Synthesis and biological evaluations of A-ring isomers of 26,26,26,27,27,27-hexafluoro-1,25-dihydroxyvitamin D3

Article abstract of DOI:10.1016/S0968-0896(00)00142-5

The activated vitamin D₃ derivative 26,27-F₆-1α,25(OH)₂D₃ (2a), its three A-ring diastereomers (2b, 2c, 2d), and 5,6-trans isomer (2e) were prepared. Two analogues (2b, 2c) of these isomers were synthesized by a palladium catalyzed coupling reaction using vinyl bromide 5 and enynes (6a, 6b), which were derived from readily commercially available 2S-(+)-glycidyl p-toluenesulfonate 7, as a common starting material. Competitive vitamin D receptor (VDR) binding affinities of these diastereomers of 2a were evaluated. Interestingly, the stereochemical effects at C-1,3 of 2a were considerably more moderate than those of 1α,25(OH)₂D₃ (1). In particular, isomerization at the 5,6-double bond of 2a only slightly reduced VDR affinity, whereas 5,6-trans-1α,25(OH)₂D₃ had a significantly lower binding affinity than 1. Copyright (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(00)00142-5

Bioorganic & Medicinal Chemistry 8 (2000) 2157±2166
Synthesis and Biological Evaluations of A-Ring Isomers of
26,26,26,27,27,27-Hexa¯uoro-1,25-dihydroxyvitamin D3
Masahiko Ikeda,* Kazuhiko Takahashi, Akihito Dan, Kohji Koyama, Katsumi Kubota,
Tomoyuki Tanaka and Masaji Hayashi
Sumitomo Pharmaceuticals Research Center, 1-98, Kasugadenaka 3-chome, Konohana-ku, Osaka 554-0022, Japan
Received 26 April 2000; accepted 31 May 2000
AbstractÐThe activated vitamin D3 derivative 26,27-F6-1a,25(OH)2D3 (2a), its three A-ring diastereomers (2b, 2c, 2d), and 5,6-
trans isomer (2e) were prepared. Two analogues (2b, 2c) of these isomers were synthesized by a palladium catalyzed coupling
reaction using vinyl bromide 5 and enynes (6a, 6b), which were derived from readily commercially available 2S-(+)-glycidyl p-
toluenesulfonate 7, as a common starting material. Competitive vitamin D receptor (VDR) binding anities of these diastereomers
of 2a were evaluated. Interestingly, the stereochemical e€ects at C-1,3 of 2a were considerably more moderate than those of
1a,25(OH)2D3 (1). In particular, isomerization at the 5,6-double bond of 2a only slightly reduced VDR anity, whereas 5,6-trans-
1a,25(OH)2D3 had a signi®cantly lower binding anity than 1. # 2000 Elsevier Science Ltd. All rights reserved.
Introduction
geometric e€ect of the 5,6-double bond of 2a. This report
concerns a synthetic method to A-ring diastereomers and
5,6-trans isomer of 2a and their VDR binding anities.
1a,25-Dihydroxyvitamin D3 (1; 1a,25(OH)2D3), the
hormonally active form of vitamin D3, plays important
roles in regulating cell proliferation and di€erentiation
of a variety of cell types, keratinocytes, immunological
and malignant cells.1 These actions are believed to be
mediated via binding to the vitamin D receptor (VDR),
which belongs to the superfamily of nuclear receptors
for glucocorticoids, estrogens and retinoic acid. E€orts
are currently aimed at the development of more thera-
peutically useful analogues of 1a,25(OH)2D3, the e€ects
of which on cell proliferation and di€erentiation may be
useful for treatment of disorders ranging from psoriasis
to cancer.2 A large number of side-chain-modi®ed ana-
logues have been described during the last decade.3 In the
A-ring and the triene system, several isomers possessing
unnatural con®gurations and geometries of 1a,25
(OH)2D3 (1)4 8 were synthesized and their biological
activities including VDR anities were evaluated. On
the other hand, previous studies have demonstrated that
26,26,26,27,27,27-hexa¯uoro-1a,25-dihydroxyvitamin D3
(2a; 26,27-F6-1a,25(OH)2D3) showed potentiation of
various aspects of some biological activities of vitamin
D3.9,10 Therefore, we decided to investigate the extent to
which A-ring stereochemical modi®cation of 2a in¯u-
ences VDR anities. In addition, we also con®rmed the
Results and Discussion
Synthesis
Our synthetic plan for the synthesis of these desired
analogues is described in Scheme 1. For the preparation
of 2c and 2d, the convergent method developed by Trost
et al.11 was thought to be useful. The enynes 6a, 6b
could possibly be derived from 7. As for the preparation
of 2a and bromoole®n 5, we decided to apply the method,
which was recently developed by our laboratory,12 to
prepare 26,27-F6 vitamin D3 analogues via aldol reaction
using hexa¯uoroacetone (HFA). In practice, 2a was
prepared in the following method, as shown in Scheme 2.
Treatment of aldehyde 4, which was prepared from
readily available vitamin D2,13 with methylmagnesium
bromide followed by TPAP oxidation14 gave methyl-
ketone 8. HFA aldol reaction and subsequent reduction
of the 23-keto group by NaBH4 a€orded 23R-alcohol
10 as the major product.12 Conversion to the acetate 11,
using Ac2O/pyridine, was followed by the introduction
of the MOM group at the 25-OH group and subsequent
removal of the acetyl group gave secondary alcohol 13.
Formation of thionocarbonate 14 and reduction by tri-
butylstannane15 a€orded 3. Deprotection of 3 with
*Corresponding author. Tel.: +81-797-74-2067; fax: +81-797-74-
2142; e-mail: ikeda@sumitomopharm.co.jp
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00142-5

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M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
Figure 1.
Scheme 1. Synthetic plan for the synthesis of A-ring isomers of 2a.

M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
2159
Scheme 2. The preparation of 2a from 4. (i) MeMgBr, Et2O, 0ꢀC, 30 min; (ii) TPAP, NMO, MS4A, CH2Cl2, rt, 15 min; (iii) CF3COCF3,
LiN(TMS)2, THF, 70 ꢀC, 30 min; (iv) NaBH4, THF-MeOH, 10 ꢀC, 10 min; (v) Ac2O, pyridine, rt, 12 h; (vi) MOMCl, iPr2NEt, CHCl3, re¯ux, 1 h;
(vii) KOH-MeOH, 0 ꢀC, 1 h; (viii) NaH, CS2, rt, overnight then CH3I; (ix) nBu3SnH, toluene, re¯ux, 1 h; (x) MsOH, MeOH, rt, 2 h.
Scheme 3. The preparation of 1-epimer 2b from 2a. (i) Dess±Martin periodinane, CH3CN, rt, 4 h; (ii) NaBH4, MeOH, 0 ꢀC, 1.5h; (iii) acetone, 80ꢀC, 6 h.
methanesulfonic acid in MeOH gave 26,27-F6-
1a,25(OH)2D3 (2a). The 1-epi isomer 2b was prepared
from 2a in the same manner as described in the prepara-
tion of the 1-epi isomer of 1.4 Dess±Martin oxidation16,17
of 2a a€orded ketone 15, which upon reduction with
NaBH4 followed by thermal isomerization gave 2b
(Scheme 3).
toluenesulfonate (7), as the common starting material; the
addition of TMS-acetylene to the epoxide 7 gave 17,19
which was treated with KCN in DMSO to a€ord 18. The
protection of secondary alcohol with the t-butyldiphe-
nylsilyl (TBDPS) group followed by reduction with
DIBALH gave aldehyde 20. Grignard addition with
vinylmagnesium bromide and subsequent removal of
the TMS group of 21 under basic conditions gave a
1.8:1 ratio of the two diastereomer mixture (22a and
22b). These isomers were separated by HPLC. To assign
the stereochemistry, 22a was converted to acetonide 23
whose 13C NMR spectrum showed signals for the geminal
dimethyl group indicative of a syn-1,3-diol chem-
istry.11,20,21 Protection of the alcohol with the t-butyldi-
methylsilyl (TBS) group gave the desired 1,7-enynes 6a
and 6b, respectively. The corresponding CD-ring fragment
was derived from 3 as shown in Scheme 5. Ozonolysis of 3
gave ketone 24. The highly E-selective bromoole®nation
The other two diastereomers (2c, 2d) were synthesized by
Trost's convergent method using a palladium-catalyzed
coupling reaction.11 This method has already been
applied to the synthesis of A-ring diastereomers of 1
and its 20-epimer analogues by Takayama et al.5,6,18 In
this report, we also employed Trost's method for the
preparation of two A-ring diastereomers of 2a. However,
as for the preparation of the A-ring precursor enyne
compounds (6a and 6b), we chose a readily commercially
available chiral building block moiety, 2S-(+)-glycidyl p-

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M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
Scheme 4. The preparation of enyne compounds (6a, 6b) from 7. (i) Trimethylsilylacetylene, nBuLi, BF3 Et2O, THF, 70 ꢀC; (ii) KCN, DMSO,
40 ꢀC, 3 h; (iii) TBDPSCl, imidazole, DMF, 40 ꢀC, 2 h; (iv) DIBALH, CH2Cl2, 10 ꢀC, 30 min; (v) vinylmagnesium bromide, THF, 70 ꢀC!rt,
30 min; (vi) K2CO3, MeOH, rt, 1 h; (vii) TBSCl, imidazole, DMF, 40 ꢀC, 2 h.
Scheme 5. Preparation of the CD ring fragment 5 from 3. (i) O3, PPh3, 78 ꢀC, 30 min; (ii) LiN(TMS)2, bromotriphenylphosphonium bromide,
toluene, 78 ꢀC, 30 min.
of CD-ring ketone derivatives by bromomethyl-
triphenylphosphorane, generated using NaN(TMS)2/
THF was also reported by Trost et al.11 However, in the
case of 24, bromoole®n 5 was obtained in only 30% yield
under the identical reaction conditions. After investiga-
tions, we found that the use of LiN(TMS)2/toluene pro-
vided 5 in a slightly increased yield (34%), without
decreasing the geometrical selectivity (E:Z=100:1), along
with recovered 24 (45%). The generation of the lithium
enolate of 24 in situ would account for the recovered
ketone. As a result, due to the recovery of 24, vinyl bro-
mide 5 was obtained in 62% yield. Palladium-catalyzed
coupling reaction of vinyl bromide 5 and 1,7-enyne 6a
paration of 5,6-trans-26,27-F6-1a,25(OH)2D3 (2e), the
same procedure used for the preparation of 5,6-trans-
1,25(OH)2D3 was applied via a cheleotropic reaction.8
Biological evaluation
Biological evaluations of A-ring isomers of 3 are sum-
marized in Table 1. The binding anity to VDR was
determined by a competitive binding assay using the
cytosolic receptor from chick intestine.22
While the analogous epimerization of the 1-OH group
(2a±2b) provided a 4-fold reducing e€ect on binding
anity, that of the 3-OH group (2a±2c) had a modest
e€ect (1.5-fold reduction). Double epimerization of the
1,3-OH groups (2a±2d) led to a substantial decrease in
binding anity (7-fold reduction). These results show that
.
by Pd2(dba)3 CHCl3 followed by deprotection with
methanesulfonic acid in MeOH gave 3-epi isomer 2c.
The 1b,3a-isomer, 2d, was synthesized from 5 and 1,7-
enyne 6b by the same method as for 2c. As for the pre-

M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
2161
.
Scheme 6. Synthesis of 2c, 2d, 2e. (i) (dba)3Pd2 CHCl3, NEt3, toluene, re¯ux, 10 min; (ii) MsOH, MeOH, rt, 1 h; (iii) liqui®ed SO2, CH2Cl2, re¯ux,
30 min; (iv) NaHCO3, EtOH, re¯ux, 90 min.
Experimental
Table 1. Binding activities of four A-ring isomers of 26,27-F6-
1a,25(OH)2D3(2a)a for the 1a,25(OH)2D3 receptor in the chick small
intestine
All reactions involving oxygen- or moisture-sensitive
compounds were carried out under a dry nitrogen
atmosphere. Reaction temperatures refer to external
Modi®cation type
26,27-F6-1,25(OH)2D3
1,25(OH)2D3
bath temperatures. Reactions were monitored by thin
layer chromatography (TLC) using Merck TLC plates
(silica gel 60 F254, 0.25 mm thickness). After ultraviolet
illumination at 254 nM, the plates were visualized by
immersion in a solution of phosphomolybdic acid in
MeOH followed by heating. Column chromatography
puri®cation was performed with Merck silica gel (silica
Natural type
1-Epimer
3-Epimer
1,3-Epimer
5,6-trans-Isomer
45
11
32
6.6
41
(100)b
(23)b
(71)b
(15)b
(91)b
100
(0.8)c
(24)c
(0.22)c
(13)d
aThe results for 1a,25(OH)2D3 are normalized to 100.
bThe results for 26,27-F6-1a,25(OH)2D3 are normalized to 100.
cref 4, chick intestinal.
1
gel 60, 70-230 mesh). H NMR data were measured in
dref 7, chick intestinal.
CDCl3. Chemical shifts are reported as d units (ppm)
down®eld from tetramethylsilane (d 0.0) using the residual
solvent signal as an internal standard: d 7.26.
the inversion of stereochemistry at C1 to an unnatural
type (1b) caused a more pronounced reduction on
binding anity than that of the 3-OH group. Inversion
of the 1a-OH group of 2a resulted in a modest reduction,
whereas it has been reported that inversion of the
1a-OH group of 1, which has a higher binding anity
than 2a, lowered binding anity by roughly two orders
of magnitude.4 5,6-trans-26,27-F6-1a,25(OH)3D3 (2e)
showed a slightly decreased binding anity compared to
2a, whereas 5,6-trans-1a,25(OH)3D3 had a signi®cantly
lower binding anity than 1.7
(5Z,7E)-(1S,3R,20S)-1,3-Bis[t-butyldimethylsilyl)oxy]-20-
(propyl-23-one)-9,10-seco-5,7,10(19)-pregnatriene (8).
Methylmagnesium bromide (0.99 M in THF, 45.5 mL)
was added to the solution of 4 (19.7 g, 33.6 mmol) in
Et2O (415 mL) at 0ꢀC. After stirring for 30 min at the
same temperature, saturated NH4Cl was added, and the
mixture extracted with ethyl acetate. The organic layer
was washed with brine, and dried over MgSO4. The
®ltrate was concentrated in vacuo. The residue was
puri®ed by silica gel chromatography (ethyl acetate:
hexane, 1:5) to give an alcohol as a colorless oil (18.4 g,
91%). IR (neat): 3353 (br), 2952, 2856, 1471, 1256,
1075, 833 cm 1. 1H NMR (270 MHz) d: 0.05±0.06 (12H,
m), 0.54 and 0.56 (total 3H, 2s), 3.91 (1H, m), 4.20 (1H,
m), 4.36 (1H, m), 4.86 (1H, d, J=2.0 Hz), 5.17 (1H, br
s), 6.01 (1H, d, J=11.0 Hz), 6.24 (1H, d, J=11.0 Hz).
Tetrapropylammonium perruthenate (600 mg, 1.71 mmol)
was added in one portion to a stirred mixture of the
alcohol (10.0 g, 16.6 mmol) 4-methylmorphorine N-
oxide (2.93 g, 25.0 mmol) and powdered 4 A molecular
sieves (5.0 g) in dichloromethane (133 mL) at room
temperature. After 15 min, the reaction mixture was
passed through silica gel chromatography (dichloro-
Conclusion
26,27-F6-1a,25(OH)2D3 (2a), its three A-ring diaster-
eomers (2b, 2c, 2d), and 5,6-trans isomer 2e were synthe-
sized from aldehyde 4. Two analogues (2b, 2c) of these
isomers were synthesized by Trost's convergent method.
Comparative VDR anities of all of the isomers of 2a
were evaluated. We found that the stereochemistry of C-
1,3 position or the C-5,6 ole®nic geometry a€ects the
VDR anity less in the case of 2a, compared to the series
of 1. We considered it is because the binding anity of the
A-ring moiety of 2a to VDR is lower than that of 1. To
our knowledge, among vitamin D3 analogues, this is the
®rst case which shows such a biological pro®le.
methane only) to give 8 (9.12 g, 92%) as a colorless oil.
1
IR (neat): 2927, 2857, 1718, 1472, 1256, 1075, 829 cm
.

2162
M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
1H NMR (270 MHz) d: 0.05±0.06 (12H, 2s), 0.57 (3H,
s), 0.88 (18H, s), 0.93 (3H, d, J=6.5 Hz), 2.12 (3H, s),
4.18 (1H, m), 4.36 (1H, m), 4.86 (1H, d, J=2.5 Hz), 5.17
(1H, br s), 6.01 (1H, d, J=11.0 Hz), 6.23 (1H, d,
J=11.0 Hz). MS(EI) m/z: 600 (M+), 585 (M+-CH3),
543 (M+-tBu), 486 (M+-2tBu), 468 (M+-2tBu-H2O),
453 (M+-2tBu-H2O-CH3). HR-MS(EI) m/z: 600.4421
(M+) (Calcd for C36H64O3Si2 600.4394).
cooling, the reaction mixture was poured into ice water
and extracted with ethyl acetate. The organic layer was
washed with 5% HCl, saturated aqueous NaHCO3, and
brine, dried over MgSO4 and concentrated in vacuo.
The residue was puri®ed by silica gel chromatography
(ethyl acetate:hexane, 1:20) to give 12 (2.58 g, 89%) as a
colorless oil. IR (neat): 2931, 2857, 1747, 1461, 1216,
1
1077cm 1. H NMR (270MHz) d: 0.06 (12H, s), 0.52
(3H, s), 0.87 (18H, s), 0.99 (3H, d, J=5.9 Hz), 2.02 (3H,
s), 2.82 (1H, dd, J=13.5, 3.3 Hz), 3.47 (3H, s), 4.19 (1H,
m), 4.38 (1H, m), 4.86 (1H, br s), 4.90 (1H, d, J=6.6 Hz),
4.99 (1H, d, J=6.6 Hz), 5.18 (1H, br s), 5.41 (br s), 5.99
(1H, d, J= 11.4Hz), 6.22 (1H, d, J=11.4 Hz).
(5Z,7E)-(1S,3R)-1,3-Bis[(t-butyldimethylsilyl)oxy]-26,26,
26,27,27,27-hexa¯uoro-23-oxo-9,10-seco-5,7,10(19)-chol-
estatrien-25-ol (9). This compound was prepared as pre-
viously reported.12 IR (neat): 3306, 2929, 1709, 1462,
1258, 1089, 834 cm 1. 1H NMR (270 MHz) d: 0.04±0.06
(total 12H, 2s), 0.57 (3H, s), 0.88 (18H, s), 0.96 (3H, d,
J=6.2 Hz), 2.88 (2H, br s), 4.20 (1H, m), 4.37 (1H, m),
4.85 (1H, d, J=2.3 Hz), 5.18 (1H, br s), 6.01 (1H, d,
J=11.0 Hz), 6.23 (1H, d, J=11.0 Hz), 6.88 (1H, br s).
MS(EI) m/z: 766 (M+), 751 (M+-CH3), 709 (M+-tBu),
697 (M+-CF3), 652 (M+-2tBu), 634 (M+-2tBu-H2O),
619 (M+-2tBu-H2O-CH3). HR-MS(EI) m/z: 766.4299
(M+) (calcd for C39H64O4F6Si2 766.4247).
(5Z,7E)-(1S,3R,23R)-Bis[(t-butyldimethylsilyl)oxy]-26,26,
26,27,27,27-hexa¯uoro-25-methoxymethoxy-9,10-seco-5,
7,10(19)-cholestatrien-23-ol (13). 12 (2.58 g, 3.02 mmol)
was dissolved in 5% KOH-MeOH (30 mL) and stirred
at 0 ꢀC for 1 h. The reaction mixture was poured into ice
water and extracted with ethyl acetate. The organic
layer was washed with brine, dried over MgSO4 and
concentrated in vacuo. The residue was puri®ed by silica
gel chromatography (ethyl acetate:hexane, 1:20) to give
(5Z,7E)-(1S,3R,23R)-1,3-Bis[(t-butyldimethylsilyl)oxy]-
26,26,26,27,27,27-hexa¯uoro-9,10-seco-5,7,10(19)-chol-
estatriene-23,25-diol (10). This compound was prepared
as previously reported.12 IR (neat): 3326, 2953, 2857,
1472, 1211, 1075, 835 cm 1. 1H NMR (270 MHz) d: 0.06
(12H, s), 0.56 (3H, s), 0.88 (18H, s), 1.00 (3H, d,
J=6.3 Hz), 4.20 (1H, m), 4.37 (2H, m), 4.86 (1H, d,
J=2.3 Hz), 5.18 (1H, br s), 6.02 (1H, d, J=11.2 Hz), 6.24
(1H, d, J=11.2 Hz), 6.28 (1H, br s). MS(EI) m/z: 768
(M+), 753 (M+-CH3), 711 (M+-tBu), 636 (M+-2tBu-
H2O), 621 (M+-2tBu-CH3-H2O). HR-MS(EI) m/z:
768.4402 (M+) (calcd for C39H66O4F6Si2 768.4404).
alcohol 13 (2.31 g, 94%) as a colorless oil. IR (neat): 3505
1
(br), 2952, 2857, 1470, 1214, 835 cm
.
1H NMR
(270MHz) d: 0.04, 0.05 (each 6H, s), 0.56 (3H, s), 0.87
(18H, s), 0.97 (1H, d, J=6.6 Hz), 2.48 (1H, m), 2.58 (1H,
d, J=2.3 Hz), 2.82 (1H, m), 3.49 (3H, s), 4.19 (2H, m),
4.36 (1H, m), 4.86 (1H, br s), 5.02 (2H, s), 5.16 (1H, br s),
6.02 (1H, d, J=11.2 Hz), 6.24 (1H, d, J=11.2 Hz).
(5Z,7E)-(1S,3R,23R)-1,3-Bis[(t-butyldimethylsilyl)oxy]-
26,26,26,27,27,27-hexa¯uoro- 25-methoxymethoxy- 23-
(methylthiothiocarbonyloxy)-9,10-seco-5,7,10(19)-cholesta-
triene (14). A mixture of 13 (2.31 g, 2.84 mmol), CS2
(0.22mL, 3.66 mmol), 60% NaH (114mg, 2.85mmol),
imidazole (19 mg, 0.28 mmol) and THF (30 mL) was stir-
red overnight at room temperature. Then CH3I
(0.27 mL, 4.33 mmol) was added and stirred at room
temperature for 2 h. The reaction mixture was poured
into ice water and extracted with ethyl acetate. The
organic layer was washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was puri®ed by
silica gel chromatography (ethyl acetate:hexane, 1:20) to
give 14 (2.05 g, 80%) as a colorless oil. IR (neat): 2951,
2856, 1214, 1075cm 1. 1H NMR (270MHz) d: 0.06 (12H,
s), 0.52 (3H, s), 0.87, 0.88 (each 9H, s), 0.99 (3H, d,
J=5.6Hz), 2.55 (3H, s), 3.47 (2H, s), 4.19 (1H, m), 4.38
(1H, m), 4.86 (1H, br s), 4.94 (1H, d, J=6.6Hz), 5.00 (1H,
d, J=6.6 Hz), 5.19 (1H, br s), 6.02 (1H, d, J=11.2 Hz),
6.21 (2H, m).
(5Z,7E)-(1S,3R,23R)-23-Acetoxy-1,3-bis[(t-butyldimethyl-
silyl)oxy]-26,26,26,27,27,27-hexa¯uoro-9,10-seco-5,7,10
(19)-cholestatrien-25-ol (11). 10 (2.50 g, 3.25 mmol) was
dissolved in pyridine:Ac2O (2:1, 30mL) and stirred at
room temperature for 12h. The reaction mixture was
poured into ice water and extracted with ethyl acetate.
The organic layer was washed with 5% HCl, saturated
aqueous NaHCO3, and brine, dried over MgSO4 and
concentrated in vacuo. The residue was puri®ed by silica
gel chromatography (ethyl acetate: hexane, 1:20) to give
11 (2.56 g, 97%) as a colorless oil. IR (neat): 3301 (br),
1
2953, 2875, 1709, 1250, 1076, 835 cm
.
1H NMR
(270 MHz) d: 0.06, 0.07 (each 6H, s), 0.55 (3H, s), 0.87,
0.88 (each 9H, s), 0.92 (3H, d, J=6.6 Hz), 2.13 (3H, s),
2.44 (1H, dd, J=13.2, 3.6 Hz), 2.83 (1H, d, J=10.9 Hz),
4.19 (1H, m), 4.38 (1H, m), 4.87 (1H, br s), 4.97 (1H, br
d, J=10.2 Hz), 5.19 (1H, br s), 6.02 (1H, d, J=11.2 Hz),
6.23 (1H, d, J=11.2 Hz), 6.50 (1H, s). MS(EI) m/z: 810
(M+), 506 (M+-H2O), 488 (M+-2H2O). HR-MS(EI) m/
z: 810.4561 (M+) (calcd for C41H68O5F6Si2 810.4510).
(5Z,7E)-(1S,3R)-1,3-Bis[(t-butyldimethylsilyl)oxy]-26,26,
26,27,27,27-hexa¯uoro-25-methoxymethoxy-9,10-seco-
5,7,10(19)-cholestatriene (3). A solution of 14 (2.05 g,
2.27 mmol) in toluene (4.0 mL) was added dropwise to a
solution of nBu3SnH (1.83 mL, 6.80 mmol) in toluene
(30 mL) at 100 ꢀC and the solution was heated under
re¯ux for 1 h. After cooling, the reaction mixture was
concentrated in vacuo. The residue was puri®ed by silica
gel chromatography (hexane only!ethyl acetate:hexane,
1:10) to give 3 (1.75 g, 97%) as a colorless oil. IR (neat):
(5Z,7E)-(1S,3R,23R)-23-Acetoxy-1,3-Bis[(t-butyldimethyl-
silyl)oxy]-26,26,26,27,27,27-hexa¯uoro-25-methoxymeth-
oxy-9,10-seco-5,7,10(19)-cholestatriene (12). A solution
of 11 (2.74 g, 3.38mmol), chloromethylmethyl ether
(5.13 mL, 67.5 mmol), iPr2NEt (17.7 mL, 0.101 mol) in
CHCl3 (20 mL) was heated under re¯ux for 1 h. After
1
2953, 2857, 1212, 1082 cm 1. H NMR (270 MHz) d:

M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
2163
0.06 (12H, s), 0.53 (3H, s), 0.87 (18H, s), 0.94 (3H, d,
J=6.3Hz), 2.21 (1H, m), 2.83 (1H, d, J=9.6 Hz), 3.46
(3H, s), 4.19 (1H, m), 4.38 (1H, m), 4.86 (1H, br s), 4.92
(2H, s), 5.18 (1H, br s), 6.02 (1H, d, J=10.9 Hz), 6.24 (1H,
d, J=10.9 Hz).
(Zorbax BP SIL, f4.6 mmÂ25 cm, methanol:dichloro-
methane:hexane=3:50:50) to give 2b (1.8 mg, 82%) as
an amorphous solid. UV (EtOH): lmax 264nM. 1H NMR
(500 MHz) d: 0.55 (3H, s), 0.95 (3H, d, J=6.7 Hz), 2.48
(1H, m), 2.56 (1H, m), 2.85 (1H, m), 4.10 (1H, m), 4.35
(1H, m), 5.01 (1H, br s), 5.29 (1H, br s), 6.02 (1H, d,
J=11.3 Hz), 6.44 (1H, d, J=11.3 Hz). MS(EI) m/z: 524
(M+), 506 (M+-H2O), 491 (M+-CH3-H2O), 488 (M+-
2H2O), 473 (M+-2H2O-CH3). HR-MS(EI) m/z:
524.2698 (M+) (calcd for C27 H38O3F6 524.2725).
(5Z,7E)-(1S,3R)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (2a). Methane-
sulfonic acid (1.0 mL) was added to a solution of 3
(200 mg, 0.251 mmol) in methanol (10 mL) at room
temperature and stirred for 2 h. The reaction mixture
was quenched with ice water and extracted with ethyl
acetate. The organic layer was washed with saturated
aqueous NaHCO3, brine, and dried over MgSO4 and
concentrated in vacuo. The residue was puri®ed by silica
gel chromatography (ethyl acetate:hexane, 3:2) to give
2a (74 mg, 56%) as an amorphous powder. UV (EtOH):
(S)-2-Hydroxy-5-trimethylsilyl-1-p-toluenesulfonyloxy-4-
pentyne (17). To a solution of trimethylsilylacetylene
(642 mg, 6.54 mmol) in THF (6.0 mL) was added n-
BuLi (1.47 M in hexane, 4.7 mL) dropwise at 70 ꢀC.
.
After stirring for 30 min, BF3 Et2O (1.10 mL,
8.50mmol) and then 7 (1.34 g, 5.90mmol) was added. The
reaction mixture was stirred for 30min at the same tem-
perature, then the reaction was quenched with saturated
aqueous NH4Cl solution and the whole was extracted
with ethyl acetate. The organic layer was washed with
water, brine, dried over MgSO4 concentrated in vacuo.
The residue was puri®ed by silica gel chromatography
(ethyl acetate:hexane, 1:3) to give 17 (1.52 g, 79%) as a
1
lmax 265 nM. H NMR (270 MHz) d: 0.55 (3H, s), 0.94
(3H, d, J=6.3 Hz), 2.93 (1H, br s), 4.24 (1H, m), 4.37
(1H, m), 5.00 (1H, br s), 5.33 (1H, br s), 6.01 (1H, d,
J=11.3 Hz), 6.38 (1H, d, J=11.3 Hz). MS(EI) m/z: 524
(M+), 506 (M+-H2O), 491 (M+-CH3-H2O), 488 (M+-
2H2O), 473 (M+-2H2O-CH3).
1
(6Z)-(3R)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-5(10),
6,8-cholestatriene-3,25-diol-1-one (15). Dess±Martin
periodinane reagent (10 mg, 23.5 mmol) was added to a
solution of 2a (5.0 mg, 9.5 mmol) in CH3CN (2.5 mL) at
room temperature and stirred for 4 h. The reaction
mixture was quenched with a 1:1 mixture of saturated
aqueous Na2S2O3 and NaHCO3 solution. The organic
layer was dried over MgSO4 and concentrated in vacuo.
The residue was puri®ed by silica gel chromatography
(ethyl acetate: hexane, 1:2) to give 15 (3.3 mg, 66%) as
colorless oil. H NMR (270 MHz) d: 0.12 (9H, s), 2.46
(3H, s), 2.45±2.48 (2H, m), 3.94±4.20 (3H, m), 7.36 (2H,
d, J=8.4 Hz), 7.81 (2H, d, J=8.4 Hz).
(S)-1-Cyano-2-hydroxy-5-trimethylsilyl-4-pentyne (18).
To the suspension of 17 (400 mg, 1.23 mmol) in DMSO
(8 mL) was added potassium cyanide (96 mg, 1.48 mmol)
and the mixture was heated at 40 ꢀC for 3 h. The reaction
mixture was cooled in an ice bath, diluted with water,
and extracted with ethyl acetate. The organic layer was
washed with water, brine, dried over MgSO4 con-
centrated in vacuo. The residue was puri®ed by silica gel
chromatography (ethyl acetate:hexane, 1:3) to give 18
(160mg, 72%) as a colorless oil. IR (neat): 3458(br), 2961,
2901, 2256, 2178, 1250, 1029 cm 1. 1H NMR (270 MHz)
d: 0.17 (9H, s), 2.40 (1H, d, J=5.7 Hz), 2.59 (2H, d,
J=5.9 Hz), 2.68 (2H, d, J=5.5 Hz), 4.08 (1H, m).
MS(EI) m/z: 166 (M+-CH3), 148 (M+-CH3-H2O). HR-
MS(EI) m/z: 166.0672 (M+-CH3) (calcd for C8H12NOSi
166.0688).
1
an amorphous solid. H NMR (270 MHz) d: 0.72 (3H,
s), 0.97 (3H, d, J=6.6 Hz), 1.79 (3H, s), 2.40±2.60 (2H,
m), 2.72±2.86 (2H, m), 2.96 (1H, m), 3.17±3.29 (2H, m),
4.23 (1H, m), 5.49 (1H, m), 6.03 (1H, d, J=11.9 Hz),
6.14 (1H, d, J=11.9 Hz).
(6Z)-(1R,3R)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5(10),6,8-cholestatriene-1ꢀ,3ꢀ,25-triol (16). NaBH4
(11.2 mg, 89.3 mmol) was added to a solution of 15
(3.3 mg, 6.31 mmol) in methanol (0.5 mL) at 0 ꢀC and
stirred for 1.5 h. The reaction mixture was quenched
with 1N HCl solution, and extracted with ethyl acetate.
The organic layer was washed with 1N HCl, saturated
aqueous NaHCO3, brine and dried over MgSO4 and
concentrated in vacuo. The residue was puri®ed by silica
gel chromatography (ethyl acetate:hexane, 1:1) to give 16
(2.2 mg, 66%) as an amorphous solid. 1H NMR
(270 MHz) d: 0.71 (3H, s), 0.97 (3H, d, J=6.6 Hz), 1.81
(3H, s), 2.64 (1H, m), 2.92 (1H, m), 4.02 (1H, m), 4.24
(1H, m), 5.56 (1H, m), 5.79 (1H, d, J=12.2 Hz), 5.96
(1H, d, J=12.2 Hz).
(S)-2-[(t-Butyldiphenylsilyl)oxy]-1-cyano-5-trimethylsilyl-
4-pentyne (19). To a solution of 18 (239mg, 1.32 mmol) in
DMF (2.0 mL) was added t-butyldiphenylsilyl chloride
(TBDPSCl, 797 mg, 2.90mmol), imidazole (272mg,
4.0 mmol) and stirred at 40 ꢀC for 2 h. The reaction mix-
ture was poured into water and extracted with water. The
organic layer was washed with water and brine, dried over
MgSO4 and concentrated in vacuo. The residue was pur-
i®ed by silica gel chromatography (ethyl acetate/hexane,
1:10) to give 19 (497mg, 90%) as a colorless oil. IR (neat):
1
3072, 2959, 2859, 2253, 2179, 1428cm
.
1H NMR
(5Z,7E)-(1R,3R)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (2b). A solution of
16 (2.2 mg, 4.19 mmol) in acetone (1 mL) was placed in a
sealed tube and heated at 80 ꢀC for 6 h. After cooling,
the reaction mixture was concentrated in vacuo. The
crude product was puri®ed by silica gel chromatography
(ethyl acetate:hexane, 1:1) and further HPLC puri®cation
(270MHz) d: 0.10 (9H, s), 1.06 (9H, s), 2.44±2.65 (4H, m),
4.05 (1H, m), 7.35±7.70 (10H, m). MS(EI) m/z: 419 (M+),
404 (M+-CH3), 362 (M+-tBu). HR-MS(EI) m/z: 419.2126
(M+) (calcd for C25H33NOSi2 419.2101).
(3S,5S)/(3R,5S)-5-[(t-Butyldiphenylsilyl)oxy]-3-hydroxy-
8-trimethylsilyl-1-octen-7-yne (21). Diisobutyl aluminium

2164
M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
hydride (DIBALH, 1.0 M, 1.5 mL) was added to a solu-
tion of 19 (323mg, 0.770 mmol) in dichloromethane
(1.5 mL) at 10ꢀC and stirred for 30min. The reaction
mixture was quenched with 1N HCl and extracted with
ethyl acetate. The organic layer was washed with 1N HCl,
saturated aqueous NaHCO3, brine, dried over MgSO4,
and concentrated in vacuo to give 20 (412mg) as a crude
product. 1H NMR (270MHz) d: 0.13 (9H, s), 1.06 (9H, s),
2.34 (2H, m), 2.68 (2H, m), 5.34 (1H, q, J=5.9 Hz), 7.41
(6H, m), 7.70 (4H, m), 9.74 (1H, t, J=2.2Hz). Vinylmag-
nesium bromide (1.0 M in THF, 7.0 mL) was added
dropwise to a solution of 20 (412 mg) in THF (4 mL) at
70 ꢀC, then warmed up to room temperature and stirred
for 30min. The reaction mixture was quenched with 1 N
HCl and extracted with ethyl acetate. The organic layer
was washed with 1N HCl, saturated aqueous NaHCO3,
brine, dried over MgSO4 and concentrated in vacuo. The
residue was puri®ed by silica gel chromatography (ethyl
acetate:hexane, 1:10) to give 21 (226 mg, 65% from 19)
as a colorless oil. 1H NMR (300 MHz) d: 0.11, 0.12
(total 9H, 2s), 1.07 (9H, s), 1.72---1.94 (2H, m), 2.27--2.52
(2H, m), 4.06, 4.13 (total 1H, 2m), 4.31, 4.41 (1H, 2m),
4.99--5.24 (2H, m), 5.70--5.85 (1H, m), 7.40 (6H, m), 7.70
(112 mg, 0.296 mmol) in DMF (1.5 mL) was added
TBSCl (89 mg, 0.591 mmol), imidazole (60 mg,
0.881 mmol), and stirred at 40 ꢀC for 2 h. The reaction
mixture was poured into water and extracted with ethyl
acetate. The organic layer was washed with water and
brine, dried over MgSO4 and concentrated in vacuo. The
residue was puri®ed by silica gel chromatography (ethyl
acetate:hexane, 1:10) to give 6a (125mg, 86%) as a color-
less oil. IR (neat): 3313, 3072, 2857, 1472, 1253cm 1. 1H
NMR (270MHz) d: 0.03 (6H, s), 0.81 (9H, s), 1.05 (9H,
s), 1.84 (2H, m), 1.91 (1H, m), 2.29 (2H, m), 3.90 (1H, m),
4.21 (1H, m), 4.86-5.00 (2H, m), 5.58 (1H, m), 7.40 (6H,
m), 7.71 (4H, m). MS(EI) m/z: 435 (M+-Bu). HR-MS(EI)
m/z: 435.2129 (M+-Bu) (calcd for C26H35O2Si2
435.2176).
(3R,5S)-[(t-Butyldimethylsilyl)oxy]-5-[(t-butyldiphenylsilyl-
oxy]-1-octen-7-yne (6b). To a solution of 22a (70 mg,
0.185 mmol) in DMF (1 mL) was added TBSCl (56 mg,
0.372 mmol), imidazole (38 mg, 0.558 mmol), and stirred
at 40 ꢀC for 2 h. The reaction mixture was treated in the
same manner described in the preparation of 7a. The
residue was puri®ed by silica gel chromatography (ethyl
acetate:hexane, 1:10) to give 6b (75 mg, 82%) as a color-
less oil. IR (neat): 3313, 3072, 2857, 1472, 1253 cm 1. 1H
NMR (270 MHz) d: 0.07 (3H, s), 0.04 (3H, s), 0.82
(9H, s), 1.06 (9H, s), 1.74 (1H, m), 1.93±2.04 (3H, m),
2.29 (2H, m), 3.93 (1H, m), 4.08 (1H, m), 4.90±5.06 (2H,
m), 5.63 (1H, ddd, J=6.9, 10.7, 17.2 Hz), 7.40 (6H, m),
7.69 (4H, m). MS(EI) m/z: 435 (M+-Bu). HR-MS(EI) m/
z: 435.2183 (M+-Bu) (calcd for C26H35O2Si2 435.2176).
(4H, m). MS(FAB+) m/z: 451 (M++H), 433 (M+
-
H2O+H). HR - MS(FAB+) m/z: 451.2457 (M++H)
(calcd for C27H39O2Si2 451.2489).
(3,S,5S)-5-[(t-Butyldiphenylsilyl)oxy]-3-hydroxy-1-octen-
7-yne (22a) and (3R,5S)-5-[(t-Butyldiphenylsilyl)oxy]-3-
hydroxy-1-octen-7-yne (22b). To a solution of 21
(226 mg, 0.501 mmol) in methanol (4 mL) was added
K2CO3 (100 mg) and stirred for 1 h at room tempera-
ture. The reaction mixture was quenched with water and
extracted with ethyl acetate. The organic layer was
washed with brine, dried over MgSO4 and concentrated
in vacuo. The residue was puri®ed by silica gel chroma-
tography (ethyl acetate:hexane, 1:10) to give a crude
product. The crude diastereomer mixture was separated
by HPLC (DAISO, IR-120-10/20-S, f50 mmÂ25 cm,
ethyl acetate:hexane, 1:30) to give 22a (104 mg, 55%,
retention time 61.3 min) and 22b (57 mg, 30%, retention
time 50.3 min).
Ketone (24). A mixture of O3 and O2 was passed into a
stirred solution of 3 (1.45 g, 1.81 mmol) in dichloro-
methane (150 mL) and MeOH (30 mL) at 78 ꢀC for
30 min. Then PPh3 (2.0 g, 7.63 mmol) was added and
stirred at 78 ꢀC for 30 min, then warmed up to 0 ꢀC and
stirred for 30 min. The resulting reaction mixture was
concentrated in vacuo. The crude mixture was puri®ed
by silica gel chromatography (ethyl acetate:hexane,
1:10±1:5) to give 24 (315 mg, 40%) as an amorphous
1
solid. IR (neat): 2960, 2881, 1714, 1212, 1049, 933 cm
.
1H NMR (270 MHz) d: 0.63 (3H, s), 0.96 (3H, d,
J=5.9 Hz), 2.45 (1H, dd, J=11.5, 7.6 Hz), 3.45 (3H, s),
4.91 (2H, s). MS(EI) m/z: 432 (M+), 417 (M+-CH3),
389 (M+-CH3-CO), 387 (M+-CH2OCH3). HR-MS(EI)
m/z: 432.2120 (M+) (calcd for C20H30O3F6 432.2099).
1
(22a) IR (neat): 3452, 3308, 2932, 1472, 1427 cm 1. H
NMR (270 MHz) d: 1.07 (9H, s), 1.85±1.90 (2H, m),
1.94 (1H, t, J=2.7 Hz), 2.23±2.33 (2H, m), 4.06 (1H, m),
4.32 (1H. m), 5.03 (1H, d, J=10.6 Hz), 5.15 (1H, d,
J=17.2 Hz), 5.77 (1H, ddd, J=5.6, 10.6, 17.2 Hz), 7.43
(6H, m), 7.69 (4H, m). MS(FAB+) m/z: 379 (M++H),
361 (M+-H2O+H). HR-MS(FAB+) m/z: 379.2084
(M++H) (calcd for C24H31O2Si 379.2093).
Vinyl bromide (5). To a solution of hexamethyldisila-
zane (0.16 mL, 0.757 mmol) in toluene (1.2 mL) was
added n-BuLi (1.6 M in hexane, 0.39 mL, 0.624 mmol)
at 70 ꢀC. After stirring for 10 min, bromomethyl-
triphenylphosphonium bromide (338 mg, 0.775 mmol)
was added to the solution and the mixture was warmed
up to 0 ꢀC and stirred for 10 min. After cooling to
70 ꢀC, a solution of 24 (67 mg, 0.155 mmol) in toluene
(0.5 mL) was added dropwise to the mixture at the same
temperature. After stirring for 30 min, NH4Cl solution
was added and warmed up to room temperature. The
resulting mixture was extracted with ethyl acetate. The
organic layer was washed with brine, dried over MgSO4
and concentrated in vacuo. The residue was puri®ed by
silica gel chromatography (ethyl acetate: hexane, 1:10)
1
(22b) IR (neat): 3452, 3308, 2932, 1472, 1427cm 1. H
NMR (270MHz) d: 1.08 (9H, s), 1.74±1.90 (2H, m), 1.93
(1H, t, J=2.6 Hz), 2.23±2.45 (2H, m), 4.12 (1H, m), 4.38
(1H, m), 5.05 (1H, d, J=10.2 Hz), 5.21 (1H, d,
J=17.2 Hz), 5.81 (1H, ddd, J=5.6, 10.2, 17.2 Hz), 7.43
(6H, m), 7.69 (4H, m). MS(FAB+) m/z: 379 (M++H),
361 (M+-H2O+H). HR-MS(FAB+) m/z: 379.2105
(M++H) (calcd for C24H31O2Si 379.2093).
(3S,5R)-3-[(t-Butyldimethylsilyl)oxy]-5-[(t-butyldiphenyl-
silyl)oxy]-1-octen-7-yne (6a). To a solution of 22a

M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
2165
to give vinyl bromide 5 (27 mg, 34%) as a colorless oil
and starting material 24 (30 mg, 45% recovery). IR
(neat): 2951, 1467, 1377, 1213 cm
(270 MHz) d: 0.57 (3H, s), 0.94 (1H, d, J=6.3Hz), 2.86
(1H, m), 3.46 (3H, s), 4.92 (2H, s), 5.65 (1H, s). MS(EI) m/z:
508 (M+), 463 (M+-CH3OCH2). HR-MS(EI) m/z:
508.1406 (M+) (calcd for C21H31O2F6Br 508.1412).
(5E,7E)-(1S,3R)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (2e). A solution of
2a (50 mg, 9.53 mmol) in dichloromethane (3 mL) was
cooled to 15 ꢀC, and then liqui®ed SO2 (10 mL) was
added. The solution was stirred under re¯ux for 30 min.
The SO2 was distilled o€, and the residue was dried in
vacuo. The following puri®cation by silica gel chroma-
tography (ethyl acetate only) a€ord a mixture of (6S)
and (6R) SO2-adducts as an amorphous powder. The
SO2 adduct was dissolved in EtOH (5 mL), and NaHCO3
(100 mg) was added to the solution. The reaction mixture
was heated under re¯ux for 90 min. The solvent was
removed, and the crude product was puri®ed by silica gel
chromatography (ethyl acetate:hexane, 2:1) to give 2e
(40 mg, 80% from 2a) as an amorphous solid. UV
(EtOH): lmax 274 nM. 1H NMR (270 MHz) d: 0.57 (3H,
s), 0.95 (3H, d, J=6.7 Hz), 2.28 (1H, m), 2.87 (2H, m),
4.23 (1H, m), 4.50 (br s), 4.98 (1H, br s), 5.13 (1H, br s),
5.88 (1H, d, J=11.6 Hz), 6.58 (1H, d, J=11.6 Hz).
MS(EI) m/z: 524 (M+), 506 (M+-H2O), 488 (M+-
2H2O). HR-MS(EI) m/z: 524.2700 (M+) (calcd for
C27H38O3F6 524.2725).
1
.
1H NMR
(5Z,7E)-(1S,3S)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (2c). A solution of
.
(dba)3 Pd2 CHCl3 (4 mg, 3.86mmol), triphenylphosphine
(11 mg, 41.9 mmol) in triethylamine (0.4mL) toluene
(0.4 mL) was stirred at room temperature for 10min. A
solution of 5 (16 mg, 31.4 mmol) and 6a (20 mg, 40.6 mmol)
in toluene (0.4 mL) was added dropwise and heated under
re¯ux for 1 h. The reaction mixture was concentrated in
vacuo, and puri®ed by silica gel chromatography (ethyl
acetate: hexane, 1:20) to give silylated 3-epimer (24 mg).
Methansulfonic acid (0.1 mL) was added to a solution of
the product (24 mg) in methanol (5 mL) at room tem-
perature and stirred for 1 h. The reaction mixture was
poured into ice water and extracted with ethyl acetate.
The organic layer was washed with saturated aqueous
NaHCO3, brine, dried over MgSO4 and concentrated in
vacuo. The residue was puri®ed by silica gel chromato-
graphy (ethyl acetate:hexane, 3:2) to give 2c (9.0 mg,
55% from 5) as an amorphous solid. UV (EtOH): lmax
264 nM. 1H NMR (500 MHz) d: 0.55 (3H, s), 0.95 (3H, d,
J=6.4 Hz), 2.56 (1H, m), 2.85 (1H, m), 4.05 (1H, m), 4.30
(1H, m), 5.00 (1H, br s), 5.30 (1H, br s), 6.02 (1H, d,
J=11.3 Hz), 6.44 (1H, d, J=11.3 Hz). MS(EI) m/z: 524
(M+), 506 (M+-H2O), 491 (M+-CH3-H2O), 488 (M+-
2H2O), 473 (M+-2H2O-CH3). HR-MS(EI) m/z:
524.2687 (M+) (calcd for C27H38O3F6 524.2725).
Measurement of vitamin D3 receptor binding anity.22
Chick intestinal 1a,25-dihydroxyvitamin D3 receptor
was obtained from Yamasa Biochemical (Chiba, Japan)
and dissolved in 50mM Tris±HCl bu€er (pH 7.4) con-
taining 0.25 M sucrose, 25mM KCl, 5 mM MgCl2, 1 mM
EDTA and 12mM thioglycerol just before use. The
receptor solution (0.2 mL, 0.08mg protein) was incubated
with 1.8 nM [3H]-1a,25-dihydroxyvitamin D3 (180Ci/
nmol) and 1a,25-dihydroxyvitamin D3 or analogue at
various concentration for 24 h at 4 ꢀC. The bound and
free [3H]-1a,25-dihydroxyvitamin D3 were separated by
treatment with dextran-coated charcoal and centrifuged
at 3000 rpm for 15 min at 4 ꢀC. The radioactivity of
supernatant with ACS-II (Amersham, UK) was counted.
(5Z,7E)-(1R,3S)-26,26,26,27,27,27-Hexa¯uoro-9,10-seco-
5,7,10(19)-cholestatriene-1,3,25-triol (2d). A solution of
.
(dba)3 Pd2 CHCl3 (4 mg, 3.86mmol), triphenylphosphine
(11 mg, 41.9 mmol) in triethylamine (0.4mL) toluene
(0.4 mL) was stirred at room temperature for 10 min. A
solution of 5 (20.1 mg, 39.3mmol) and 6b (22 mg,
44.6 mmol) in toluene (0.4 mL) was added dropwise and
heated under re¯ux for 1 h. The reaction mixture was
concentrated in vacuo, and puri®ed by silica gel chro-
matography (ethyl acetate: hexane, 1:20) to give sily-
lated 1,3-epimer (24 mg). Methansulfonic acid (0.1 mL)
was added to a solution of the product (24 mg) in
methanol (3.0 mL) at room temperature, and stirred for
1 h. The reaction mixture was poured into ice water and
extracted with ethyl acetate. The organic layer was
washed with saturated aqueous NaHCO3, brine, dried
over MgSO4 and concentrated in vacuo. The residue
was puri®ed by silica gel chromatography (ethyl acet-
ate:hexane, 3:2) to give 2d (10.1 mg, 49% from 5) as an
References and Notes
1. Ettinger, R. A.; DeLuca, H. F. Advances in Drug Research
1996, 28, 269.
2. DeLuca, H. F.; Burmester, J.; Darwish, H.; Krisinger, J.
Comprehensive Medical Chemistry, Pergamon, New York
1990, vol. 3, 1129.
3. Bouillon, R.; Okamura, W. H.; Norman, A. W. Endocr.
Rev. 1995, 16, 200.
4. Muralidharan, K. R.; de Lera, A. R.; Isae€, S. D.; Nor-
man, A. W.; Okamura, W. H. J. Org. Chem. 1993, 58, 1895.
5. Konno, K.; Maki, S.; Fujishima, T.; Liu, Z.; Miura, D.;
Chokki, M.; Takayama, H. Bioorg. Med. Chem. Lett. 1998, 8,
151.
6. Fujishima, T.; Konno, K.; Nakagawa, K.; Kurobe, M.;
Okano, T.; Takayama, H. Bioorg. Med. Chem. 2000, 8, 123.
7. Wecksler, W. R.; Norman, A. W. Steroids 1980, 35, 419.
8. VanAlstyne, E. M.; Norman, A. W.; Okamura, W. H. J.
Am. Chem. Soc. 1994, 116, 6207.
9. Inaba, M.; Okuno, S.; Nishizawa, Y.; Imanishi, Y.; Katsu-
mata, T.; Sugata, I.; Mori, H. Biochemical Pharmacology
1993, 45, 2331.
10. Honda, A.; Nakashima, N.; Shida, Y.; Mori, Y.; Nagata,
A.; Ishizuka, S. Biochem. J. 1993, 295, 509.
1
amorphous solid. H NMR (500MHz) d: 0.55 (3H, s),
0.94 (3H, d, J=6.7Hz), 2.30 (1H, dd, J=13.3, 7.5 Hz),
2.62 (1H, dd, J=13.3, 4.0 Hz), 2.83 (1H, dd, J=11.8,
4.0 Hz), 4.20 (1H, m), 4.42 (1H, m), 5.01 (1H, br s), 5.32
(1H, m), 6.01 (1H, d, J=11.3 Hz), 6.39 (1H, d,
J=11.3 Hz). UV (EtOH): lmax 264 nM. MS(EI) m/z: 524
(M+), 506 (M+-H2O), 491 (M+-CH3), 488 (M+-2H2O),
473 (M+-CH3-H2O). HR-MS(EI) m/z: 524.2691 (M+)
(calcd for C27H38O3F6 524.2725).
11. Trost, B. M.; Dumas, J.; Villa, M. J. Am. Chem. Soc.
1992, 114, 9836.

2166
M. Ikeda et al. / Bioorg. Med. Chem. 8 (2000) 2157±2166
12. Ikeda, M.; Matsumura, H.; Sawada, N.; Hashimoto, K.;
Tanaka, T.; Noguchi, T.; Hayashi, M. Bioorg. Med. Chem.
2000, 8, 1809.
18. Fujishima, T.; Liu, Z.; Miura, D.; Chokki, M.; Ishizuka,
S.; Konno, K.; Takayama, H. Bioorg. Med. Chem. Lett. 1998,
8, 2145.
13. Ando, K.; Kondo, F.; Koike, F.; Takayama, H. Chem.
Pharm. Bull 1992, 40, 1662.
14. Grith, W. P.; Ley, S. V.; Whitcombe, G. P.; White, A.
D. J. Chem. Soc., Chem. Commun. 1987, 1625.
15. Barton, D. H. R.; McCombie, S. W. J. Chem. Soc., Perkin
Trans. 1 1975, 1574.
19. Brase, S.; Schomenauer, S.; McGan, G.; Stolle, A.;
Meijere, A. D. Chem. Eur. J. 1996, 2, 545.
20. Rychnovsky, S. D.; Skalitzky, D. J. Tetrahedron Lett.
1990, 31, 945.
21. Evans, D. A.; Rieger, D. L.; Gage, J. R. Tetrahedron Lett.
1990, 31, 7099.
16. Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4156.
17. Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113,
7277.
22. Seino, Y.; Yamaoka, K.; Ishida, M.; Yabuuchi, H.; Ichi-
kawa, M.; Ishige, H.; Yoshino, H.; Avioli, L. V. Calci®ed
Tissue International 1982, 34, 265.