Synthesis of 2,2-dimethyl-1,25-dihydroxyvitamin D3: A-ring structural motif that modulates interactions of vitamin D receptor with transcriptional coactivators

Article abstract of DOI:10.1039/b302017g

A concise synthesis of all four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D₃ and characterization of their distinct transcriptional features, which appear to have been inherited from the corresponding 2α-methyl derivatives, is reported.

Full text of DOI:10.1039/b302017g

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Synthesis of 2,2-dimethyl-1,25-dihydroxyvitamin D3:
A-ring structural motif that modulates interactions of vitamin D
receptor with transcriptional coactivators
Toshie Fujishima,a Atsushi Kittaka,a Kazuyoshi Yamaoka,b Ken-ichi Takeyama,b Shigeaki Katob
and Hiroaki Takayama*a
a Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan
b Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113-0032, Japan
Received 24th February 2003, Accepted 2nd April 2003
First published as an Advance Article on the web 17th April 2003
A concise synthesis of all four possible A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D3 and
characterization of their distinct transcriptional features, which appear to have been inherited from the
corresponding 2α-methyl derivatives, is reported.
additional space.5h,6c Our study of all eight possible A-ring
Introduction
stereoisomers of 2-methyl-1,25-dihydroxyvitamin D3 revealed
that 2α-methyl-1α,25-dihydroxyvitamin D3 (2) was a fourfold
better binder to VDR, whereas its 2-epimer, 2β-methyl-1α,25-
dihydroxyvitamin D3 (3) showed one-eighth of the affinity of
1.5a–c In view of these important results, we have now synthesised
all four possible A-ring stereoisomers of 2,2-dimethyl-1,25-di-
hydroxyvitamin D3 (4a–d) as A-ring analogues having methyl
substituents projecting in both directions in the cavity, to investi-
gate how the second methyl group affects the activity profiles of
the parent compounds.
Cholecalciferol, known as vitamin D3, is metabolized to afford
the hormonally active form, 1α,25-dihydroxyvitamin D3 (1),
through 25-hydroxyvitamin D3. In addition to its classical role
in calcium and phosphorus homeostasis, 1α,25-dihydroxy-
vitamin D3 dominates the cell cycle in many malignant cells,
regulating proliferation, differentiation and apoptosis.1 The
broad spectrum of biological activities of 1 is considered to be
mediated by a ligand-inducible transcriptional factor, vitamin
D receptor (VDR), which belongs to the nuclear receptor
superfamily.2 The specific interaction of ligands with the ligand-
binding domain (LBD) of VDR has been a major focus of
attention in recent years, since the X-ray crystal structure of
deletion mutant VDR complexed with the natural ligand 1 was
solved in 2000.3 Insight into the structure–function relation-
ships of a variety of ligands is essential to understand how the
subtype-free, singular VDR can deliver the diverse biological
activities of 1, as well as to allow the development of poten-
tial therapeutic agents with selective activity profiles for the
treatment of cancer or osteoporosis.
Results and discussion
Synthesis was carried out by using a convergent method7
pioneered by Trost et al.8 The synthetic route to the 2,2-di-
substituted A-ring precursors (12a,b) is shown in Scheme 1.
Protection of the primary alcohol in methyl 3-hydroxy-2,2-di-
methylpropionate (5) with a p-anisyl group gave 6 in excellent
yield.9 Reduction of the methyl ester 6 using LiAlH4, followed
by PDC oxidation of the resultant alcohol 7 furnished the
Modification of 1 in the A-ring, which possesses two critical
hydroxy groups at C1 and C3, has become of interest in recent
years, because the other three A-ring stereoisomers of 1 have
proven to exhibit unique activity profiles, being different from
the natural hormone 1.4 In addition, some of our synthesised
2-substituted analogues exhibited remarkably high affinity for
VDR.5,6 The X-ray crystal structure of VDR complexed with 1
indicated, in addition to the cavity in which the flexible C17
side-chain moiety of 1 is accommodated, the presence of an
extra space in the vicinity of the A-ring.3a It was suggested that
substituents of synthetic A-ring analogues could occupy this
Scheme 1 Reagents and conditions: (a) 4-methoxyphenol, DEAD,
Ph3P–THF, 98%; (b) LiAlH4–THF; 97%; (c) PDC, 4 Å MS–CH2Cl2,
89%; (d) allenylmagnesium bromide–ether, 68%; (e) TBSOTf, 2,6-
lutidine–CH2Cl2, 81% for 9, quant. for 12a,b; (f ) CAN–CH3CN, H2O,
77%; (g) TPAP, NMO, 4 Å MS–CH2Cl2, 69%; (h) vinylmagnesium
bromide–toluene, 60%.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
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Table 1 Vitamin D receptor binding affinity of 2,2-dimethyl-1,25-
dihydroxyvitamin D3 (4a–d)a
corresponding aldehyde, which was reacted with allenyl-
magnesium bromide10 to give an enantiomeric mixture of the
propargyl alcohol ( )-8. Protection of the secondary alcohol of
( )-8 with a tert-butyldimethylsilyl (TBS) group afforded ( )-9
in 81% yield, and deprotection of the p-anisyl group with
cerium() diammonium nitrate (CAN) furnished the primary
alcohol ( )-10 in a satisfactory yield. Introduction of a vinyl
group into the aldehyde, obtained from ( )-10 with tetra-
propylammonium perruthenate (TPAP) in 69% yield, pro-
ceeded smoothly to give the allylic alcohols ( )-11a,b in 60%
yield as an approximately 1 : 2 mixture of the diastereomers.
These isomers were readily separable by silica gel column
chromatography and the relative stereochemistry of the 1,3-diol
in each enantiomeric mixture was determined by 13C NMR
analysis of the acetonides (Scheme 2).11 The data for the
dimethyl groups, in addition to the acetal methyl groups,
supported the conformational preference.12
Compounds
VDRb affinity
1
100
2
400c
13c
3
4a
4b
4c
4d
3
0.005
<0.001
0.06
a The potency of 1α,25-dihydroxyvitamin D3 (1) is taken as 100.
b Bovine thymus vitamin D receptor. c Ref 5a,c.
were obtained, and each of them was purified by using recycling
reversed-phase HPLC for biological evaluation.
Table 1 summarizes the relative VDR binding affinity of the
synthesised compounds (4a–d) in comparison with the natural
hormone 1, together with the 2-methyl analogues (2, 3).5 The
analogue 4a showed 3% of the affinity of 1, which suggests that
introduction of the second methyl group into 2 results in an
approximately 100-fold reduction of the affinity to VDR. Other
diastereomers (4b–d) showed weaker affinity compared with
their mono-methyl parents.5
Transcriptional machinery of the nuclear receptors is highly
conserved among the nuclear receptor superfamily.2 The modu-
lation of the transcriptional function by ligands depends on
conformational changes of the transactivation domain of the
receptor C-terminal region (AF-2), which provides an interface
for binding of cofactors.15,16 Modifications that can influence
the interface of VDR should include those in the side chain
moiety of 1, which would be embedded in the vicinity of the
AF-2 region.15a,16a In particular, 22-oxa-1α,25-dihydroxy-
vitamin D3 (OCT) used to treat psoriasis, was reported to
induce selective interaction of VDR with coactivator TIF2,
which could be responsible for its unique activity profile.15
Using the A-ring analogues, we investigated ligand-specific
potentiation of VDR transcriptional activity with two coactiv-
ators, TIF2 and SRC-1, both of which belong to the 160 kDa
protein family of coactivators (Fig. 2).15 Treatment with 1 at
10Ϫ8 M in the presence of TIF2 induced approximately twice
the transcriptional potency compared with the result with no
Scheme 2 Determination of relative stereochemistry of the 1,3-diols
by 13C NMR analysis. Reagents and conditions: (a) TBAF–THF;
(b) 2,2-dimethoxypropane, CSA.
Coupling reaction of the protected A-ring enynes ( )-12a,b
with the CD-ring portion 148 in the presence of the tetra-
kis(triphenylphosphine)palladium, followed by deprotection
with TBAF,13 proceeded smoothly to give the vitamin ana-
logues 4a,b and 4c,d, respectively (Scheme 3). The absolute
stereochemistry of the diols was determined by 1H NMR
analyses of their bis-MTPA esters (Fig. 1).14 In this way, four
stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin D3 (4a–d)
Scheme 3 Reagents and conditions: (a) (Ph3P)4Pd, Et3N–toluene, 63–66%; (b) TBAF–THF, 29–63%.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
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Fig. 1 Determination of absolute configuration at the C1 and C3 positions of 4a–d by 1H NMR analysis of their bis-MTPA esters.
spectra were recorded on a Jasco FT/IR-8000 spectrometer and
are expressed in cmϪ1. Ultraviolet spectra were recorded with a
Shimadzu UV-1600 spectrophotometer. Recycling preparative
HPLC was performed on a Waters LC equipped with a 510
HPLC pump and a 484 tunable absorbance detector.
Methyl 3-(4-methoxyphenoxy)-2,2-dimethylpropionate (6). A
solution of methyl hydroxypivalate 5 (3.00 g, 22.7 mmol),
4-methoxyphenol (8.45 g, 68.1 mmol) and triphenylphosphine
(7.74 g, 29.5 mmol) in THF (50 mL) was treated with diethyl
azodicarboxylate (DEAD) (40% in toluene solution, 13 mL) at
0 ЊC under an argon atmosphere. The resulting homogeneous
solution was heated at reflux for 2 h, cooled to room temper-
ature and concentrated under reduced pressure. The residue
was subjected to silica gel column chromatography (ethyl
acetate–n-hexane = 1 : 9) to afford 6 (5.30 g) as a colourless
oil in 98% yield.
Fig. 2 Ligand-specific potentiation of VDR transactivation function
by coactivators. COS-1 cells were transfected with a reporter plasmid
[heptadecamer(×2)-β-globin-luciferase]
and
pM(GAL4-DBD)-
VDR(DEF) with or without TIF2 or SRC-1 expression vectors in the
presence or absence of 1α,25-dihydroxyvitamin D3 (1), or its analogues
(2,3,4a) and harvested for luciferase assay at 24 h post-transfection.
6: 1H NMR (400 MHz, CDCl3) δ 1.30 (6 H, s), 3.69 (3 H, s),
3.76 (3 H, s), 3.91 (2 H, s), 6.82 (4 H, m); 13C NMR (100 MHz,
CDCl3) δ 22.4 (q), 43.4 (s), 52.0 (q), 55.7 (q), 75.3 (t), 114.6 (d),
115.7 (d), 153.3 (s), 176.5 (s); IR (neat) 2951, 2835, 1734, 1510,
coactivator (control), whereas the treatment had little effect in
the presence of SRC-1. On the other hand, in the presence of
SRC-1, the 2α-methyl analogue 2 at 10Ϫ8 M potentiated the
VDR transcription function by 35% compared with its control.
The 2β-methyl analogue 3 showed a weaker potency, but exhib-
ited a significant preference for TIF2 over SRC-1. Treatment
with 4a at 10Ϫ7 M in the presence of SRC-1 caused a 32%
increase of the transcriptional potency, which is similar to the
result with 2 at 10Ϫ8 M. The character of 4a may be inherited
from the 2α-methyl compound 2.
The present results suggest that ligands with a modification
in the A-ring, which appears to be located far from AF-2, can
alter the VDR-coactivator interaction, resulting in selective
potentiation of the transcription function. This strategy to
modulate the functions of the receptor based on an altered
protein–protein interaction by A-ring modification may prove
to be valuable.
In summary, we have efficiently synthesised all four possible
A-ring stereoisomers of 2,2-dimethyl-1,25-dihydroxyvitamin
D3 (4a–d) by employing a convergent method using a palladium
catalyst. The transcriptional function of the dimethyl analogue
4a in the presence of coactivators of the 160 kDa protein family
suggested that 4a retained the unique activity afforded by 2α-
methyl substitution, despite the reduced VDR binding affinity.
Differential recruitment of coactivators could be an interesting
approach for the development of selective therapeutic agents.
1471, 1396, 1367, 1232, 1151, 1107, 1049, 1003, 825, 742 cmϪ1
;
MS m/z 238 (M)ϩ, 207 (M Ϫ OMe)ϩ; HRMS calcd for
C13H18O4 238.1205, found 238.1206.
3-(4-Methoxyphenoxy)-2,2-dimethylpropanol (7). A solution
of 6 (2.07 g, 8.39 mmol) in THF (15 mL) was added dropwise to
a suspension of lithium aluminium hydride (478 mg, 12.6
mmol) in THF (10 mL) at 0 ЊC. The mixture was stirred for
1.5 h at this temperature, then ethyl acetate, followed by water,
was added, and the whole was filtered over CeliteTM. The filtrate
was extracted with ethyl acetate. The organic layer was dried
over magnesium sulfate, filtered and concentrated. The residue
was subjected to silica gel column chromatography (ethyl
acetate–n-hexane = 1 : 3) to afford 7 (1.71 g) as a colourless solid
in 97% yield.
7: 1H NMR (400 MHz, CDCl3) δ 1.02 (6 H, s), 2.01 (1 H, br
s), 3.54 (2 H, m), 3.73 (2 H, s), 3.77 (3 H, s), 6.84 (4 H, m); IR
(neat) 3265, 2959, 2937, 2909, 2874, 2835, 1516, 1460, 1359,
1292, 1242, 1180, 1111, 1035, 821 cmϪ1; MS m/z 210 (M)ϩ;
HRMS calcd for C12H18O3 210.1256, found 210.1265.
1-(4-Methoxyphenoxy)-2,2-dimethylhex-5-yn-3-ol (8).
A
stirred mixture of 7 (1.67 g, 7.94 mmol) and powdered 4 Å MS
(500 mg) in CH2Cl2 (20 mL) was treated with PDC (7.45 g, 19.9
mmol) at 0 ЊC under an argon atmosphere. The resulting mix-
ture was stirred overnight at room temperature, and separated
by silica gel column chromatography (ethyl acetate–n-hexane =
1 : 3) to give the corresponding aldehyde (1.47 g) as a colourless
oil in 89% yield. The aldehyde was used immediately in the
following step.
Experimental
General
NMR spectra were recorded on a JEOL ECP-600 or an AL-400
spectrometer. Chemical shifts are expressed in ppm relative to
tetramethylsilane. Mass spectra (MS) and high-resolution mass
spectra (HRMS) were recorded on a JMS-SX 102A. Infrared
3-(4-Methoxyphenoxy)-2,2-dimethylpropanal. 1H NMR (600
MHz, CDCl3) δ 1.20 (6 H, s), 3.77 (3 H, s), 3.91 (2 H, s), 6.82
(4 H, s), 9.64 (1 H, s); 13C NMR (150 MHz, CDCl3) δ 19.0 (q),
21.8 (q), 46.8 (s), 55.7 (q), 73.3 (t), 114.6 (d), 115.5 (d), 152.9 (s),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
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154.0 (s), 204.6 (d); IR (neat) 2964, 2910, 2874, 2835, 1734,
1510, 1471, 1442, 1402, 1377, 1290, 1230, 1182, 1107, 1049,
1005, 929, 898, 825 cmϪ1; MS m/z 208 (M)ϩ; HRMS calcd for
C12H16O3 208.1099, found 208.1079.
To a solution of the aldehyde (4.73 g, 22.7 mmol) in ether
(40 mL) was added with stirring allenylmagnesium bromide
(ca. 2 M in ether, 66 mL) at Ϫ78 ЊC under argon. The reaction
mixture was stirred for 1.5 h, and the reaction was quenched
by addition of saturated aqueous NH4Cl. After extraction
with ethyl acetate, the organic layer was washed with brine,
dried over magnesium sulfate and filtered. Evaporation of the
solvent afforded a residue, from which the 3-epimeric alcohols 8
(3.82 g) were obtained in 68% total yield.
mmol), 4-methylmorpholine N-oxide (NMO) (1.79 g, 2.6 eq.)
and powdered 4 Å MS (150 mg) in CH2Cl2 (15 mL) at room
temperature under argon. The resulting mixture was stirred for
1.5 h, and separated by silica gel column chromatography (ethyl
acetate–n-hexane = 1 : 9) to give the corresponding aldehyde
(1.03 g) as a colourless oil in 69% yield. The aldehyde was used
immediately in the following step.
3-(tert-Butyldimethylsilyl)oxy-2,2-dimethylhex-5-ynal.
1H
NMR (600 MHz, CDCl3) δ 0.09 (3 H, s), 0.15 (3 H, s), 0.87
(9 H, s), 1.08 (3 H, s), 1.09 (3 H, s), 2.02 (1 H, t, J = 2.8 Hz), 2.33
(1H, ddd, J = 17.6, 4.9, 2.8 Hz), 2.45 (1 H, ddd, J = 17.6, 6.0,
2.8 Hz), 3.97 (1 H, t, J = 5.5 Hz), 9.67 (1 H, s); 13C NMR
(150 MHz, CDCl3) δ Ϫ4.8 (q), Ϫ4.0 (q), 18.05 (q), 18.09 (s),
18.9 (q), 23.8 (t), 25.8 (q), 51.4 (s), 71.7 (d), 75.0 (d), 81.4 (s),
205.4 (d); IR (neat) 3314, 2957, 2932, 2887, 2858, 2710, 2121,
8: 1H NMR (600 MHz, CDCl3) δ 1.03 (3 H, s), 1.04 (3 H, s),
2.04 (1 H, t, J = 2.8 Hz), 2.38 (1H, ddd, J = 16.5, 9.3, 2.8 Hz),
2.50 (1 H, dt, J = 16.5, 2.8 Hz), 2.63 (1 H, br d, J = 2.8 Hz), 3.68
(1 H, d, J = 8.8 Hz), 3.77 (3 H, s), 3.83 (1 H, dt, J = 9.3, 2.8 Hz),
3.84 (1 H, d, J = 8.8 Hz), 6.83 (4 H, m); 13C NMR (150 MHz,
CDCl3) δ 19.6 (q), 21.9 (q), 22.5 (t), 38.5 (s), 55.7 (q), 70.3 (s),
74.8 (d), 75.8 (t), 82.2 (d), 114.6 (d), 115.4 (d), 153.0 (s), 153.9
(s); MS m/z 248 (M)ϩ; HRMS calcd for C15H20O3 248.1413,
found 248.1408.
1730, 1469, 1388, 1361, 1255, 1101, 1006, 918, 837, 777 cmϪ1
;
MS m/z 239 (M Ϫ Me)ϩ; HRMS calcd for C13H23O2Si 239.1468,
found 239.1472.
To a solution of the aldehyde (230 mg, 0.91 mmol) in toluene
(2 mL) was added with stirring vinylmagnesium bromide
(1.6 M in THF, 0.57 mL, 0.91 mmol) at Ϫ78 ЊC under argon.
The reaction mixture was stirred for 1 h, and the reaction was
quenched by addition of saturated aqueous NH4Cl. After
extraction with ethyl acetate, the organic layer was washed with
brine, dried over magnesium sulfate and filtered. Evaporation
of the solvent afforded a residue, from which 11a (53 mg, 20%,
less polar) and 11b (102 mg, 40%, more polar) were obtained by
silica gel column chromatography (ethyl acetate–n-hexane =
1 : 9), both as colourless oils.
4-(tert-Butyldimethylsilyl)oxy-6-(4-methoxyphenoxy)-5,5-di-
methylhex-1-yne (9). To a stirred mixture of 8 (3.77 g, 15 mmol)
and 2,6-lutidine (4.2 mL, 45 mmol) in CH2Cl2 (30 mL) was
added TBSOTf (6.5 mL, 23 mmol) at 0 ЊC under argon. The
resulting mixture was stirred at 0 ЊC for 5 min, diluted with
ethyl acetate and washed with water. The organic layer was
dried over magnesium sulfate and filtered. Removal of the sol-
vent afforded a residue, from which 9 (4.45 g) was separated by
silica gel column chromatography (ethyl acetate–n-hexane =
1 : 12) as a colourless oil in 81% yield.
11a: 1H NMR (600 MHz, CDCl3) δ 0.15 (3 H, s), 0.20
(3 H, s), 0.82 (3 H, s), 0.93 (9 H, s), 0.98 (3 H, s), 2.04 (1 H, t,
J = 2.7 Hz), 2.41 (1H, ddd, J = 17.6, 4.9, 2.7 Hz), 2.66 (1 H, ddd,
J = 17.6, 4.9, 2.7 Hz), 3.76 (1 H, t, J = 4.9 Hz), 3.86 (1 H, br s),
4.31 (1 H, dt, J = 6.3, 1.1 Hz), 5.18 (1H, ddd, J = 10.4, 1.9, 1.1
Hz), 5.28 (1H, ddd, J = 17.0, 1.9, 1.1 Hz), 5.84 (1H, ddd,
J = 17.0, 10.4, 6.3 Hz); 13C NMR (150 MHz, CDCl3) δ Ϫ4.8 (q),
Ϫ4.1 (q), 18.1 (s), 20.1 (q), 22.5 (q), 23.0 (t), 25.9 (q), 41.3 (s),
70.8 (s), 77.2 (d), 80.6 (d), 82.3 (d), 116.8 (t), 137.1 (d); IR (neat)
3464, 3314, 2957, 2932, 2885, 2858, 2120, 1639, 1471, 1425,
1390, 1361, 1255, 1082, 1005, 922, 837, 810, 777 cmϪ1; MS m/z
282 (M)ϩ; HRMS calcd for C16H30O2Si 282.2015, found
282.2012.
9: 1H NMR (600 MHz, CDCl3) δ Ϫ0.01 (3 H, s), 0.15 (3 H, s),
0.88 (9 H, s), 1.00 (3 H, s), 1.04 (3 H, s), 1.98 (1 H, t, J = 2.8 Hz),
2.28 (1H, ddd, J = 17.0, 4.9, 2.8 Hz), 2.57 (1 H, ddd, J = 17.0,
4.9, 2.8 Hz), 3.57 (1 H, d, J = 8.8 Hz), 3.74 (1 H, d, J = 8.8 Hz),
3.76 (1 H, s), 3.93 (1 H, t, J = 4.9 Hz), 6.81 (4 H, m); 13C NMR
(150 MHz, CDCl3) δ Ϫ4.8 (q), Ϫ3.8 (q), 18.2 (s), 19.8 (q), 21.9
(q), 23.2 (t), 26.0 (q), 40.1 (s), 55.7 (q), 70.1 (s), 74.5 (t), 74.8 (d),
83.3 (d), 114.5 (d), 115.1 (d), 153.4 (s), 153.5 (s); MS m/z 362
t
(M)ϩ, 347 (M Ϫ Me)ϩ, 305 (M Ϫ Bu)ϩ; HRMS calcd for
C21H34O3Si 362.2278, found 362.2285.
11b: 1H NMR (600 MHz, CDCl3) δ 0.12 (3 H, s), 0.17 (3 H,
s), 0.85 (3 H, s), 0.92 (9 H, s), 0.93 (3 H, s), 2.04 (1 H, t, J = 2.8
Hz), 2.30 (1H, ddd, J = 17.6, 4.4, 2.8 Hz), 2.34 (1 H, br d, J = 3.8
Hz), 2.63 (1 H, ddd, J = 17.6, 6.0, 2.8 Hz), 3.82 (1 H, dd, J = 6.0,
4.4 Hz), 4.14 (1 H, m), 5.19 (1H, ddd, J = 10.4, 1.7, 1.1 Hz), 5.27
(1H, dt, J = 17.0, 1.7 Hz), 5.94 (1H, ddd, J = 17.0, 10.4, 6.3 Hz);
13C NMR (150 MHz, CDCl3) δ Ϫ4.8 (q), Ϫ3.8 (q), 18.2 (s),
18.4 (q), 20.3 (q), 23.6 (t), 26.0 (q), 42.7 (s), 70.4 (s), 77.5 (d),
77.6 (d), 83.3 (d), 116.3 (t), 137.6 (d); IR (neat) 3454, 3312,
2957, 2932, 2885, 2858, 2120, 1732, 1641, 1471, 1425, 1388,
1363, 1255, 1087, 1005, 924, 837, 810, 775 cmϪ1; MS
m/z 282 (M)ϩ; HRMS calcd for C16H30O2Si 282.2015, found
282.1994.
3-(tert-Butyldimethylsilyl)oxy-2,2-dimethylhex-5-yn-1-ol (10).
A solution of 9 (2.38 g, 6.6 mmol) in CH3CN (68 mL) and
water (17 mL) was treated with diammonium cerium nitrate
(CAN) (8.66 g, 15.8 mmol) at 0 ЊC. After 15 min, ethyl acetate
(50 mL) and brine (30 mL) were added to the solution. The
aqueous layer was extracted with ethyl acetate. The combined
organic layer was washed with saturated aqueous NaHCO3,
dried over magnesium sulfate and filtered. Evaporation of the
filtrate afforded a residue, from which 10 (1.30 g) was separated
by silica gel column chromatography (ethyl acetate–n-hexane =
1 : 9) as a pale yellow oil in 77% yield.
10: 1H NMR (600 MHz, CDCl3) δ 0.12 (3 H, s), 0.17 (3 H, s),
0.87 (3 H, s), 0.92 (9 H, s), 1.03 (3 H, s), 2.04 (1 H, t, J = 2.7 Hz),
2.34 (1H, ddd, J = 17.6, 4.4, 2.7 Hz), 2.58 (1 H, ddd, J = 17.6,
6.0, 2.7 Hz), 3.35 (1 H, dd, J = 11.0, 6.0 Hz), 3.70 (1 H, d,
J = 11.0 Hz), 3.72 (1 H, dd, J = 6.0, 4.4 Hz); 13C NMR
(150 MHz, CDCl3) δ Ϫ4.9 (q), Ϫ4.1 (q), 18.1 (s), 21.6 (q), 22.8
(q), 23.3 (t), 25.9 (q), 39.9 (s), 69.7 (t), 70.6 (s), 78.1 (d), 82.5 (d);
MS m/z 199 (M Ϫ tBu)ϩ; HRMS calcd for C10H19O2Si 199.1154,
found 199.1156.
(3RS,5RS )-Bis[(tert-butyldimethylsilyl)oxy]-4,4-dimethyloct-
1-en-7-yne (12a). To a stirred mixture of 11a (91 mg, 0.32
mmol) and 2,6-lutidine (90 µL, 0.96 mmol) in CH2Cl2 (1 mL)
was added TBSOTf (140 µL, 0.48 mmol) at 0 ЊC under argon.
The resulting mixture was stirred at 0 ЊC for 1 h, diluted with
ethyl acetate and washed with water. The organic layer was
dried over magnesium sulfate and filtered. Removal of the sol-
vent afforded a residue, from which 12a (126 mg) was separated
by silica gel column chromatography (ethyl acetate–n-hexane =
1 : 12) as a colourless oil in quantitative yield.
(3RS,5RS )-5-(tert-Butyldimethylsilyl)oxy-4,4-dimethyloct-1-
en-7-yn-3-ol (11a: 1,3-anti-diol) and (3RS,5SR)-5-(tert-butyl-
dimethylsilyl)oxy-4,4-dimethyloct-1-en-7-yn-3-ol (11b: 1,3-syn-
diol). Solid tetrapropylammonium perruthenate (TPAP) (483
mg, 0.23 eq.) was added to a stirred mixture of 10 (1.50 g, 5.86
12a: 1H NMR (600 MHz, CDCl3) δ 0.00 (3 H, s), 0.04 (3 H,
s), 0.08 (3 H, s), 0.15 (3 H, s), 0.82 (3 H, s), 0.86 (3 H, s), 0.89
(9 H, s), 0.91 (9 H, s), 1.97 (1 H, t, J = 3.1 Hz), 2.22 (1H, ddd,
J = 17.3, 5.8, 3.1 Hz), 2.56 (1 H, ddd, J = 17.3, 4.1, 3.1 Hz), 3.75
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
1866

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(1 H, dd, J = 5.8, 4.1 Hz), 4.01 (1 H, d, J = 8.0 Hz), 5.12 (1H, d,
J = 18.7 Hz), 5.13 (1H, d, J = 10.4 Hz), 5.83 (1H, ddd, J = 18.7,
10.4, 8.0 Hz); 13C NMR (150 MHz, CDCl3) δ Ϫ4.7 (q), Ϫ4.5
(q), Ϫ3.6 (q), Ϫ3.4 (q), 18.2 (s), 18.3 (s), 19.6 (q), 19.9 (q), 23.6
(t), 25.9 (q), 26.1 (q), 44.1 (s), 70.1 (s), 75.7 (d), 78.9 (d), 83.8
(d), 116.6 (t), 138.7 (d); IR (neat) 3314, 3078, 2957, 2932, 2887,
2858, 2121, 1471, 1388, 1361, 1253, 1074, 1005, 925 cmϪ1; MS
m/z 396 (M)ϩ, 381 (M Ϫ Me)ϩ, 339 (M Ϫ tBu)ϩ; HRMS calcd
for C22H44O2Si2 396.2880, found 396.2910.
chromatography (ethyl acetate–n-hexane = 1 : 9) gave the corre-
sponding acetonide as a colourless oil in 52–54% yield.
1
Acetonide of 13a. H NMR (600 MHz, CDCl3) δ 0.82 (3 H,
s), 0.84 (3 H, s), 1.38 (3 H, s), 1.41 (3 H, s), 1.98 (1 H, t, J = 2.8
Hz), 2.29 (1H, ddd, J = 17.1, 8.0, 2.8 Hz), 2.32 (1 H,
ddd, J = 17.1, 5.8, 2.8 Hz), 3.63 (1 H, dd, J = 8.0, 5.8 Hz), 3.84
(1 H, d, J = 6.9 Hz), 5.19 (1H, d, J = 10.7 Hz), 5.24 (1H, d,
J = 17.0 Hz), 5.78 (1H, ddd, J = 17.0, 10.7, 6.9 Hz); 13C NMR
(150 MHz, CDCl3) δ 19.0 (q), 20.0 (t), 20.5 (q), 23.8 (q), 24.1
(q), 39.8 (s), 69.1 (s), 74.9 (d), 77.7 (d), 82.0 (d), 101.3 (s), 117.0
(t), 134.1 (d); IR (neat) 3314, 3080, 2988, 2961, 2928, 2856,
2365, 2123, 1736, 1649, 1541, 1466, 1379, 1228, 1176, 1128,
1082, 1059, 1014, 924 cmϪ1; MS m/z 208 (M)ϩ, 193 (M Ϫ Me)ϩ;
HRMS calcd for C13H20O2 208.1464, found 208.1492.
(3RS,5SR)-Bis[(tert-butyldimethylsilyl)oxy]-4,4-dimethyloct-
1-en-7-yne (12b). This compound was obtained by the same
procedure as described for 12a, starting from 11b instead of
11a.
12b: 1H NMR (600 MHz, CDCl3) δ Ϫ 0.01 (3 H, s), 0.04 (3 H,
s), 0.09 (3 H, s), 0.17 (3 H, s), 0.76 (3 H, s), 0.86 (3 H, s), 0.90
(9 H, s), 0.92 (9 H, s), 1.96 (1 H, t, J = 2.7 Hz), 2.20 (1H, ddd,
J = 17.3, 6.3, 2.7 Hz), 2.60 (1 H, dt, J = 17.3, 2.7 Hz), 3.80 (1 H,
dd, J = 6.3, 2.7 Hz), 4.03 (1 H, d, J = 7.1 Hz), 5.13 (1H, d,
J = 10.4 Hz), 5.14 (1H, d, J = 17.3 Hz), 5.81 (1H, ddd, J = 17.3,
10.4, 7.1 Hz); 13C NMR (150 MHz, CDCl3) δ Ϫ4.9 (q), Ϫ4.6
(q), Ϫ3.7 (q), Ϫ3.3 (q), 18.2 (s), 18.4 (s), 18.5 (q), 20.0 (q), 23.3
(t), 25.9 (q), 26.2 (q), 43.8 (s), 70.0 (s), 75.7 (d), 78.2 (d), 84.1
(d), 116.3 (t), 138.4 (d); IR (neat) 3316, 3078, 2957, 2932, 2887,
2858, 2121, 1471, 1386, 1361, 1253, 1084, 1005, 924 cmϪ1; MS
Acetonide of 13b. 1H NMR (600 MHz, CDCl3) δ 0.81
(3 H, s), 0.84 (3 H, s), 1.45 (3 H, s), 1.49 (3 H, s), 1.96 (1 H, t,
J = 2.8 Hz), 2.23 (1H, ddd, J = 17.0, 8.8, 2.8 Hz), 2.40 (1 H, dt,
J = 17.0, 2.8 Hz), 3.77 (1 H, dd, J = 8.8, 2.8 Hz), 3.99 (1 H, d,
J = 6.6 Hz), 5.23 (1H, d, J = 9.3 Hz), 5.26 (1H, d, J = 16.0 Hz),
5.77 (1H, ddd, J = 16.0, 9.3, 6.6 Hz); 13C NMR (150 MHz,
CDCl3) δ 12.8 (q), 19.3 (q), 20.2 (t), 20.9 (q), 30.0 (q), 35.9 (s),
68.9 (s), 77.2 (d), 79.5 (d), 82.4 (d), 99.0 (s), 118.3 (t), 134.0 (d);
IR (neat) 3310, 3080, 2991, 2968, 2939, 2874, 2123, 1861, 1740,
1645, 1468, 1425, 1381, 1263, 1201, 1174, 1126, 1089, 1060,
1037, 993 cmϪ1; MS m/z 208 (M)ϩ, 193 (M Ϫ Me)ϩ; HRMS
calcd for C13H20O2 208.1464, found 208.1484.
t
m/z 396 (M)ϩ, 339 (M Ϫ Bu)ϩ; HRMS calcd for C22H44O2Si2
396.2880, found 396.2889.
(3RS,5RS )-4,4-Dimethyloct-1-en-7-yne-3,5-diol (13a). To a
stirred solution of 11a (50 mg, 0.18 mmol) in THF (1 mL) was
added TBAF (1.0 M in THF, 0.27 mL, 0.27 mmol), and the
resulting mixture was stirred at 0 ЊC for 30 min. The reaction
mixture was diluted with ethyl acetate and washed with water.
The organic layer was dried over magnesium sulfate and fil-
tered. Evaporation of the filtrate afforded a residue, from which
13a (29 mg) was separated by silica gel column chromatography
(ethyl acetate–n-hexane = 2 : 3) as a colourless oil in 94% yield.
13a: 1H NMR (600 MHz, CDCl3) δ 0.89 (3 H, s), 0.95 (3 H,
s), 2.06 (1 H, t, J = 2.7 Hz), 2.37 (1H, ddd, J = 16.5, 9.3, 2.7 Hz),
2.42 (1 H, ddd, J = 16.5, 3.6, 2.7 Hz), 3.78 (1 H, dd, J = 9.3, 3.6
Hz), 3.99 (1 H, br d, J = 6.3 Hz), 5.23 (1H, dt, J = 10.4, 1.7 Hz),
5.30 (1H, dt, J = 17.3, 1.7 Hz), 5.96 (1H, ddd, J = 17.3, 10.4, 6.4
Hz); 13C NMR (150 MHz, CDCl3) δ 19.9 (q), 21.2 (q), 22.5 (t),
40.2 (s), 70.6 (s), 75.9 (d), 80.1 (d), 81.8 (d), 116.9 (t), 137.0 (d);
IR (neat) 3354, 3308, 3080, 2966, 2928, 2880, 2120, 1734, 1641,
(5Z,7E )-(1R,3R)-2,2-Dimethyl-9,10-seco-5,7,10(19)-choles-
tatriene-1,3,25-triol (4a) and (5Z,7E )-(1S,3S )-2,2-dimethyl-
9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (4b). A mixture
of the CD-ring portion 14 (57 mg, 0.16 mmol), tetrakis-
(triphenylphosphine)palladium (55 mg, 48 µmol) and triethyl-
amine (2.5 mL) in toluene (1 mL) was stirred for 20 min at room
temperature, then a solution of the A-ring enyne precursor 12a
(63 mg, 0.16 mmol) in toluene (2 mL) was added. After having
been heated at reflux for 1 h and diluted with ether, the reaction
mixture was filtered through a pad of silica gel with ether. After
evaporation of the solvent, the residue was subjected to silica
gel preparative TLC (ethyl acetate–n-hexane = 1 : 3) to give a
mixture of the protected vitamins (71 mg, 66%). The crude
vitamins dissolved in THF (3 mL) were treated with tetrabutyl-
ammonium fluoride (TBAF) (1.0 M in THF, 0.5 mmol) at room
temperature for 15 h. Brine was added to the reaction mixture
and the whole was extracted with ethyl acetate. The combined
organic layer was dried over magnesium sulfate, filtered and
concentrated. The residue was purified by silica gel preparative
TLC (ethyl acetate–n-hexane = 1 : 2) to give 4a,b (14 mg, 29%)
with recovery of mono-silylated alcohols (21 mg, 34%). Separ-
ation and further purification of 4a,b were conducted by using
a reversed-phase recycling HPLC (YMC-Pack ODS column,
20 × 150 mm, 9.0 mL minϪ1, acetonitrile–water = 9 : 1).
1469, 1425, 1392, 1369, 1280, 1203, 1115, 1043, 955, 927 cmϪ1
;
MS m/z 150 (M Ϫ H2O)ϩ; HRMS calcd for C10H14O 150.1044,
found 150.1048.
(3RS,5SR)-4,4-Dimethyloct-1-en-7-yne-3,5-diol (13b). This
compound was obtained by the same procedure as described for
13a, starting from 11b instead of 11a.
13b: 1H NMR (600 MHz, CDCl3) δ 0.77 (3 H, s), 0.91 (3 H,
s), 2.06 (1 H, t, J = 2.7 Hz), 2.34 (1H, ddd, J = 16.5, 9.6, 2.7 Hz),
2.47 (1 H, dt, J = 16.5, 2.7 Hz), 3.76 (1 H, dd, J = 9.6, 2.7 Hz),
4.04 (1 H, dt, J = 7.1, 1.1 Hz), 5.20 (1H, ddd, J = 10.4, 1.9, 1.1
Hz), 5.26 (1H, ddd, J = 17.0, 1.9, 1.1 Hz), 5.90 (1H, ddd,
J = 17.0, 10.4, 7.1 Hz); 13C NMR (150 MHz, CDCl3) δ 14.5 (q),
21.4 (q), 22.7 (t), 40.6 (s), 70.6 (s), 77.5 (d), 80.9 (d), 81.9 (d),
117.4 (t), 137.1 (d); IR (neat) 3368, 3304, 3080, 2972, 2937,
2880, 2118, 1734, 1639, 1463, 1423, 1394, 1373, 1273, 1116,
1060, 995, 929 cmϪ1; MS m/z 168 (M)ϩ; HRMS calcd for
C10H16O2 168.1150, found 168.1144.
1
4a: UV (EtOH) λmax 268 nm, λmin 229 nm; H NMR (600
MHz, CDCl3) δ 0.54 (3 H, s), 0.93 (3 H, d, J = 6.6 Hz), 0.98
(3 H, s), 1.04 (3 H, s), 1.21 (6 H, s), 1.48 (1 H, d, J = 6.0 Hz),
1.49 (1 H, d, J = 5.8 Hz), 2.28 (1 H, dd, J = 14.0, 6.6 Hz), 2.64
(1 H, dd, J = 14.0, 3.6 Hz), 2.81 (1 H, dd, J = 12.4, 4.4 Hz), 3.76
(1 H, dt, J = 3.8, 6.3 Hz), 3.99 (1 H, d, J = 5.5 Hz), 5.05 (1 H, t,
J = 1.7 Hz), 5.31 (1 H, t, J = 1.7 Hz), 6.03 (1 H, d, J = 11.3 Hz),
6.36 (1 H, d, J = 11.3 Hz); 13C NMR (150 MHz, CDCl3) δ 12.0,
18.8, 20.5, 20.8, 21.0, 22.3, 23.6, 27.6, 29.1, 29.2, 29.4, 36.1,
36.4, 40.5, 40.8, 41.4, 44.4, 45.9, 56.3, 56.5, 71.1, 74.8, 78.8,
113.1, 116.9, 124.8, 133.1, 143.1, 145.0; MS m/z 444 (M)ϩ, 426
(M Ϫ H2O)ϩ, 408 (M Ϫ 2H2O)ϩ, 393 (M Ϫ 2H2O Ϫ Me)ϩ, 390
(M Ϫ 3H2O)ϩ, 375 (M Ϫ 3H2O Ϫ Me)ϩ; HRMS calcd for
C29H48O3 444.3604, found 444.3610.
General procedure for synthesis of acetonides. To each of the
above enyne compounds (25 mg, 0.15 mmol) dissolved in
dimethoxypropane (0.5 mL) was added CSA (7 mg, 0.2 eq.)
at room temperature under argon. The resulting mixture
was stirred overnight at room temperature. Evaporation of
the solvent, followed by separation using silica gel column
1
4b: UV (EtOH) λmax 268 nm, λmin 229 nm; H NMR (600
MHz, CDCl3) δ 0.54 (3 H, s), 0.93 (3 H, d, J = 6.6 Hz), 1.01
(3 H, s), 1.02 (3 H, s), 1.21 (6 H, s), 1.45 (1 H, d, J = 4.9 Hz),
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
1867

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1.49 (1 H, d, J = 6.0 Hz), 2.30 (1 H, dd, J = 14.0, 7.4 Hz), 2.60
(1 H, dd, J = 14.0, 3.8 Hz), 2.82 (1 H, dd, J = 12.4, 4.4 Hz), 3.78
(1 H, ddd, J = 7.7, 6.0, 4.4 Hz), 3.96 (1 H, d, J = 5.2 Hz), 5.05
(1 H, dd, J = 1.9, 1.1 Hz), 5.29 (1 H, dd, J = 1.9, 1.1 Hz), 6.02
(1 H, d, J = 11.3 Hz), 6.37 (1 H, d, J = 11.3 Hz); 13C NMR (150
MHz, CDCl3) δ 12.0, 18.8, 20.3, 20.8, 21.0, 22.3, 23.6, 27.6,
29.0, 29.2, 29.4, 36.1, 36.4, 40.5, 40.9, 41.3, 44.4, 45.9, 56.3,
56.5, 71.1, 74.3, 79.6, 113.8, 116.9, 124.9, 133.0, 143.1, 145.7;
MS m/z 426 (M Ϫ H2O)ϩ, 408 (M Ϫ 2H2O)ϩ, 393 (M Ϫ 2H2O
Ϫ Me)ϩ, 390 (M Ϫ 3H2O)ϩ, 375 (M Ϫ 3H2O Ϫ Me)ϩ; HRMS
calcd for C29H46O2 426.3498, found 426.3498.
n-hexane = 1 : 2) without pretreatment to afford the corre-
sponding MTPA ester.
(S )-MTPA ester of 4a. 1H NMR (600 MHz, CDCl3) δ 0.439
(3 H, s), 0.609 (3 H, s), 0.908 (3 H, s), 0.929 (3 H, d, J = 6.6 Hz),
1.219 (6 H, s), 2.507 (1 H, t, J = 11.8 Hz), 2.729 (1 H, dd,
J = 13.7, 4.9 Hz), 2.806 (1 H, dd, J = 12.9, 4.1 Hz), 3.485 (3 H,
s), 3.516 (3 H, s), 5.047 (1 H, dd, J = 10.4, 4.9 Hz), 5.178 (1 H,
s), 5.264 (1 H, s), 5.498 (1 H, s), 5.918 (1 H, d, J = 11.5 Hz),
6.449 (1 H, d, J = 11.5 Hz), 7.35–7.53 (10 H, m); FABMS
m/z 900 (M ϩ Na)ϩ, 877 (M ϩ H)ϩ; HRFABMS calcd for
C49H62O7F6Na 899.4297, found 899.4299.
(5Z,7E )-(1S,3R)-2,2-Dimethyl-9,10-seco-5,7,10(19)-choles-
tatriene-1,3,25-triol (4c) and (5Z,7E )-(1R,3S )-2,2-dimethyl-
9,10-seco-5,7,10(19)-cholestatriene-1,3,25-triol (4d). A mixture
of the CD-ring portion 14 (57 mg, 0.16 mmol), tetrakis(tri-
phenylphosphine)palladium (55 mg, 48 µmol) and triethyl-
amine (2.5 mL) in toluene (1 mL) was stirred for 20 min at room
temperature, then a solution of the A-ring enyne precursor 12b
(63 mg, 0.16 mmol) in toluene (2 mL) was added. After having
been heated at reflux for 1 h and diluted with ether, the reaction
mixture was filtered through a pad of silica gel with ether. After
evaporation of the solvent, the residue was subjected to silica
gel preparative TLC (ethyl acetate–n-hexane = 1 : 3) to give a
mixture of the protected vitamins (68 mg, 63%). The crude
vitamins dissolved in THF (3 mL) were treated with tetrabutyl-
ammonium fluoride (TBAF) (1.0 M in THF, 0.5 mmol) at room
temperature for 19 h. Brine was added to the reaction mixture
and the whole was extracted with ethyl acetate. The combined
organic layer was dried over magnesium sulfate, filtered and
concentrated. The residue was purified by silica gel preparative
TLC (ethyl acetate–n-hexane = 3 : 2) to give 4c,d (28 mg, 63%).
Separation and further purification of 4c,d were conducted by
using a reversed-phase recycling HPLC (YMC-Pack ODS
column, 20 × 150 mm, 9.0 mL minϪ1, acetonitrile–water =
9 : 1).
(R)-MTPA ester of 4a. 1H NMR (600 MHz, CDCl3) δ 0.270
(3 H, s), 0.911 (3 H, d, J = 6.3 Hz), 0.953 (6 H, s), 1.231 (6 H, s),
2.388 (1 H, t, J = 11.8 Hz), 2.679 (1 H, dd, J = 13.7, 4.9 Hz),
2.759 (1 H, dd, J = 13.5, 4.4 Hz), 3.394 (3 H, s), 3.513 (3 H, s),
5.076 (1 H, dd, J = 10.2, 4.9 Hz), 5.187 (1 H, s), 5.257 (1 H, s),
5.428 (1 H, s), 5.804 (1 H, d, J = 11.3 Hz), 6.362 (1 H, d, J = 11.5
Hz), 7.39–7.56 (10 H, m); FABMS m/z 900 (M ϩ Na)ϩ;
HRFABMS calcd for C49H62O7F6Na 899.4297, found 899.4298.
(S )-MTPA ester of 4b. 1H NMR (600 MHz, CDCl3) δ 0.513
(3 H, s), 0.934 (3 H, d, J = 6.3 Hz), 0.939 (3 H, s), 0.978 (3 H, s),
1.220 (6 H, s), 2.380 (1 H, dd, J = 12.4, 11.3 Hz), 2.662 (1 H, dd,
J = 13.7, 4.7 Hz), 2.742 (1 H, dd, J = 13.5, 4.9 Hz), 3.428 (3 H,
s), 3.503 (3 H, s), 5.068 (1 H, dd, J = 9.9, 4.7 Hz), 5.204 (1 H, s),
5.220 (1 H, s), 5.425 (1 H, s), 5.890 (1 H, d, J = 11.3 Hz), 6.320
(1 H, d, J = 11.3 Hz), 7.35–7.43 (6 H, m), 7.48–7.50 (4 H, m);
FABMS m/z 900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62-
O7F6Na 899.4297, found 899.4303.
(R)-MTPA ester of 4b. 1H NMR (600 MHz, CDCl3) δ 0.539
(3 H, s), 0.718 (3 H, s), 0.926 (3 H, s), 0.939 (3 H, d, J = 6.3 Hz),
1.217 (6 H, s), 2.518 (1 H, t, J = 11.8 Hz), 2.732 (1 H, dd,
J = 13.7, 4.9 Hz), 2.807 (1 H, dd, J = 12.4, 4.1 Hz), 3.472 (3 H,
s), 3.528 (3 H, s), 5.107 (1 H, dd, J = 10.7, 4.9 Hz), 5.194 (1 H,
s), 5.306 (1 H, s), 5.530 (1 H, s), 5.966 (1 H, d, J = 11.3 Hz),
6.446 (1 H, d, J = 11.3 Hz), 7.29–7.50 (10 H, m); FABMS m/z
900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62O7F6Na 899.4297,
found 899.4308.
1
4c: UV (EtOH) λmax 266 nm, λmin 228 nm; H NMR (600
MHz, CDCl3) δ 0.55 (3 H, s), 0.94 (3 H, d, J = 6.6 Hz), 0.96
(3 H, s), 1.17 (3 H, s), 1.21 (6 H, s), 2.24 (1 H, d, J = 5.0 Hz),
2.39 (1 H, dd, J = 14.6, 4.4 Hz), 2.71 (1 H, br d, J = 14.0 Hz),
2.81 (1 H, d, J = 8.8 Hz), 2.84 (1 H, dd, J = 11.5, 3.3 Hz), 3.57
(1 H, dd, J = 8.8, 4.4 Hz), 3.82 (1 H, d, J = 4.1 Hz), 5.07 (1 H, d,
J = 2.2 Hz), 5.26 (1 H, d, J = 1.9 Hz), 6.07 (1 H, d, J = 11.3 Hz),
6.46 (1 H, d, J = 11.3 Hz); 13C NMR (150 MHz, CDCl3) δ 11.9,
18.8, 20.7, 20.8, 22.4, 23.7, 24.5, 27.6, 29.1, 29.2, 29.3, 36.1,
36.4, 39.9, 40.5, 41.2, 44.4, 45.9, 56.4, 56.5, 71.1, 76.5, 82.4,
115.0, 116.9, 125.8, 131.1, 143.0, 145.6; MS m/z 444 (M)ϩ, 426
(M Ϫ H2O)ϩ, 408 (M Ϫ 2H2O)ϩ, 393 (M Ϫ 2H2O Ϫ Me)ϩ, 390
(M Ϫ 3H2O)ϩ, 375 (M Ϫ 3H2O Ϫ Me)ϩ; HRMS calcd for
C29H48O3 444.3604, found 444.3610.
(S )-MTPA ester of 4c. 1H NMR (600 MHz, CDCl3) δ 0.553
(3 H, s), 0.850 (3 H, s), 0.857 (3 H, s), 0.940 (3 H, d, J = 6.3 Hz),
1.220 (6 H, s), 2.484 (1 H, t, J = 11.8 Hz), 2.655 (1 H, dd,
J = 13.2, 4.9 Hz), 2.790 (1 H, dd, J = 12.6, 3.9 Hz), 3.520 (3 H,
s), 3.535 (3 H, s), 4.896 (1 H, s), 4.912 (1 H, s), 4.943 (1 H, dd,
J = 11.3, 5.0 Hz), 5.235 (1 H, s), 5.888 (1 H, d, J = 11.3 Hz),
6.402 (1 H, d, J = 11.3 Hz), 7.38–7.43 (6 H, m), 7.51–7.55 (4 H,
m); FABMS m/z 900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62-
O7F6Na 899.4297, found 899.4304.
1
4d: UV (EtOH) λmax 267 nm, λmin 229 nm; H NMR (600
MHz, CDCl3) δ 0.53 (3 H, s), 0.93 (3 H, d, J = 6.6 Hz), 0.98
(3 H, s), 1.13 (3 H, s), 1.21 (6 H, s), 2.12 (1 H, d, J = 5.2 Hz),
2.40 (1 H, dd, J = 14.3, 5.2 Hz), 2.66 (1 H, dd, J = 14.3, 2.2 Hz),
2.71 (1 H, d, J = 7.1 Hz), 2.84 (1 H, dd, J = 11.3, 2.8 Hz), 3.56 (1
H, ddd, J = 7.1, 5.2, 2.2 Hz), 3.80 (1 H, d, J = 4.9 Hz), 5.04 (1 H,
d, J = 2.2 Hz), 5.26 (1 H, d, J = 2.2 Hz), 6.03 (1 H, d, J = 11.3
Hz), 6.43 (1 H, d, J = 11.3 Hz); 13C NMR (150 MHz, CDCl3) δ
12.0, 18.8, 19.6, 20.8, 22.2, 23.5, 24.5, 27.7, 29.1, 29.2, 29.4,
36.1, 36.4, 40.2, 40.5, 41.3, 44.4, 45.9, 56.3, 56.5, 71.1, 76.4,
81.8, 114.5, 117.0, 125.6, 131.1, 143.0, 145.6; MS m/z 444 (M)ϩ,
426 (M Ϫ H2O)ϩ, 408 (M Ϫ 2H2O)ϩ, 393 (M Ϫ 2H2O Ϫ Me)ϩ,
390 (M Ϫ 3H2O)ϩ, 375 (M Ϫ 3H2O Ϫ Me)ϩ; HRMS calcd for
C29H48O3 444.3604, found 444.3611.
(R)-MTPA ester of 4c. 1H NMR (600 MHz, CDCl3) δ 0.543
(3 H, s), 0.806 (3 H, s), 0.940 (3 H, d, J = 6.3 Hz), 0.968 (3 H, s),
1.220 (6 H, s), 2.356 (1 H, dd, J = 12.4, 10.7 Hz), 2.619 (1 H,
dd, J = 12.7, 4.7 Hz), 2.758 (1 H, dd, J = 13.7, 4.9 Hz), 3.425
(6 H, s), 4.941 (1 H, dd, J = 9.6, 4.7 Hz), 4.992 (1 H, s), 5.076
(1 H, s), 5.281 (1 H, s), 5.857 (1 H, d, J = 11.3 Hz), 6.280 (1 H, d,
J = 11.3 Hz), 7.39–7.43 (6 H, m), 7.49–7.55 (4 H, m); FABMS
m/z 900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62O7F6Na
899.4297, found 899.4312.
(S )-MTPA ester of 4d. 1H NMR (600 MHz, CDCl3) δ 0.513
(3 H, s), 0.934 (3 H, d, J = 6.3 Hz), 0.939 (3 H, s), 0.978 (3 H, s),
1.220 (6 H, s), 2.380 (1 H, dd, J = 12.4, 11.3 Hz), 2.662 (1 H, dd,
J = 13.7, 4.7 Hz), 2.742 (1 H, dd, J = 13.5, 4.9 Hz), 3.428 (3 H,
s), 3.503 (3 H, s), 5.068 (1 H, dd, J = 9.9, 4.7 Hz), 5.204 (1 H, s),
5.220 (1 H, s), 5.425 (1 H, s), 5.890 (1 H, d, J = 11.3 Hz), 6.320
(1 H, d, J = 11.3 Hz), 7.35–7.43 (6 H, m), 7.48–7.50 (4 H, m);
General procedure for synthesis of MTPA esters14. A solution
of each of the above vitamins dissolved in dry CH2Cl2 was
treated with DMAP (4 eq.) and (R)- or (S)-MTPACl (2.5 eq.) at
room temperature under an argon atmosphere. The reaction
mixture was purified by preparative TLC (ethyl acetate–
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 8 6 3 – 1 8 6 9
1868

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FABMS m/z 900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62O7-
4 (a) A. W. Norman, R. Bouillon, M. C. Farach-Carson, J. E. Bishop,
L.-X. Zhou, L. Nemere, J. Zhao, K. R. Muralidoharan and
W. H. Okamura, J. Biol. Chem., 1993, 268, 20022; (b) K. R.
Muralidoharan, A. R. de Lera, S. D. Isaeff, A. W. Norman and
W. H. Okamura, J. Org. Chem., 1993, 58, 1895; (c) M. G. Bischof,
M.-L. Siu-Caldera, A. Weiskopf, P. Vouros, H. S. Cross, M. Peterlik
and G. S. Reddy, Exp. Cell. Res., 1998, 241, 194.
F6Na 899.4297, found 899.4303.
(R)-MTPA ester of 4d. 1H NMR (600 MHz, CDCl3) δ 0.539
(3 H, s), 0.718 (3 H, s), 0.926 (3 H, s), 0.939 (3 H, d, J = 6.3 Hz),
1.217 (6 H, s), 2.518 (1 H, t, J = 11.8 Hz), 2.732 (1 H, dd,
J = 13.7, 4.9 Hz), 2.807 (1 H, dd, J = 12.4, 4.1 Hz), 3.472 (3 H,
s), 3.528 (3 H, s), 5.107 (1 H, dd, J = 10.7, 4.9 Hz), 5.194 (1 H,
s), 5.306 (1 H, s), 5.530 (1 H, s), 5.966 (1 H, d, J = 11.3 Hz),
6.446 (1 H, d, J = 11.3 Hz), 7.29–7.50 (10 H, m); FABMS m/z
900 (M ϩ Na)ϩ; HRFABMS calcd for C49H62O7F6Na 899.4297,
found 899.4308.
5 (a) K. Konno, S. Maki, T. Fujishima, Z.-P. Liu, D. Miura,
M. Chokki and H. Takayama, Bioorg., Med. Chem. Lett., 1998, 8,
151; (b) T. Fujishima, Z.-P. Liu, D. Miura, M. Chokki, S. Ishizuka,
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Chem., 2000, 43, 4247; (d ) K. Nakagawa, M. Kurobe, K. Ozono,
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8, 123; (g) T. Fujishima, Z.-P. Liu, K. Konno, K. Nakagawa,
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T. Okano, M. Kurihara, N. Miyata and H. Takayama, Chem. Biol.,
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M. Kurihara and H. Takayama, Org. Lett., 2000, 2, 2619; (b) Y.
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A. Kittaka, K. Yamaguchi, T. Fujishima, K. Konno, N. Miyata and
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7 For reviews of synthesis of vitamin D compounds, see: (a) G.-D.
Zhu and W. H. Okamura, Chem. Rev., 1995, 95, 1877; (b) H. Dai and
G. H. Posner, Synthesis, 1994, 1383.
Vitamin D receptor (VDR) binding assay. Bovine thymus
VDR receptor was obtained from Yamasa Biochemical (Chiba,
Japan) and dissolved in 0.05 M phosphate buffer (pH 7.4)
containing 0.3 M KCl and 5 mM dithiothreitol just before use.
The receptor solution (500 µL) was pre-incubated with 50 µL
ethanol solution of 1α,25-dihydroxyvitamin D3 or an analogue
at various concentrations for 60 min at 25 ЊC. Then, the
receptor mixture was left to stand overnight with 0.1 nM [3H]-
1α,25-dihydroxyvitamin D3 at 4 ЊC. The bound and free [3H]-
1α,25-dihydroxyvitamin D3 were separated by treatment with
dextran-coated charcoal for 30 min at 4 ЊC and centrifuged
at 3000 rpm for 10 min. The supernatant (500 µL) was mixed
with ACS-II (9.5 mL) (Amersham, UK) and the radioactivity
was counted. The relative potency of the analogues was calcu-
lated from their concentration needed to displace 50% of
[3H]-1α,25-dihydroxyvitamin D3 from the receptor compared
with the activity of 1α,25-dihydroxyvitamin D3 (assigned a
100% value).
8 (a) B. M. Trost, J. Dumas and M. Villa, J. Am. Chem. Soc., 1992,
114, 9836; (b) B. M. Trost and J. Dumas, J. Am. Chem. Soc., 1992,
114, 1924.
Transactivation assay.15 COS-1 cells were maintained in
Dulbecco’s modified Eagle’s medium without phenol red,
supplemented with 5% fetal calf stripped with dextran-coated
charcoal. Cells were transfected by means of lipofection. The
following plasmids were used for transfection: reporter plasmid
pGL-GAL4-UAS (0.5 µg) containing the GAL4 upstream acti-
vation sequence (UAS) (heptadecamer ×2, β-globin promoter,
and luciferase) cotransfected with 0.1 µg of pM(GAL4-DBD)-
VDR(DEF) with or without TIF2 or SRC-1 expression vector
(0.1 µg). As a reference plasmid for normalization, 0.1 ng of
plasmid pRL-CMV plasmid (Promega) was used. Either 1 or
an analogue was added to the medium at 6 h after transfection.
After 24 h, firefly luciferase activity (from GAL4-UAS) was
used to measure transfection efficiency by Renilla luciferase
activity (from pRL-CMV).
9 An attempt using p-methoxybenzyl, instead of p-anisyl, as a
protective group of the primary alcohol in 5 gave a similar result.
10 H. Hoph, I. Böhm and J. Kleinschroth, in Organic Synthesis
Collective Volume VII, ed. J. P. Freeman, Wiley, New York, 1990,
pp. 485–490.
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945; (b) S. D. Rychnovsky, B. Rogers and G. Yang, J. Org. Chem.,
1993, 58, 3511.
12 The anti 1,3-diol acetonide adopts a twist-boat conformation,
whereas the syn counterpart adopts a chair conformation: See ref
11b.
13 Deprotection of the TBS groups in the anti 1,3-diols was rather slow
compared with that of their syn counterparts. Using this character,
we have separated anti and syn 1,3-diols during deprotection of TBS
in the synthesis of 1-methyl and 3-methyl vitamin D analogues:
T. Fujishima, Y. Harada, K. Konno, K. Nakagawa, M. Tanaka,
T. Okano and H. Takayama, In Vitamin D Endocrine System:
Structural, Biological, Genetic and Clinical Aspects, Proceedings
of the 11th Workshop on Vitamin D, Nashville, May 27-June 1,
eds. A. W. Norman, R. Bouillon, M. Thomasset, University of
California, Printings and Reprographics: Riverside, 2000, pp. 93–96.
14 The modified Mosher’s method using a simple derivation of
secondary alchohols to their methoxy-trifluoromethyl-phenylacetic
acid (MTPA) esters was shown to be applicable to determine the
absolute configuration of 1,3-diols by their bis-MTPA esters:
K. Konno, T. Fujishima, Z.-P. Liu and H. Takayama, Chirality,
2002, 14, 72.
15 (a) K. Takeyama, Y. Masuhiro, H. Fuse, H. Endoh, A. Murayama,
S. Kitanaka, M. Suzawa, J. Yanagisawa and S. Kato, Mol. Cell.
Biol., 1999, 19, 1049; (b) Y. Kodera, K. Takeyama, A. Murayama,
M. Suzawa, Y. Masuhiro and S. Kato, J. Biol. Chem., 2000, 275,
33201.
16 (a) S. Väisänen, M. Peräkylä, J. Kärkkäinen, A. Steinmeyer and
C. Carlberg, J. Mol. Biol., 2002, 315, 229; (b) H. E. Xu, T. B. Stanley,
V. G. Montana, M. H. Lambert, B. G. Shearer, J. E. Cobb,
D. D. McKee, C. M. Galardi, K. D. Plunket, R. T. Nolte, D. J. Parks,
J. T. Moore, S. A. Kiliewer, T. M. Willson and J. B. Stimmel, Nature,
2002, 415, 813.
Acknowledgements
This work was supported in part by a Grant from the Kihara
Memorial Yokohama Foundation for the Advancement of Life
Sciences.
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