼4.0, 9.2 Hz), 3.15 (1H, dd, J¼8.0, 9.2 Hz), 3.47 (1H, dt, J¼6.8,
10.0 Hz), 3.57 (1H, dt, J¼6.4, 10.0 Hz), 3.84 (1H, ddd, J¼4.0, 4.0,
8.0 Hz), 3.92 (1H, dt, J¼6.4, 10.8 Hz), 3.96 (1H, dt, J¼6.4, 10.8 Hz),
7.22–7.31 (10H, m), 7.41–7.43 (5H, m). 13C NMR (100 MHz, CDCl3)
CDCl3) d 17.4, 22.8, 27.3, 28.5, 38.8, 39.2, 45.0, 64.5, 65.6, 73.6, 114.6,
142.3, 178.5. IR (neat): 912, 1034, 1055, 1287, 1368, 1399, 1462, 1640,
1728, 2934, 3428 cmꢁ1. MS m/z: 272 (Mþ). HRMS calcd for
24
d
ꢁ5.2, ꢁ5.2, 16.8, 18.3, 22.7, 26.0, 27.3, 28.2, 30.5, 37.2, 38.7, 44.6,
[C15H28O4] 272.1948, found 272.2003. [
a
]
þ13.1 (c 1.28, CHCl3).
D
61.6, 64.6, 66.9, 71.1, 86.8, 127.1, 127.8, 128.5, 143.8, 178.4. IR (neat):
704, 774, 835, 1093, 1161, 1254, 1287, 1390, 1450, 1460, 1728, 2932,
4.18. (4R,5R)-4-[(S)-1-Hydroxy-2-(p-toluenesulfonyloxy)-
ethyl]-5-methylhept-6-enyl pivalate (28)
2955, 3501 cmꢁ1. FABMS m/z: 669 (MþNa)þ. HRFABMS calcd for
23
[C40H58O5SiNa] 669.3951, found 669.3940. [
a]
ꢁ0.4 (c 0.89, CHCl3).
D
Under an Ar atmosphere, to a cold (0 ꢀC) solution of 27 (67 mg,
0.24 mmol) in CH2Cl2 (1 mL) were added n-Bu2SnO (1 mg,
4.16. (4R,5R)-7-Hydroxy-4-[(S)-1-hydroxy-2-trityloxyethyl]-5-
methylheptyl pivalate (26)
4.8 mmol), Et3N (33 mL, 0.24 mmol) and TsCl (46 mg, 0.24 mmol),
and stirred at room temperature for 2 h. TsCl (23 mg, 0.12 mmol)
was added further, and the mixture was stirred at the same tem-
perature for 2.3 h. The mixture was filtered through Celite, and the
filtrate was concentrated. Purification by silica gel column chro-
matography (hexane/AcOEt (6:1)) gave the tosylate 28 (87 mg, 85%)
as a pale yellow syrupy oil.
Under an Ar atmosphere, to a cold (0 ꢀC) solution of the above
pivalate (734 mg, 1.13 mmol) in THF (10 mL) was added TBAF (1 M
in THF, 2.3 mL, 2.3 mmol), and stirred at room temperature for
1.5 h. The reaction mixture was partitioned between water (20 mL)
and AcOEt (150 mL), and the organic layer was washed with brine
(10 mL), dried (MgSO4) and concentrated. Purification by silica gel
column chromatography (hexane/AcOEt (4:1)) gave the diol 26
(583 mg, 97%) as a colorless oil.
1H NMR (400 MHz, CDCl3)
d
1.01 (3H, d, J¼6.8 Hz), 1.18 (9H, s),
1.34–1.45 (3H, m), 1.57–1.68 (2H, m), 1.91 (1H, br s), 2.28–2.35 (1H,
m), 2.46 (3H, s), 3.92 (1H, ddd, J¼3.8, 3.8, 9.6 Hz), 3.96 (1H, dd,
J¼6.4, 10.4 Hz), 3.97–4.00 (2H, m), 4.03 (1H, dd, J¼3.8, 10.4 Hz), 4.98
(1H, ddd, J¼1.2,1.2, 17.6 Hz), 4.99 (1H, ddd, J¼1.2, 1.2,10.0 Hz), 5.64–
5.73 (1H, m), 7.36 (2H, d, J¼8.0 Hz), 7.80 (2H, d, J¼8.0 Hz). 13C NMR
1H NMR (400 MHz, CDCl3)
d
0.88 (3H, d, J¼6.8 Hz), 1.16 (9H, s),
1.16–1.65 (8H, m), 3.14 (2H, d, J¼6.0 Hz), 3.49 (1H, dt, J¼7.2,10.4 Hz),
3.62 (1H, dt, J¼6.0,10.4 Hz), 3.85 (1H, dt, J¼4.0, 6.0 Hz), 3.93 (1H, dt,
J¼6.8,10.8 Hz), 3.96 (1H, dt, J¼6.8,10.8 Hz), 7.22–7.32 (10H, m), 7.42–
(100 MHz, CDCl3)
d 17.3, 21.7, 22.7, 27.2, 28.2, 38.7, 38.8, 44.6, 64.3,
7.44 (5H, m). 13C NMR (100 MHz, CDCl3)
d
17.0, 22.7, 27.3, 27.3, 28.2,
70.6, 73.1, 114.8,127.9, 129.9, 132.6, 141.8, 145.0, 178.4. IR (neat): 814,
30.5, 36.9, 44.5, 61.3, 64.5, 66.8, 71.4, 86.9, 127.1, 127.8, 128.5, 143.7,
178.5. IR (neat): 704, 1076, 1154, 1254, 1289, 1480, 1724, 2878, 2955,
970, 1178, 1289, 1362, 1480, 1725, 2973, 3507 cmꢁ1. FABMS m/z: 449
(MþNa)þ. HRFABMS calcd for [C22H34O6SNa] 449.1962, found
26
3456 cmꢁ1
[C34H44O5Na] 555.3086, found 555.3083. [
.
FABMS m/z: 555 (MþNa)þ. HRFABMS calcd for
449.1983. [
a
]
þ15.9 (c 0.96, CHCl3).
D
24
a
]
ꢁ0.9 (c 0.99, CHCl3).
D
4.19. (4R,5R)-5-Methyl-4-[(S)-oxiranyl]-hept-6-enyl
4.17. (4R,5R)-4-[(S)-1,2-Dihydroxyethyl]-5-methylhept-6-enyl
pivalate (29)
pivalate (27)
Under an Ar atmosphere, to a cooled (ꢁ78 ꢀC) solution of the
tosylate 28 (85 mg, 0.20 mmol) in THF (2 mL) was added LiHMDS
(1 M in THF, 0.24 mL, 0.24 mmol), and stirred at the same tem-
perature for 40 min, and then at room temperature for 20 min. The
reaction was cooled in an ice bath, and saturated aqueous NH4Cl
solution (5 mL) was added. The mixture was extracted with Et2O
(40 mL), and the organic layer was washed with brine (5 mL), dried
(MgSO4) and concentrated. Purification by silica gel column chro-
matography (hexane/AcOEt (5:1)) gave the epoxide 29 (46 mg, 91%)
as a colorless syrupy oil.
Under an Ar atmosphere, to a cold (0 ꢀC) solution of 27 (242 mg,
0.45 mmol) and 2-nitrophenyl selenocyanate (204 mg, 0.90 mmol)
in THF (4 mL) was added n-Bu3P (0.22 mL, 0.90 mmol), and the
mixture was stirred at the same temperature for 35 min, and then at
room temperature for 35 min. 2-Nitrophenyl selenocyanate (51 mg,
0.23 mmol) and n-Bu3P (0.05 mL, 0.23 mmol) were added further at
0 ꢀC. Reaction temperature was raised gradually up to room tem-
perature, and the mixture was stirred overnight. The reaction was
quenched by adding water (10 mL), and the mixture was extracted
with Et2O (80 mL). The organic layer was washed with brine (10 mL),
dried (MgSO4) and concentrated. The residue was dissolved in THF
(4 mL), and the mixturewas cooled in an ice bath. Tothe mixturewas
added 30% aqueous H2O2 (2.5 mL), and the mixture was stirred at
the same temperature for 10 min, and then at room temperature for
2.3 h. The mixture was cooled in an ice bath, and 10% aqueous
Na2S2O3 solution (20 mL) was added. The mixture was extracted
with Et2O (80 mL), and the organic layer was washed with brine
(10 mL), dried (MgSO4) and concentrated. The residue was dissolved
1H NMR (400 MHz, CDCl3)
d
1.05 (3H, d, J¼7.2 Hz), 1.20 (9H, s),
1.45–1.54 (1H, m), 1.58–1.64 (1H, m), 1.63–1.71 (1H, m), 1.68–1.76
(1H, m),1.78–1.89 (1H, m), 2.30 (1H, m), 2.49 (1H, dd, J¼2.8, 4.8 Hz),
2.72–2.78 (2H, m), 4.06 (2H, t, J¼6.4 Hz), 5.00 (1H, ddd, J¼0.8, 1.2,
10.4 Hz), 5.01 (1H, ddd, J¼1.2, 2.0,17.2 Hz), 5.73 (1H, ddd, J¼8.0,10.4,
17.2 Hz).13C NMR (100 MHz, CDCl3)
d 17.2, 26.3, 26.8, 27.2, 38.6, 40.0,
46.1, 46.9, 54.8, 64.4, 114.2, 142.0, 178.4. IR (neat): 916, 1159, 1285,
1460, 1482, 1730, 2971 cmꢁ1. MS m/z: 254 (Mþ). HRMS calcd for
25
[C15H26O3] 254.1883, found 254.1881. [
a
]
þ10.4 (c 1.25, CHCl3).
D

7144
S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
4.20. (4R,5R)-4-[(S)-1-Methylprop-2-enyl]-oct-7-yne-
1,5-diol (30)
(10:1)). The residue was dissolved inTHF (2 mL), and cooled in an ice
bath. TBAF (1 M in THF, 0.3 mL, 0.3 mmol) was added, and the
mixture was stirred at room temperature for 5 h. The mixture was
diluted with Et2O (10 mL) and water (5 mL), and extracted with Et2O
(30 mL). The organic layer was washed with brine (5 mL), dried
(MgSO4) and concentrated. Purification by preparative TLC (hexane/
AcOEt (1:1)) gave the target 4 (6 mg, 29% for 2 steps) as a white solid.
This compound was further purified by preparative reverse phase
Under an Ar atmosphere, to a cooled (ꢁ78 ꢀC) solution of
ethynyltrimethylsilane (0.13 mL, 0.90 mmol) in THF (2 mL) was
added n-BuLi (1.45 M in hexane, 1.86 mL, 2.70 mmol), and the
mixture was stirred at the same temperature for 10 min. To this was
added the epoxide 29 (7 mg, 0.30 mmol) in THF (3 mL) via cannula,
and BF3$OEt2 (0.11 mL, 0.90 mmol) was added. The mixture was
stirred at the same temperature for 10 min, and then at room
temperature for 3.3 h. The reaction was quenched by adding water
(5 mL), and the mixture was extracted with Et2O (50 mL). The or-
ganic layer was washed with brine (5 mL), dried (MgSO4) and
concentrated. The residue was dissolved in MeOH (5 mL), and to
this was added NaOMe (49 mg, 0.90 mmol). The mixture was stir-
red at 40 ꢀC overnight, and cooled in an ice bath. Saturated aqueous
NH4Cl solution (5 mL) was added, and the mixture was extracted
with AcOEt (50 mL). The organic layer was washed with brine
(5 mL), dried (MgSO4), and concentrated. Purification by silica gel
flash column chromatography (hexane/AcOEt (3:1)) gave the diol
30 (54 mg, 92%) as a colorless syrupy oil.
HPLC. HPLC (YMC-Pack ODS column, 20ꢂ150 mm, 10 mL minꢁ1
,
acetonitrile/water¼95:5). UV (EtOH) lmax 264 nm, lmin 228 nm. 1H
NMR (400 MHz, CDCl3)
d
0.53 (3H, s), 0.94 (3H, d, J¼6.0 Hz), 0.94 (3H,
d, J¼7.2 Hz),1.05–1.06 (1H, m),1.22 (6H, s),1.25–1.70 (19H, m),1.84–
1.91 (1H, m),1.69–1.97 (1H, m),1.99–2.01 (1H, m), 2.22 (1H, dd, J¼7.8,
12.6 Hz), 2.61–2.64 (1H, m), 2.64 (1H, dd, J¼3.6, 7.8 Hz), 2.81 (1H, dd,
J¼4.8,12.6 Hz), 3.67 (2H, t, J¼6.0 Hz), 3.77 (1H, dt, J¼4.2, 7.8 Hz), 4.80
(1H, d, J¼2.4 Hz), 5.00 (1H, d, J¼1.8 Hz), 5.98 (1H, d, J¼10.8 Hz), 6.27
(1H, d, J¼10.8 Hz). 13C NMR (100 MHz, CDCl3)
d 11.9, 14.3, 18.9, 20.9,
22.3, 23.6, 23.8, 27.7, 29.1, 29.3, 29.4, 30.6, 36.1, 36.4, 38.9, 40.6, 44.4,
44.6, 45.9, 47.9, 56.3, 56.5, 63.1, 70.7, 71.1, 110.9, 117.4, 122.8, 135.0
141.9, 149.4. IR (film): 605, 731, 909, 1038, 1377, 1455, 1472, 1632,
2934, 3401 cmꢁ1. MS m/z: 472 (Mþ). HRMS calcd for [C31H52O3]
23
1H NMR (400 MHz, CDCl3)
d
1.06 (3H, d, J¼6.8 Hz),1.40–1.73 (5H,
472.3907, found 472.3925. [
a]
þ44.1 (c 0.22, CHCl3).
D
m), 2.05 (1H, t, J¼2.4 Hz), 2.35–2.38 (1H, m), 2.42 (2H, dd, J¼2.4,
6.0 Hz), 3.64 (1H, ddd, J¼6.8, 10.4, 16.8 Hz), 3.65 (1H, ddd, J¼6.8,
10.4, 16.8 Hz), 3.80 (1H, dt, J¼6.0, 11.2 Hz), 5.00 (1H, ddd, J¼1.6, 1.6,
10.4 Hz), 5.03 (1H, ddd, J¼1.6,1.6,17.6 Hz), 5.77 (1H, ddd, J¼8.0,10.4,
4.23. Reporter assays using luciferase as a reporter
Human breast cancer cell line MCF7 cells were grown at 37 ꢀC in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S)
in an atmosphere of 95% air and 5% CO2. Cells were collected, sus-
pended in the DMEM supplemented with 5% FBS (stripped with
dextran-coated charcoal) and 1% P/S without phenol red, and
plated in 24-well plate (2.5ꢂ104 cells/well). Cells were incubated in
CO2 incubator at 37 ꢀC overnight. Ligand stock solutions were
prepared at various concentrations in DMSO (10ꢁ7–10ꢁ3 M). DMSO
itself was used as vesicle. Plasmids used in our assays were as fol-
lows; receptor plasmids pM(GAL4-hVDR(DEF)) for wild type hVDR,
and pM(GAL4-hVDR(R274L)(DEF)) for mutant hVDR, the latter
prepared by site-directed mutagenesis using QuikChange II XL Site-
Directed Mutagenesis Kits (Stratagene), reporter plasmid (17M2-G-
Luc) and internal standard plasmid (pRL-CMV). Plasmids were
diluted in OPTI-MEM medium at concentrations of 50 ng/well for
17.6 Hz). 13C NMR (100 MHz, CDCl3)
d 17.8, 22.1, 25.8, 32.4, 39.3,
46.6, 62.7, 70.8, 71.6, 81.2, 114.6, 141.9. IR (neat): 916, 1036, 1059,
1418, 1451, 1640, 2932, 3308 cmꢁ1. MS m/z: 169 (Mþ). HRMS calcd
25
for [C12H20O2] 196.1447, found 196.1467. [
a
]
þ15.0 (c 1.27, CHCl3).
D
4.21. (3R,4R,5R)-5-(tert-Butyldimethylsilyloxy)-4-[3-(tert-
butyldimethylsilyloxy)propyl]-3-methyloct-1-en-7-yne (31)
Under an Ar atmosphere, to a cold (ꢁ78 ꢀC) solution of diol 30
(6 mg, 33
264
mmol) in CH2Cl2 (0.5 mL) were added 2,6-lutidine (31 mL,
mol) and TBSOTf (30 mL, 132 mmol), and stirred at the same
m
temperature for 50 min, and then at 0 ꢀC for 45 min. The reaction
was quenched by adding saturated aqueous NaHCO3 solution
(5 mL), and the mixture was extracted with CH2Cl2 (30 mL). The
organic layer was washed with brine (5 mL), dried (MgSO4) and
concentrated. Purification by silica gel flash column chromatogra-
phy (hexane/AcOEt (150:1)) gave the bisTBS ether 31 (9 mg, 62%) as
a colorless syrupy oil.
receptor plasmid, 0.2 mg/well for reporter plasmid, and 2.5 ng/well
for internal plasmid. Transfections were carried out by using
TransFast reagent (Promega) according to the manufacture’s in-
struction. After 3–6 h of transfection, ligand stock solutions were
added at the final concentrations of 10ꢁ10–10ꢁ6 M, and cells were
further incubated overnight. Luciferase assays were performed by
using Dual-Luciferase Reporter Assay System Kit (Promega). All
experiments were carried out at least 3 times.
1H NMR (400 MHz, CDCl3)
d 0.04 (6H, s), 0.07 (3H, s), 0.08 (3H,
s), 0.89 (9H, s), 0.89 (9H, s), 1.02 (3H, d, J¼6.8 Hz), 1.34–1.60 (5H, m),
1.96 (1H, t, J¼2.4 Hz), 2.30–2.35 (1H, m), 2.37 (2H, dd, J¼2.4, 6.4 Hz),
3.56 (2H, t, J¼6.8 Hz), 3.95 (1H, dt, J¼3.2, 6.4 Hz), 4.95 (1H, ddd,
J¼1.2, 1.2, 10.0 Hz), 4.99 (1H, ddd, J¼1.2, 1.2, 17.2 Hz), 5.74 (1H, ddd,
J¼8.0, 10.0, 17.2 Hz). 13C NMR (100 MHz, CDCl3)
d
ꢁ5.2, ꢁ4.5, ꢁ3.9,
18.1, 18.4, 18.8, 23.1, 25.4, 25.9, 26.0, 32.4, 38.8, 47.0, 63.6, 69.9, 72.0,
82.1, 113.6, 143.3. IR (neat): 775, 835, 916, 1005, 1096, 1256, 1362,
4.24. Molecular modeling
1389, 1462, 2240, 2363, 2957 cmꢁ1. MS m/z: 424 (Mþ). HRMS calcd
for [C24H48O2Si2] 424.3181, found 424.3201. [
CHCl3).
The mutant binding-site model was generated by changing the
side chain of Arg274 to that of Leu from wild type VDR(1DB1), and
the docking model of ligands bound to mutant VDR(Arg274Leu)
was constructed by conformational search with MacroModel (ver.
8.1). Conformational search was performed by fixing receptor
24
a
]
ꢁ2.9 (c 0.91,
D
4.22. 2a-(3-Hydroxypropyl)-25-hydroxy-1a-
methylvitamin D3 (4)
protein without ligands and Leu274 residue. AMBER
*
was used as
force field.
Under an Ar atmosphere, a mixture of A-ring enyne 31 (26 mg,
mol), CD-ring bromoolefin 518 (33 mg, 91
mol), Pd(PPh3)4
(35 mg, 31 mol) in Et3N (2 mL) and toluene (2 mL) was stirred at
61
m
m
Acknowledgements
m
room temperature for 10 min, and then at 80 ꢀC for 1 h. After cooled
to room temperature, the mixture was diluted with Et2O (10 mL),
and filtered through Celite. The filtrate was concentrated, and par-
tially purified by silica gel column chromatography (hexane/AcOEt
We are grateful to Ms. Junko Shimode and Ms. Akiko Tonoki
(Teikyo University) for the spectroscopic measurements. This work
has been supported in part by Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan

S. Honzawa et al. / Tetrahedron 65 (2009) 7135–7145
7145
14. (a) Saito, N.; Suhara, Y.; Kurihara, M.; Fujishima, T.; Honzawa, S.; Takayanagi, H.;
Kozono, T.; Matsumoto, M.; Ohmori, M.; Miyata, N.; Takayama, H.; Kittaka, A. J.
Org. Chem. 2004, 64, 7463; (b) Suhara, Y.; Nihei, K.; Kurihara, M.; Kittaka, A.;
Yamaguchi, K.; Fujishima, T.; Konno, K.; Miyata, N.; Takayama, H. J. Org. Chem.
2001, 66, 8760; (c) Suhara, Y.; Nihei, K.; Tanigawa, H.; Fujishima, T.; Konno, K.;
Nakagawa, K.; Okano, T.; Takayama, H. Bioorg. Med. Chem. Lett. 2000, 10, 1129;
(d) Posner, G. H.; Johnson, N. J. Org. Chem. 1994, 59, 7855; (e) Takahashi, E.;
Nakagawa, K.; Suhara, Y.; Kittaka, A.; Nihei, K.; Konno, K.; Takayama, H.; Ozono,
K.; Okano, T. Biol. Pharm. Bull. 2006, 29, 2246.
15. For VDR antagonists, see: (a) Saito, N.; Kittaka, A. ChemBioChem 2006, 7, 1478;
(b) Saito, N.; Matsunaga, T.; Saito, H.; Anzai, M.; Takenouchi, K.; Miura, D.;
Namekawa, J.; Ishizuka, S.; Kittaka, A. J. Med. Chem. 2006, 49, 7063; (c) Saito, N.;
Masuda, M.; Saito, H.; Takenouchi, K.; Ishizuka, S.; Namekawa, J.; Takimoto-
Kamimura, M.; Kittaka, A. Synthesis 2005, 2533; (d) Saito, N.; Masuda, M.;
Matsunaga, T.; Saito, H.; Anzai, M.; Takenouchi, K.; Miura, D.; Ishizuka, S.;
Takimoto-Kamimura, M.; Kittaka, A. Tetrahedron 2004, 60, 7951; (e) 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; (f) Saito,
N.; Matsunaga, T.; Saito, H.; Anzai, M.; Takenouchi, K.; Miura, D.; Ishizuka, S.;
Takayama, H.; Kittaka, A. Heterocycles 2006, 67, 311.
(#17790095 to S.H.), and by Grant-in-Aid from Japan Society for the
Promotion of Science (#17590012 to A.K.). A.K. gratefully ac-
knowledges Uehara Memorial Foundation.
References and notes
1. (a) Patrick, G. L. An Introduction to Medicinal Chemistry, 2nd ed.; Oxford Uni-
versity Press: Oxford, 2001; pp 20–36; (b) Jeffrey, G. A. An Introduction to Hy-
drogen Bonding; Oxford University Press: Oxford, 1997; (c) Hydrogen
BondingdA Theoretical Perspective; Scheiner, S., Ed.; Oxford University Press:
Oxford, 1997.
2. (a) See, Ref. 1a; (b) Otto, S.; Engberts, J. B. F. N. Org. Bioorg. Chem. 2003, 1, 1809;
(c) Southall, N. T.; Dill, K. A.; Haymet, A. D. J. J. Phys. Chem. B 2002, 106, 521; (d)
Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Mem-
brane, 2nd ed.; Wiley: New York, NY, 1980.
3. (a) Vitamin D, 2nd ed.; Feldman, D., Pike, J. W., Glorieux, F. H., Eds.; Elsevier
Academic: New York, NY, 2005; New vitamin D analogues are described in
chapters 80–88; (b) Bouillon, R. W.; Okamura, H.; Norman, A. W. Endocr. Rev.
1995, 16, 200; (c) Zhu, G. D.; Okamura, W. H. Chem. Rev. 1995, 95, 1877; (d)
DeLuca, H. F. Nutr. Rev. 2008, 66, S73; (e) Ettinger, R. A.; DeLuca, H. F. Adv. Drug
Res. 1996, 28, 269; (f) Brown, A. J.; Slatopolsky, E. Mol. Aspects Med. 2008, 29,
433; (g) Laverny, G.; Penna, G.; Uskokovic, M.; Marczak, S.; Maehr, H.; Jan-
kowski, P.; Ceailles, C.; Vouros, P.; Smith, B.; Robinson, M.; Reddy, G. S.; Adorini,
L. J. Med. Chem. 2009, 52, 2204.
4. (a) Evans, R. M. Science 1988, 240, 889; (b) Chambon, P. Mol. Endocrinol. 2005,19,1418.
5. Rochel, N.; Wurtz, J. M.; Mitschler, A.; Klahoz, B.; Moras, D. Mol. Cell 2000, 5, 173.
6. Nakabayashi, M.; Yamada, S.; Yoshimoto, N.; Tanaka, T.; Igarashi, M.; Ikura, T.;
Ito, N.; Makishima, M.; Tokiwa, H.; DeLuca, H. F.; Shimizu, M. J. Med. Chem.
2008, 51, 5320.
16. For 19-norvitamin
D analogues, 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.
17. (a) Kittaka, A.; Kurihara, M.; Peleg, S.; Suhara, Y.; Takayama, H. Chem. Pharm.
Bull. 2003, 51, 357; (b) Analogues which possess 2
a
-aromatic ring substituent
-benzyl analogue was
were tested for the mutant VDR (Arg274Leu), and 2
a
shown to have similar activity as the natural hormone, 1. Honzawa, S.; Hirasaka,
K.; Yamamoto, Y.; Peleg, S.; Fujishima, T.; Kurihara, M.; Saito, N.; Kishimoto, S.;
Sugiura, T.; Waku, K.; Takayama, H.; Kittaka, A. Tetrahedron 2005, 61, 11253.
18. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc. 1992, 114, 9836.
19. A part of this work has been published as proceedings of 13th Workshop on
Vitamin D (Victoria, Canada, April 2006): Kittaka, A.; Saito, N.; Honzawa, S.;
Takenouchi, K.; Ishizuka, S.; Chen, T. C.; Peleg, S.; Kato, S.; Arai, M. A. J. Steroid
Biochem. Mol. Biol. 2007, 103, 269.
7. (a) Yamamoto, K.; Abe, D.; Yoshimoto, N.; Choi, M.; Yamagishi, K.; Tokiwa, H.;
Shimizu, M.; Makishima, M.; Yamada, S. J. Med. Chem. 2006, 49, 1313; (b) Choi,
M.; Yamamoto, K.; Itoh, T.; Makishima, M.; Mangelsdorf, D. J.; Moras, D.;
DeLuca, H. F.; Yamada, S. Chem. Biol. 2003, 10, 261.
20. (a) Gros, E. G.; Deulofeu, V. J. Org. Chem. 1964, 29, 3647; (b) Zimmermann, P.;
Schmidt, R. R. Liebigs Ann. Chem. 1998, 663.
8. Malloy, P. J.; Pike, J. W.; Feldman, D. Endocr. Rev. 1999, 20, 156.
9. Kristjansson, K.; Rut, A. R.; Hewison, M.; O’Riordan, J. L.; Hughes, M. R. J. Clin.
Invest. 1993, 92, 12.
10. (a) Swann, S. L.; Bergh, J. J.; Farach-Carson, M. C.; Koh, J. T. Org. Lett. 2002, 4,
3863; (b) Non-seco steroid type analogues were reported by the same research
group. Swann, S. L.; Bergh, J. J.; Farach-Carson, M. C.; Ocasio, C. A.; Koh, J. T.
J. Am. Chem. Soc. 2002, 124, 13795.
11. Honzawa, S.; Yamamoto, Y.; Yamashita, A.; Sugiura, T.; Kurihara, M.; Arai, M. A.;
Kato, S.; Kittaka, A. Bioorg. Med. Chem. 2008, 16, 3002.
12. (a) Gardezi, S. A.; Nguyen, C.; Malloy, P. J.; Posner, G. H.; Feldman, D.; Peleg, S. J.
Biol. Chem. 2001, 276, 29148; (b) At first, Posner et al. reported that the ana-
21. (a) Breit, B.; Schmidt, Y. Chem. Rev. 2008, 108, 2928; (b) Modern Organocopper
Chemistry; Krause, N., Ed.; Wiley-VCH: Weinheim, 2002; p 188.
22. Taber, D. F.; Green, J. H.; Geremia, J. M. J. Org. Chem. 1997, 62, 9342.
23. Grieco, P. A.; Gilman, S.; Nishizawa, M. J. Org. Chem. 1976, 41, 1485.
24. Martinelli, M. J.; Nayyar, N. K.; Moher, E. D.; Dhokte, U. P.; Pawlak, J. M.;
Vaidyanathan, R. Org. Lett. 1999, 1, 447.
25. (a) Yamaguchi, M.; Hirao, I. Tetrahedron Lett. 1983, 24, 391; (b) Yamaguchi, M.;
Hirao, I. J. Chem. Soc., Chem. Commun. 1984, 202.
26. Hourai, S.; Fujishima, T.; Kittaka, A.; Suhara, Y.; Takayama, H.; Rochel, N.; Moras,
D. J. Med. Chem. 2006, 49, 5199.
logue has 1
b
,3
a
stereochemistry, but recently, they revised this stereochem-
27. 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.
istry was 1
a
,3
b
Posner, G. H.; Jeon, H. B.; Sarjeant, A.; Riccio, E. S.; Doppalapudi,
R. S.; Kapetanovic, I. M.; Saha, U.; Dolan, P.; Kensler, T. W. Steroids 2004, 69, 757.
13. Forareviewsee:Saito,N.;Honzawa,S.;Kittaka, A.Curr. Top. Med. Chem.2006, 6,1273.