Synthesis of putative metabolites of 1α,25-dihydroxy-2β-(3-hydroxypropoxy)vitamin D3 (ED-71)

Article abstract of DOI:10.1016/j.steroids.2005.11.001

1α,25-Dihydroxy-2β-(3-hydroxypropoxy)vitamin D₃ (ED-71), an analog of active vitamin D₃, 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] is under phase III clinical trials in Japan for the treatment of osteop

Full text of DOI:10.1016/j.steroids.2005.11.001

steroids 7 1 ( 2 0 0 6 ) 529–540
available at www.sciencedirect.com
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Synthesis of putative metabolites of
1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71)
Yoshiyuki Onoa,∗, Hiroyoshi Watanabea, Ikuo Tairab, Keisuke Takahashib,
Jun Ishiharab, Susumi Hatakeyamab, Noboru Kuboderaa
a
Chemistry Research Department I, Chugai Pharmaceutical Co., Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan
Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki 852-8521, Japan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
1␣,25-Dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71), an analog of active vitamin D3,
1␣,25-dihydroxyvitamin D3 [1,25(OH)2D3] is under phase III clinical trials in Japan for the
treatment of osteoporosis and bone fracture prevention. Since ED-71 has a substituent at
the 2␤-position of the A-ring, it is recognized that the metabolic pathway of ED-71 might be
more complicated than 1,25(OH)2D3 because of metabolism at the 2␤-position substituent in
addition to the inherent metabolism of the side chain. To clarify the metabolism of hydroxy-
propoxy substituent of the 2␤-positon and a combination of metabolism between side chain
and 2␤-positon, four putative metabolites of ED-71 have been prepared as authentic sam-
ples. The metabolites at the 2␤-positon, the methyl ester derivative considered as an ester
standard of the oxidized metabolite and the tetraol derivative as the truncated metabolite
were synthesized from ␣-epoxide, a key intermediate of ED-71 synthesis. The combination
metabolites between side chain and 2␤-positon, the 24(S)- and 24(R)-pentaols were synthe-
sized using Trost’s convergent method.
Received 28 June 2005
Received in revised form 10 October
2005
Accepted 4 November 2005
Published on line 25 April 2006
Keywords:
Active vitamin D3
1␣,25-Dihydroxyvitamin D3
1␣,25-Dihydroxy-2␤-(3-
hydroxypropoxy)vitamin
D3
© 2006 Elsevier Inc. All rights reserved.
ED-71
Putative metabolites
Convergent method
1.
Introduction
24-hydroxylated 1,25(OH)2D3 [8–12]. On the assumption that
hydroxylation pathway of ED-71 (1) and 2 would be similar, we,
1␣,25-Dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71, 1)
[1–5], an analog of active vitamin D3, 1␣,25-dihydroxyvitamin
D3 [1,25(OH)2D3, 2] [6] containing a hydroxypropoxy sub-
stituent at the 2␤-position of 2, has potent effects on bone ther-
apy. ED-71 is currently under phase III clinical trials in Japan as
a promising candidate for the treatment of osteoporosis and
bone fracture prevention [7] (Fig. 1). During the development
of ED-71 (1), it was necessary to synthesize possible metabo-
lites of 1 for pharmacokinetic and metabolic studies. It is well
known that 1,25(OH)2D3 (2) is hydroxylated at the 24-position
of the side chain as a first step of its metabolism to produce
therefore, reported the synthesis of 24-hydroxylated ED-71 in
24(S) and 24(R) forms (3 and 4) in our previous paper [13]. In
the case of 1,25(OH)2D3 (2), 24-hydroxylated 1,25(OH)2D3 is fur-
ther hydroxylated at the 23- and 26-positions of the side chain
and oxidized to a keto-alcohol, lactone (calcitriol lactone), or
carboxylic acid (calcitroic acid) [8–12]. Since ED-71 (1) has a
substituent at the 2␤-position of the A-ring, it is recognized
that the metabolic pathway of 1 might be more complicated
than 1,25(OH)2D3 (2) because of metabolism at the 2␤-position
substituent in addition to the inherent metabolism of the side
chain. In fact, in our preliminary metabolic studies of ED-71 (1),
∗
Corresponding author. Tel.: +81 550 87 6742; fax: +81 550 87 5329.
E-mail address: onoysy@chugai-pharm.co.jp (Y. Ono).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.11.001

530
steroids 7 1 ( 2 0 0 6 ) 529–540
CDCl3 as a solvent. Chemical shifts are reported in parts per
million (ppm) downfield from tetramethylsilane or calibrated
from CHCl3. Mass spectra (MS) were measured with JEOL JMS-
DX303 (electron ionization method (EI)), JEOL JMS-700N (EI),
Shimadzu GCMS QP-1000 (EI), and Waters micromassZQ (elec-
trospray ionization method (ESI)) instruments. High resolution
mass spectra (HRMS) were recorded on JEOL JMS-AX500 (EI)
and VG Auto Spec Q (ESI) instruments. Ultra violet (UV) spec-
tra were obtained with Shimadzu UV-1600PC instrument using
ethanol as a solvent. All reactions were carried out under an
atmosphere of argon or nitrogen unless otherwise noted. All
extracts were dried over MgSO4 and evaporated under reduced
pressure with a rotary evaporator. Chromatographic purifica-
tion was carried out with Merck silica gel 60 (column) or Merck
silica gel 60 PF254 (preparative TLC).
Fig. 1 – Structures of ED-71 and 1,25(OH)2D3.
24-hydroxylated ED-71 (3 and 4) were found as metabolites of
1 which were accompanied by several other metabolic asso-
ciates that may be derived from metabolism of the hydroxy-
propoxy substituent of the 2␤-position and a combination of
metabolism between the side chain and the 2␤-position. In
order to assist in the confirmation and structure elucidation
of metabolic derivatives of ED-71 (1), the synthesis of putative
metabolites of 1 was undertaken.
In this paper, we describe the synthesis of postulated
metabolites of ED-71 (1), namely the methyl ester derivative
(6) as an ester standard of the oxidized metabolite (5) at the
2␤-position, the tetraol derivative (7) as a truncated metabo-
lite at the 2␤-position, and pentaols (8 and 9) in 24(S) and 24
(R) forms that arise from the combination metabolism of the
side chain and the 2␤-position (Fig. 2).
2.1.
2ˇ-(2-Acetyloxyethoxy)-1˛,3ˇ,25-
trihydroxycholesta-5,7-diene (11)
To a mixture of 10 (306 mg, 0.643 mmol), 4-dimethylamino-
pyridine (DMAP) (10 mg), and pyridine (0.60 ml) in CH2Cl2
(30 ml), acetic anhydride (0.150 ml, 1.59 mmol) was added at
0 ◦C and the mixture was stirred at 0 ◦C for 1.5 h, poured
into dilute hydrochloric acid, and extracted with CH2Cl2.
The extract was washed with saturated NaHCO3, dried, and
evaporated. The residue was chromatographed on column
(CH2Cl2/EtOH = 50/3) to give 11 (215 mg, 65%) with a recovery of
10 (105 mg, 34%); IR (neat): 3415 (br), 2930, 1740 cm−1 1H NMR
;
ı: 0.63 (3H, s), 0.96 (3H, d, J = 6.3 Hz), 1.05 (3H, s), 1.12 (6H, s),
2.07 (3H, s), 3.64–3.77 (2H, m), 3.84 (1H, brs), 3.89–4.00 (2H, m),
4.17–4.34 (2H, m), 5.33–5.41 (1H, m), 5.70 (1H, d, J = 3.6 Hz); UV
ꢀmax: 293, 282, 271 nm; MS (EI (m/z)): 518 (M+), 87 (100%).
2.
Experimental
2.2.
2ˇ-(2-Acetyloxyethoxy)-1˛,3ˇ,25-
Optical rotations were measured with JASCO DIP-370
polarimeter. Infrared (IR) spectra were obtained using JASCO
FT/IR-230 and HORIBA FT-730 spectrophotometers. 1H and
13C NMR spectra were recorded on VARIAN Gemini-300, VAR-
IAN Unity Plus 500, and JEOL JNM-EX270 spectrometers using
tris(triethylsilyloxy)cholesta-5,7-diene (12)
To a mixture of 11 (260 mg, 0.502 mmol) and 2,6-lutidine
(0.887 ml, 7.53 mmol) in CH2Cl2 (30 ml), triethylsilyl trifluo-
romethanesulfonate (TESOTf) (1.14 ml, 5.02 mmol) was added
Fig. 2 – Putative metabolic pathway of ED-71.

steroids 7 1 ( 2 0 0 6 ) 529–540
531
at 0 ◦C and the mixture was stirred at 0 ◦C for 1.5 h and evap-
2.6.
PTAD adduct of 2ˇ-(2-cyanoethoxy)-1˛,3ˇ,25-
orated. The residue was chromatographed on column (hex-
ane/AcOEt = 20/1) to give 12 (327 mg, 76%) as a colorless oil; IR
(neat): 2950, 1750 cm−1; 1H NMR ı: 0.52–0.72 (21H, m), 0.88–1.03
(33H, m), 1.18 (6H, s), 2.04 (3H, s), 3.58 (1H, brs), 3.63–3.74
(1H, m), 3.75 (1H, d, J = 3.6 Hz), 3.94–4.11 (2H, m), 4.21 (2H, t,
J = 5.0 Hz), 5.28–5.35 (1H, m), 5.57–5.62 (1H, m); UV ꢀmax: 293,
282, 271 nm; MS (EI (m/z)): 860 (M+), 87 (100%).
tris(triethylsilyloxy)cholesta-5,7-diene (16)
To a solution of 15 (180 mg, 0.163 mmol) in dimethyl sulfoxide
(DMSO) (26 ml) and THF (15 ml), a solution of sodium cyanide
(NaCN) (8.0 mg, 0.163 mmol) in DMSO (8 ml) was added at room
temperature. The mixture was stirred at 50 ◦C for 3.5 h, poured
into ice, and extracted with hexane/AcOEt (10:1). The extract
was washed with H2O, dried, and evaporated. The residue was
chromatographed on preparative TLC (hexane/AcOEt = 10/1)
to give 16 (141 mg, 87%) as a colorless oil; IR (neat): 2955,
2.3.
2ˇ-(2-Hydroxyethoxy)-1˛,3ˇ,25-
tris(triethylsilyloxy)cholesta-5,7-diene (13)
2250, 1750, 1700 cm−1 1H NMR ı: 0.51–0.75 (18H, m), 0.80 (3H,
;
To a solution of 12 (327 mg, 0.380 mmol) in THF (5 ml), 1N
KOH in MeOH (10 ml) was added at room temperature and
the mixture was stirred at room temperature for 1 h. To the
mixture, acetic acid (0.15 ml) was added and the mixture was
poured into water and extracted with hexane/AcOEt (1:1). The
extract was washed with saturated NaHCO3 and brine, dried,
and evaporated. The residue was chromatographed on column
(hexane/AcOEt = 12/1) to give 13 (176 mg, 56%) as a colorless
s), 0.89–1.02 (30H, m), 1.05 (3H, s), 1.19 (6H, s), 2.98 (1H, dd,
J = 13.9, 4.6 Hz), 3.66 (1H, t, J = 3.3 Hz), 3.71–3.79 (1H, m), 3.93
(1H, d, J = 3.0 Hz), 4.06–4.13 (1H, m), 4.90–4.98 (1H, m), 6.23 (1H,
d, J = 8.3 Hz), 6.35 (1H, d, J = 8.3 Hz), 7.22–7.49 (5H, m); UV ꢀmax
258, 204 nm; MS (EI (m/z)): 827 ([M − PTAD]+), 75 (100%).
:
2.7.
PTAD adduct of 2ˇ-(2-methoxycarbonylethyloxy)-
1˛,3ˇ,25-trihydroxycholesta-5,7-diene (17)
oil; IR (neat): 3465 (br), 2955 cm−1 1H NMR ı: 0.51–0.71 (21H,
;
m), 0.89–1.07 (33H, m), 1.19 (6H, s), 3.48–3.71 (4H, m), 3.76 (1H,
brs, J = 3.6 Hz), 3.82–3.91 (1H, m), 4.02–4.13 (1H, m), 5.30–5.36
(1H, m), 5.61 (1H, m); UV ꢀmax: 293, 282, 271 nm; MS (EI (m/z)):
818 (M+), 75 (100%).
To a solution of 16 (180 mg, 0.163 mol) in Et2O (6.6 ml),
methanolic hydrochloric acid (concd. HCl/MeOH) (3.3 ml) was
added at room temperature and the mixture was stirred at
room temperature for 6.5 h and evaporated. The residue was
chromatographed on preparative TLC (CH2Cl2/EtOH = 10/1) to
give 17 (13 mg, 25%) as a colorless oil; IR (neat): 3450 (br), 2950,
2.4.
PTAD adduct of 2ˇ-(2-hydroxyethoxy)-1˛,3ˇ,25-
1740, 1690 cm−1 1H NMR ı: 0.80 (3H, s), 0.96 (3H, d, J = 6.6 Hz),
;
tris(triethylsilyloxy)cholesta-5,7-diene (14)
1.00 (3H, s), 1.21 (6H, s), 2.59 (2H, t, J = 5.6 Hz), 3.08–3.18 (1H, m),
3.70 (3H, s), 3.78–3.98 (4H, m), 6.20 (1H, d, J = 8.3 Hz), 6.40 (1H,
d, J = 8.3 Hz), 7.25–7.42 (5H, m); MS (EI (m/z)): 518 ([M − PTAD]+),
60 (100%).
To a solution of 13 (173 mg, 0.211 mmol) in CH2Cl2 (5 ml), 4-
phenyl-1,2,4-triazoline-3,5-dione (PTAD) (36.9 mg, 0.211 mmol)
was added at room temperature and the mixture was stirred at
room temperature for 30 min and evaporated. The residue was
chromatographed on preparative TLC (hexane/AcOEt = 1/3) to
give 14 (173 mg, 82%) as a yellow oil; IR (neat): 3580 (br),
2.8.
2ˇ-(2-Methoxycarbonylethyloxy)-1˛,3ˇ,25-
trihydroxycholesta-5,7-diene (18)
2950, 1750, 1695 cm−1 1H NMR ı: 0.51–0.82 (21H, m), 0.89–1.01
;
(30H, m), 1.05 (3H, s), 1.19 (6H, s), 3.00 (1H, dd, J = 13.9, 4.6 Hz),
3.58–3.71 (4H, m), 3.83–3.89 (2H, m), 3.91 (1H, d, J = 3.0 Hz),
4.90–4.99 (1H, m), 6.23 (1H, d, J = 8.3 Hz), 6.36 (1H, d, J = 8.3 Hz),
7.23–7.49 (5H, m); UV ꢀmax: 258, 218, 206 nm; MS (EI (m/z)): 818
([M − PTAD]+), 103 (100%).
A
solution of 17 (18 mg, 0.026 mmol) in 1,3-dimethyl-2-
imidazolidinone (DMI) (3 ml) was stirred at 140 ◦C for 1 h.
The mixture was poured into H2O and extracted with hex-
ane/AcOEt (1:4). The extract was washed with H2O, dried, and
evaporated. The residue was chromatographed on prepara-
tive TLC (EtOH/AcOEt = 1/100) to give 18 (8 mg, 59%) as a white
powder; IR (neat): 3420 (br), 2960, 1725 cm−1 1H NMR ı: 0.63
;
2.5.
PTAD adduct of 2ˇ-(2-iodoethoxy)-1˛,3ˇ,25-
tris(triethylsilyloxy)cholesta-5,7-diene (15)
(3H, s), 0.96 (3H, d, J = 6.6 Hz), 1.00 (3H, s), 1.20 (6H, s), 2.61 (2H,
t, J = 5.8 Hz), 3.64–3.85 (4H, m), 3.74 (3H, s), 3.90–4.01 (1H, m),
5.31–5.39 (1H, m), 5.62–5.69 (1H, m); UV ꢀmax: 293, 282, 271 nm;
MS (EI (m/z)): 518 (M+), 60 (100%).
To a mixture of 14 (173 mg, 0.174 mmol), triphenyl phosphine
(Ph3P) (123 mg, 0.469 ml), and imidazole (32 mg, 0.470 mmol)
in CH2Cl2 (7 ml), iodide (75.2 mg, 0.296 mmol) was added at
room temperature and the mixture was stirred at room tem-
perature for 80 min, poured into 10% Na2S2O3, and extracted
with CH2Cl2. The extract was dried and evaporated. The
residue was chromatographed on preparative TLC (hex-
ane/AcOEt = 10/1) to give 15 (188 mg, 98%) as a colorless oil;
2.9.
2ˇ-(2-Methoxycarbonylethoxy)-1˛,3ˇ,25-
trihydroxy-9,10-secocholesta-5,7,10(19)-triene (6)
A solution of 18 (8.0 mg, 0.015 mmol) in EtOH (200 ml) was irra-
diated using a 400 W high pressure mercury lamp with Vycor®
filter at 0 ◦C for 95 s and the mixture was refluxed for 2 h and
evaporated. The residue was chromatographed on preparative
TLC (EtOH/AcOEt = 1/100) to give 6 (2.0 mg, 25%) as a color-
less oil; 1H NMR ı: 0.55 (3H, s), 0.93 (3H, d, J = 6.3 Hz), 1.22 (6H,
s), 2.66 (2H, t, J = 5.1 Hz), 3.26 (1H, dd, J = 9.1, 2.8 Hz), 3.74–3.83
(1H, m), 3.92–4.02 (1H, m), 4.24–4.32 (2H, m), 5.08 (1H, s), 5.53
IR (neat): 2980, 1765, 1715 cm−1 1H NMR ı: 0.52–0.73 (18H,
;
m), 0.81 (3H, s), 0.88–1.01 (30H, m), 1.07 (3H, s), 1.19 (6H, s),
2.97 (1H, dd, J = 13.9, 4.6 Hz), 3.20–3.34 (2H, m), 3.66 (1H, brt,
J = 3.3 Hz), 3.69–3.81 (1H, m), 3.97 (1H, d, J = 3.3 Hz), 4.11–4.22
(1H, m), 4.86–4.96 (1H, m), 6.23 (1H, d, J = 8.3 Hz), 6.35 (1H, d,
J = 8.3 Hz), 7.22–7.49 (5H, m); UV ꢀmax: 256, 204 nm; MS (EI (m/z)):
928 ([M − PTAD]+), 75 (100%).
(1H, s), 6.06 (1H, d, J = 11.4 Hz), 6.36 (1H, d, J = 11.4 Hz); UV ꢀmax
:

532
steroids 7 1 ( 2 0 0 6 ) 529–540
264 nm, ꢀmin: 229 nm; MS (EI (m/z)): 518 (M+), 60 (100%); MS
(ESI): 536([M + NH4]+), 1054 ([2M + NH4]+); HRMS (ESI) calcd for
C31H54NO6 [(M + NH4)+]: 536.3951, found: 536.3937.
for 2 h and evaporated. The residue was chromatographed on
preparative TLC (CH2Cl2/EtOH = 10/1) to give 7 (0.48 mg, 5%) as
a white powder; 1H NMR ı: 0.55 (3H, s), 0.94 (3H, d, J = 5.9 Hz),
1.22 (6H, s), 2.49–2.55 (2H, m), 2.78–2.83 (1H, m), 3.51 (1H, dd,
J = 9.1, 2.8 Hz), 4.15–4.15 (2H, m), 5.09 (1H, s), 5.46 (1H, s), 6.02
(1H, d, J = 11.4 Hz), 6.36 (1H, d, J = 11.4 Hz); UV ꢀmax: 264 nm,
ꢀmin 226 nm; MS (EI (m/z)): 432 (M+), 324 (100%); MS (ESI): 450
([M + NH4]+), 882([2M + NH4]+), 431 ([M − H]−), 863 ([2M − H]−);
HRMS (ESI) calcd for C27H44NaO4 [(M + Na)+]: 455.3137, found:
455.3127.
2.10. PTAD adduct of 3-acetoxy-1˛,2˛-epoxy-25-
hydroxycholesta-5,7-diene (20)
To a solution of 19 (100 mg, 0.171 mmol) in pyridine (3 ml),
acetic anhydride (0.323 ml, 3.42 mmol) was added at room
temperature and the mixture was stirred at 50 ◦C for 16 h
and extracted with AcOEt. The extract was washed with
1N hydrochloric acid (HCl) and brine, dried, and evapo-
rated. The residue was chromatographed on preparative TLC
(CH2Cl2/EtOH = 10/1) to give 20 (88 mg, 82%) as a colorless oil;
2.14. (1S,2S)-1,2-Bis((R)-2,2-dimethyl-1,3-
dioxolan-4-yl)ethane-1,2-dipivalate (25)
IR (neat): 3500 (br), 3000, 2900, 1760, 1700 cm−1
;
1H NMR ı: 0.90
To a solution of 24 (13.6 g, 51.8 mmol) in pyridine (52 ml),
pivaloyl chloride (tert-BuCOCl) (16 ml, 129.5 mmol) and DMAP
(0.63 g, 5.5 mmol) were added at room temperature and the
mixture was refluxed for 24 h. To the mixture, MeOH (6.3 ml)
was added and the mixture was filtrated through Celite® and
evaporated. The residue was extracted with AcOEt. The extract
was washed with cold H2O, dried, and evaporated. The residue
was recrystallized from AcOEt to give 25 (16 g, 72%) as colorless
crystals; [˛]2D0 + 33.8 (c 1.08, CHCl3); IR (film) 1735, 1479, 1371,
(3H, s), 0.95 (3H, d, J = 5.9 Hz), 1.10 (3H, s), 1.21 (6H, s), 2.10 (3H,
s), 2.67–2.75 (1H, m), 3.35–3.44 (1H, m), 5.92–5.97 (1H, m), 6.20
(1H, d, J = 8.5 Hz), 6.45 (1H, d, J = 8.5 Hz), 7.27–7.47 (5H, m); MS
(EI (m/z)): 454 ([M − PTAD]+), 364 (100%).
2.11. 3ˇ-Acetoxy-1˛,2ˇ,
25-trihydroxycholesta-5,7-diene (22)
To a solution of 20 (88 mg, 0.139 mmol) in THF (4 ml), boron
trifluoride-diethyl etherate (BF3·OEt2) (0.0159 ml, 0.139 mmol)
was added at room temperature and the mixture was stirred
at 60 ◦C for 16 h and extracted with AcOEt. The extract was
washed with brine, dried, and evaporated. The residue was
chromatographed on preparative TLC (CH2Cl2/EtOH = 10/1).
The resultant 21 was dissolved in DMI (6.5 ml) and the mixture
was stirred at 140 ◦C for 2 h and extracted with hexane/AcOEt
(1:100). The extract was washed with H2O, dried, and evapo-
rated. The residue was chromatographed on preparative TLC
(CH2Cl2/EtOH = 10/1) to give 22 (29 mg, 44%) as a colorless oil;
IR (neat): 3450 (br), 2960, 2900, 1730, 1390, 1260, 1060, 930,
1228, 1155, 1074 cm−1 1H NMR ı: 1.23 (9H, s), 1.31 (3H, s), 1.38
;
(3H, s), 3.77 (1H, dd, J = 7.8, 6.3 Hz), 3.9 (1H, dd, J = 7.8, 6.3 Hz),
4.09 (1H, q, J = 6.3 Hz), 5.31 (1H, d, J = 6.0 Hz); 13C NMR ı: 25.2,
26.5, 27.3, 39.1, 66.2, 71.4, 74.5, 109.4, 117.0; HRMS (EI) calcd for
C
21H35O8 [(M − Me)+]: 415.2332, found: 415.2312.
2.15. (2R,3R,4S)-5-((R)-2,2-Dimethyl-1,3-
dioxolan-4-yl)ethane-3,4-dipivaloxy-1,2-diol (26)
To a solution of 25 (10.6 g, 24.6 mmol) in THF (100 ml), 1N
HCl (98 ml) was added at 0 ◦C and the mixture was stirred at
room temperature for 20 h. To the mixture, NaHCO3 (10.5 g)
was added to be neutralized. The mixture was evaporated.
The residue was extracted with CH2Cl2. The extract was dried
and evaporated. The residue was chromatographed on col-
umn (CHCl3/MeOH = 20/1) to give 26 (5.3 g, 50%) and 27 (2.9 g,
34%) as a clear oil respectively with a recovery of 25 (1.5 g,
16%); [˛]2D0 + 42.7 (c 1.01, CHCl3); IR (film) 3498, 1745, 1481, 1371,
750 cm−1 1H NMR ı: 0.62 (3H, s), 0.95 (3H, d, J = 5.9 Hz), 1.01
;
(3H, s), 1.22 (6H, s), 2.10 (3H, s), 3.77–3.84 (1H, m), 4.16–4.23 (1H,
m), 5.11–5.19 (1H, m), 5.32–5.40 (1H, m), 5.65–5.73 (1H, m); UV
ꢀmax: 293, 281, 270 nm; MS (EI (m/z)): 456 (M+), 412 (100%).
2.12. 1˛,2ˇ,3ˇ,25-Tetahydroxycholesta-5,7-diene (23)
1174, 1070 cm−1 1H NMR ı: 1.24 (9H, s), 1.25 (9H, s), 1.31 (3H,
;
To a solution of 22 (29 mg, 0.061 mmol) in THF (10 ml), lithium
aluminum hydride (LiAlH4) (2.3 mg, 0.122 mmol) was added
at room temperature and the mixture was refluxed for 1 h
and extracted with AcOEt. The extract was washed with 1N
NaOH and brine, dried, and evaporated. The residue was chro-
matographed on preparative TLC (CH2Cl2/EtOH = 10/1) to give
23 (9 mg, 34%) as a white powder; IR (neat): 3450 (br), 2950, 2870,
s), 1.40 (3H, s), 2.66 (1H, d, J = 6.3 Hz), 2.45 (2H, m), 3.6 (2H, m),
3.77 (1H, dd, J = 8.4, 6.0 Hz), 3.96 (1H, t, J = 8.4 Hz), 4.11 (1H, q,
J = 6.3 Hz), 5.11 (1H, d, J = 9.6 Hz), 5.29 (1H, d, J = 7.2 Hz); 13C NMR
ı: 25.2, 26.6, 27.2, 39.2, 39.2, 62.6, 66.6, 69.1, 70.9, 71.6, 73.9, 109.6,
178.1, 179.0; HRMS (EI) calcd for C18H31O8 [(M − Me)+]: 375.2019,
found: 375.2008.
1270, 1140, 1050, 820 cm−1
;
1H NMR ı: 0.64 (3H, s), 0.96 (3H, d,
2.16. (1S,2S)-1-((R)-2-Ethoxy-1,3-dioxolan-4-yl)-2-
((R)-2,2-dimethyl-1,3-dioxolan-4-yl)ethane-1,2-dipivalate
(28)
J = 6.6 Hz), 1.22 (6H, s), 2.10 (3H, s), 3.75–4.10 (1H, m), 5.35–5.40
(1H, m), 5.70–5.75 (1H, m); UV ꢀmax: 294, 281, 271 nm; MS (EI
(m/z)): 432 (M+), 324 (100%).
To a solution of 26 (4.76 g, 12.2 mmol) in CH2Cl2 (61 ml),
triethyl orthoformate (HC(OEt)3) ((3.0 ml, 18.3 mmol) and
p-toluenesulfonic acid monohydrate (TsOH·H2O) (16.2 mg,
0.085 mmol) were added at 0 ◦C and the mixture was stirred
at room temperature for 3 h. To the mixture, triethylamine
(NEt3) (5.7 ml) was added and the mixture was extracted
with CH2Cl2. The extract was washed with H2O, dried, and
2.13. 1˛,2ˇ,3ˇ,25-Tetrahydroxy-9,10-secochoresta-
5,7,10(19)-triene (7)
A solution of 23 (150 mg, 0.20 mmol) in EtOH (230 ml) was
irradiated using a 400 W high pressure mercury lamp with
Vycor® filter at 0 ◦C for 130 s and the mixture was refluxed

steroids 7 1 ( 2 0 0 6 ) 529–540
533
evaporated. The residue was chromatographed on column
(hexane/AcOEt = 20/1) to give 28 (4.81 g, 88%) as a colorless oil;
[˛]1D9 + 38.8 (c 1.05, CHCl3); IR (film) 3506, 1729, 1479, 1376, 1277,
44.3, 49.4, 71.2, 72.7, 118.5, 132.0, 176.8, 176.9; HRMS (EI) calcd
for C16H26O5 (M+): 298.1781, found: 298.1752.
1151, 1066 cm−1 1H NMR ı: 1.23 (9H, s), 1.24 (9H, s), 1.30 (3H,
;
2.20. (4R,5R,6R)-5,6-Dipivaloxy-1-(trimethylsilyl)oct-
7-en-1-yn-4-ol (32)
s), 1.39 (3H, s), 3.60 (3H, m), 3.77 (2H, m), 3.91 (4H, m), 4.08 (2H,
m), 5.40 (2H, m), 5.78 (1H, s); 13C NMR ı: 14.0, 14.9, 22.6, 25.1,
26.4, 27.0, 27.1, 31.5, 38.9, 60.4, 60.7, 65.2, 65.9, 66.1, 70.9, 71.3,
71.9, 73.7, 74.1, 74.3, 109.2, 109.4, 114.9, 115.4, 176.8; HRMS (EI)
calcd for C19H31O9 [(M − C3H7)+]: 403.1968, found: 403.1963.
To a solution of trimethylsilylacetylene (2.5 ml, 17.8 mmol) in
THF (33 ml), n-butyl lithium (n-BuLi) (1.58 M in hexane; 10.8 ml,
17 mmol) was added at −78 ◦C and the mixture was stirred at
−78 ◦C for 30 min. To the mixture, BF3·OEt2 (2.2 ml, 17 mmol)
was added at −78 ◦C and the mixture was stirred at −78 ◦C for
30 min. To the mixture, a solution of 31 (1.04 g, 3.5 mmol) in
THF (97 ml) cooled to −70 ◦C was added at −78 ◦C for 20 min.
The mixture was warmed to −30 ◦C, stirred at −30 ◦C for 3 h,
quenched by addition of H2O (1.0 ml), warmed to room tem-
perature gradually, and evaporated. The residue was extracted
with CH2Cl2. The extract was washed with H2O, dried, and
evaporated. The residue was chromatographed on column
(hexane/AcOEt = 30/1 to 15/1) to give 32 (1.17 g, 85%) as a col-
orless oil; [˛]2D4 + 61.0 (c 1.03, CHCl3); IR (film) 3517, 2177, 1737,
2.17. (3S,4R)-4-((R)-2,2-Dimethyl-1,3-dioxolan-
4-yl)-3,4-dipivaloxy-1-butene (29)
A solution of 28 (4.53 g, 10.1 mmol) in acetic anhydride (51 ml)
was refluxed for 16 h and the mixture was evaporated. To the
residue, saturated NaHCO3 was added to be neutralized. The
mixture was extracted with CH2Cl2. The extract was dried
and evaporated. The residue was chromatographed on column
(hexane/AcOEt = 6/1) to give 29 (3.35 g, 93%) as a colorless oil;
[˛]2D1 + 50.0 (c 1.05, CHCl3); IR (film) 1737, 1479, 1371, 1276, 1139,
1064 cm−1
;
1H NMR ı: 1.16 (9H, s), 1.20 (9H, s), 1.26 (3H, s), 1.34
1479, 1278, 1141, 1034 cm−1 1H NMR ı: 0.16 (9H, s), 1.19 (9H, s),
;
(3H, s), 3.74 (1H, dd, J = 8.1, 6.6 Hz), 3.92 (1H, dd, J = 8.1, 6.3 Hz),
4.14 (1H, q, J = 6.3 Hz), 5.15 (1H, s), 5.44 (1H, m), 5.68 (1H, ddd,
J = 15.9, 10.8, 5.4 Hz); 13C NMR ı: 25.2, 26.6, 27.2, 38.9, 39.0, 66.1,
72.4, 72.5, 74.0, 109.3, 117.6, 132.6, 176.8, 177.1; HRMS (EI) calcd
for C19H32O6 (M+): 356.2199, found: 356.2201.
1.26 (9H, s), 2.22 (1H, dd, J = 17.1, 7.2 Hz), 2.31 (1H, dd, J = 16.8,
3.9 Hz), 2.65 (1H, d, J = 5.1 Hz), 3.57 (1H, m), 4.87 (1H, dd, J = 8.1,
2.7 Hz), 5.04 (1H, dd, J = 10.5, 1.5 Hz), 5.10 (1H, dd, J = 15.6, 1.5 Hz),
5.54 (1H, d, J = 1.5 Hz), 5.60 (1H, ddd, J = 15.3, 10.2, 4.8 Hz); 13C
NMR ı: −0.1, 25.0, 27.1, 27.2, 38.8, 39.1, 67.8, 71.8, 74.4, 88.0,
101.9, 117.1, 132.95, 177.0, 177.8; HRMS (EI) calcd for C21H36O5Si
(M+): 396.2332, found: 396.2342.
2.18. (2R,3S,4R)-3,4-Dipivaloxy-5-hexene-1,2-diol (30)
To a solution of 29 (3.35 g, 9.4 mmol) in THF (37.6 ml), 1N HCl
(37.6 ml) was added at room temperature and the mixture was
stirred at room temperature for 44 h. To the mixture, satu-
rated NaHCO3 was added and the mixture was extracted with
CH2Cl2. The extract was dried and evaporated. The residue
was chromatographed on column (CHCl3/MeOH = 15/1) to give
30 (2.73 g, 93%) as a colorless oil; [˛]2D2 + 70.3 (c 1.03, CHCl3);
2.21. (3R,4R,5R)-3,4,5-Tris(triethylsilyloxy)-oct-
1-en-7-yne (33)
To a solution of 32 (1.07 g, 2.69 mmol) in MeOH (11.7 ml), 1N
NaOH (11.7 ml) was added at 0 ◦C and the mixture was stirred
at room temperature for 6 h, neutralized by addition of 1N HCl,
and evaporated. The residue was extracted with CH2Cl2. The
extract was dried and evaporated. The residue was dissolved
in CH2Cl2 (13.4 ml). To the solution, NEt3 (3.0 ml, 21.5 mmol)
and TESOTf (2.4 ml, 10.7 mmol) were added at −40 ◦C and the
mixture was stirred at −40 ◦C for 11 h, quenched by addition of
H2O (1 ml), and extracted with Et2O. The extract was washed
with 1N NaOH, dried, and evaporated. The residue was chro-
matographed on column (hexane) to give 33 (1.25 g, 93%) as
a colorless oil; [˛]2D4 + 33.7 (c 1.02, CHCl3); IR (film) 3313, 1458,
IR (film) 3482, 1737, 1481, 1398, 1280, 1160 cm−1 1H NMR ı:
;
1.21 (9H, s), 1.28 (9H, s), 2.36 (1H, dd, J = 9.6, 4.2 Hz), 3.37 (1H, d,
J = 6.0 Hz), 3.48 (2H, m), 3.62 (1H, m), 4.95 (1H, d, J = 7.5 Hz), 5.23
(1H, dd, J = 10.5, 1.5 Hz), 5.32 (1H, dd, J = 16.8, 1.5 Hz), 5.70–5.80
(2H, m); 13C NMR ı: 27.2, 27.2, 39.0, 39.2, 62.7, 69.3, 71.7, 72.5,
100.7, 117.4, 132.8, 178.1, 178.6; HRMS (EI) calcd for C16H28O6
(M+): 316.1886, found: 316.1880.
2.19. (R)-2-((1S,2R)-1,2-Dipivaloxy-3-butenyl)oxirane
(31)
1415, 1240, 1132, 1007 cm−1 1H NMR ı: 0.61 (18H, m), 0.96 (27H,
;
m), 1.90 (1H, t, J = 2.7 Hz), 2.26 (1H, ddd, J = 17.4, 8.4, 2.7 Hz), 2.43
(1H, dt, J = 17.4, 2.7 Hz), 3.72 (1H, d, J = 5.1 Hz), 4.0 (1H, dd, J = 8.1,
3.3 Hz), 4.09 (1H, t, J = 5.7 Hz), 5.13 (1H, d, J = 10.5 Hz), 5.23 (1H,
dt, J = 16.8, 1.8 Hz), 5.88 (1H, ddd, J = 16.8, 10.5, 5.7 Hz); 13C NMR
ı: 4.9, 5.0, 5.1, 6.8, 6.9, 7.0, 23.1, 69.0, 71.6, 75.2, 80.3, 83.8, 115.6,
137.8; HRMS (EI) calcd for C26H54O5Si3 (M+): 498.3381, found:
498.3378.
To a solution of 30 (3.17 g, 10 mmol) in dioxane (140 ml),
Ph3P (7.78 g, 30 mmol) and diisopropyl azodicarboxylate (DIAD)
(5.9 ml, 30 mmol) were added at room temperature and the
mixture was refluxed for 23 h. Additionally, Ph3P (5.33 g,
20 mmol) and DIAD (4.0 ml, 20 mmol) were added at room
temperature and the mixture was refluxed for 22 h and evap-
orated. The residue was chromatographed on column (hex-
ane/AcOEt = 30/1 to 10/1) to give 31 (2.19 g, 74%) as a colorless
oil; [˛]2D5 + 55.4 (c 1.0, CHCl3); IR (film) 1745, 1481, 1396, 1367,
2.22. (1R,3aR,4S,7aR)-7a-Methyl-1-[(1S)-1-methyl-
1-(phenylsulfonyl)ethyl]octahydro-1H-inden-4-ol (36)
1278, 1164, 1033 cm−1
;
1H NMR ı: 1.19 (9H, s), 1.24 (9H, s), 2.74
To a solution of 35 (512 mg, 1.39 mmol) in acetone (9 ml),
sodium iodide (NaI) (524 mg, 3.49 mmol) was added at room
temperature and the mixture was refluxed for 2.5 h. The mix-
ture was evaporated and the residue was dissolved in N,N-
(2H, m), 3.05 (1H, m), 4.99 (1H, t, J = 4.8 Hz), 5.28 (1H, dt, J = 10.8,
1.2 Hz), 5.35 (1H, dt, J = 16.8, 1.5 Hz), 5.54 (1H, tt, J = 4.8, 1.2 Hz),
5.79 (1H, ddd, J = 16.8, 10.8, 6.0 Hz); 13C NMR ı: 27.0, 38.8, 38.9,

534
steroids 7 1 ( 2 0 0 6 ) 529–540
52.7, 54.3, 67.0, 69.1, 119.6, 121.3, 128.2, 128.6, 129.0, 129.1,
133.3; HRMS (EI) calcd for C24H36O3S (M+): 404.2386, found:
404.2386.
dimethylformamide (DMF) (2.8 ml). To the solution, benzene-
sulfinic acid sodium salt dihydrate (NaSO2Ph·2H2O) (114 mg,
0.7 mmol) was added at room temperature and the mix-
ture was stirred at room temperature for 10 h. Additionally,
NaSO2Ph·2H2O (114 mg, 0.7 mmol) was added at room temper-
ature and the mixture was stirred at room temperature for 8 h
and extracted with Et2O. The extract was washed with brine,
dried, and evaporated. The residue was chromatographed on
column (hexane/AcOEt = 10/1 to 4/1) to give 36 (399 mg, 78%)
as a colorless oil; [˛]2D5 + 48.2 (c 1.0, CHCl3); IR (film) 3535,
2.25. A 4:1 mixture of (1R,3aR,4S,7aR)-7a-methyl-1-
[(1R)-1,5-dimethyl-4-hexenyl]octahydro-1H-inden-4-ol
and (1R,3aR,4S,7aR)-7a-methyl-1-[(1R,2E)-1,5-
dimethyl-2,4-hexadienyl]octahydro-1H-inden-4-ol (39)
To
a solution of 38 (842 mg, 2.08 mmol) in THF/MeOH
3062, 1704, 1583, 1450, 1298, 1143, 1078 cm−1 1H NMR ı: 0.9
;
(3:2) (42 ml), 5% sodium mercury amalgam (Na/Hg) (12.6 g,
27.4 mmol) was added at room temperature and the mixture
was stirred at room temperature for 9 h and sonicated for
1.5 h. To the mixture, 50% MeOH solution (11 ml) was added
and the mixture was stirred for 1 h, filtrated through Celite®,
and evaporated. To the residue, saturated NH4Cl was added
and the mixture was extracted with AcOEt. The extract was
dried and evaporated. The residue was chromatographed on
column (hexane/AcOEt = 20/1 to 10/1) to give 39 (394 mg, 72%)
as a mixture of ene/diene = 4/1; IR (film) 3831, 3735, 3671, 3610,
(3H, s), 1.17 (3H, d, J = 6.3 Hz), 1.20–1.8 (10H, m), 1.95 (1H, d,
J =13.8 Hz), 2.04 (1H, m), 2.84 (1H, dd, J = 14.1, 9.6 Hz), 3.14 (1H,
dd, J = 14.1, 1.5 Hz), 4.05 (1H, d, J = 2.7 Hz), 7.54–7.68 (3H, m), 7.91
(2H, dt, J = 6.9, 1.5 Hz); 13C NMR ı: 13.3, 17.3, 20.0, 22.4, 27.1, 32.0,
35.5, 40.2, 42.1, 52.5, 55.8, 61.9, 69.0, 127.9, 129.3, 129.4, 133.6,
140.3.
2.23. (1R,3aR,4S,7aR)-7a-Methyl-1-[(1S)-1-methyl-1-
(phenylsulfonyl)ethyl]-4-(triethylsilyloxy)octahydro-
1H-indene (37)
3201, 2130, 1701, 1523, 1459 cm−1 1H NMR ı: 0.90–2.1 (37H,
;
m), 4.07 (1.3H, brs), 5.08 (0.8H, t, J = 6.9 Hz), 5.38 (0.2H, dd,
J = 15.9, 8.1 Hz), 5.74 (0.2H, d, J = 10.5 Hz), 6.14 (0.2H, dd, J = 15.9,
10.5 Hz); 13C NMR ı: 13.6, 13.8, 17.5, 17.7, 18.3, 18.5, 20.5, 22.6,
24.8, 25.9, 26.0, 27.2, 33.6, 35.2, 36.0, 40.4, 41.9, 52.7, 56.7, 69.5,
125.2, 131.0.
To a solution of 36 (1.67 g, 4.55 mmol) in DMF (9 ml), imidazole
(0.93 g, 13.67 mmol) and triethylsilyl chloride (TESCl) (1.1 ml,
6.83 mmol) were added at room temperature and the mixture
was stirred at room temperature for 4 h. To the mixture, satu-
rated NaHCO3 was added and the mixture was extracted with
Et2O. The extract was dried and evaporated. The residue was
chromatographed on column (hexane/AcOEt = 8/1) to give 37
(2.17 g, 100%) as a colorless crystals; [˛]2D4 + 54.0 (c 1.01, CHCl3);
2.26. A 4:1 mixture of (1R,3aR,4S,7aR)-
4-acetoxy-7a-methyl-1-[(1R)-1,5-dimethyl-4-
hexenyl]octahydro-1H-indene and (1R,3aR,4S,7aR)
-4-acetoxy-7a-methyl-1-[(1R,2E)-1,5-dimethyl-
2,4-hexadienyl]octahydro-1H-indene (40)
IR (film) 1701, 1454, 1307, 1238, 1151, 1081, 1018 cm−1 1H NMR
;
ı: 0.54 (6H, m), 0.86 (3H, s), 0.92 (9H, t, J = 8.1 Hz), 1.15 (3H, d,
J = 6.6 Hz), 0.97–2.06 (12H, m), 3.03 (1H, dd, J = 14.1, 9.6 Hz), 3.35
(1H, d, J = 14.1 Hz), 4.19 (1H, d, J = 2.1 Hz), 7.54–7.67 (3H, m), 7.90
(2H, d, J = 7.2 Hz); 13C NMR ı: 4.9, 7.0, 13.4, 17.6, 20.0, 22.8, 27.2,
32.0, 34.5, 40.6, 42.4, 53.0, 56.1, 62.0, 69.2, 127.9, 129.3, 133.5,
140.4.
To a solution of 39 (255 mg, 0.753 mmol) in pyridine (3.8 ml),
acetic anhydride (0.13 ml, 1.09 mmol) and DMAP (9.0 mg,
0.07 mmol) were added at 0 ◦C and the mixture was stirred
at room temperature for 20 h and extracted with Et2O. The
extract was washed with brine, dried, and evaporated. The
residue was chromatographed on column (benzene) to give 40
(212 mg, 92%) as a mixture of ene/diene = 4/1; 1H NMR ı: 0.87
(3H, s), 0.91 (3H, d, J = 6.6 Hz), 1.0–1.52 (12H, m), 1.59 (3H, s), 1.67
(3H, s), 1.71–2.01 (5H, m), 2.03 (3H, s), 5.07 (0.8H, t, J = 6.9 Hz), 5.13
(1H, brs), 5.37 (0.2H, dd, J = 14.4, 8.7 Hz), 5.73 (0.2H, d, J = 9.9 Hz),
6.13 (0.2H, dd, J = 14.4, 10.5 Hz); 13C NMR ı: 13.1, 17.7, 18.0, 18.6,
21.5, 22.7, 24.7, 25.8, 27.2, 30.6, 35.3, 35.9, 40.1, 42.1, 51.4, 56.5,
71.5, 125.2, 131.1, 171.0.
2.24. (1R,3aR,4S,7aR)-7a-Methyl-1-[(1S,2RS)-1,5-
dimethyl-2-(phenylsulfonyl)-4-hexenyl)octahydro-
1H-inden-4-ol (38)
To a solution of 37 (2.17 g, 4.55 mmol) in THF (45 ml), n-BuLi
(1.58 M in hexane; 3.75 ml, 5.9 mmol) was added at −40 ◦C and
the mixture was stirred at −40 ◦C for 30 min. To the mixture,
4-bromo-2-methyl-2-butene (0.78 ml, 6.8 mmol) was added at
−40 ◦C and the mixture was stirred at −40 ◦C for 4 h, quenched
by addition of saturated NaHCO3, warmed to room temper-
ature, and extracted with Et2O. The extract was dried and
evaporated. The residue was dissolved in THF (5 ml). To the
solution, 3N HCl (2.3 ml) was added at 0 ◦C and the mixture
was stirred at room temperature for 12 h and extracted with
CH2Cl2. The extract was dried and evaporated. The residue
was chromatographed on column (hexane/AcOEt = 8/1 to 5/1)
to give 38 (1.76 g, 96%); [˛]2D4 + 14.9 (c 1.47, CHCl3); IR (film)
2.27. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4S)-4,5-
dihydroxy-1,5-dimethylhexyl]-7a-methyloctahydro-
1H-indene (41)
To
a solution of 40 (46.4 mg, 0.151 mmol) in 50% tert-
BuOH (1.8 ml) solution, methanesulfonamide (MeSO2NH2)
(14.3 mg, 0.151 mmol) and the commercially available asym-
metric osmium tetroxide dihydroxylation reagent, AD-mix-␣
(Aldrich, 0.5% OsO4; 154 mg, 0.003 mmol) were added at 0 ◦C
and the mixture was stirred at 2 ◦C for 3 days. To the mixture,
saturated Na2S2O3 was added and the mixture was extracted
with AcOEt. The extract was dried and evaporated. The residue
was chromatographed on column (CHCl3/MeOH = 20/1). The
3540, 1668, 1448, 1378, 1297, 1143, 1079 cm−1 1H NMR ı: 0.87
;
(3H, d, J = 4.8 Hz), 1.10 (3H, d, J = 6.9 Hz), 1.52 (3H, s), 1.54 (3H,
s), 2.34–2.62 (2H, m), 3.06 (1H, m), 4.04 (1H, brs), 4.82 (1H, m),
7.51–7.61 (3H, m), 7.83–7.89 (2H, m); 13C NMR ı: 13.2, 14.6, 17.5,
17.8, 22.4, 22.6, 25.6, 27.2, 33.6, 33.6, 33.9, 36.8, 40.4, 42.1, 42.2,

steroids 7 1 ( 2 0 0 6 ) 529–540
535
resultant was dissolved in EtOH (5.0 ml) and hydrogenated
with 10% Pd/C (12 mg) at room temperature for 10 h. The mix-
ture was filtrated through Celite® and evaporated to give 41
(49.8 mg, 96%); [˛]2D5 + 21.7 (c 1.03, CHCl3); IR (film) 3448, 1735,
mixture, tetrapropylammonium perruthenate (TPAP) (2.7 mg,
0.0078 mmol) was added at room temperature and the mix-
ture was stirred at room temperature for 4 h, filtrated through
Celite® and evaporated. The residue was chromatographed
on column (hexane/AcOEt = 5/1) to give 44 (55.9 mg, 95 %);
1448, 1378, 1297, 1143, 1079 cm−1 1H NMR ı: 0.88 (3H, s), 0.93
;
(3H, d, J = 6.6 Hz), 1.16 (3H, s), 1.21 (3H, s), 2.04 (3H, s), 3.27 (1H,
d, J = 6.6 Hz), 5.14 (1H, brs); 13C NMR ı: 13.0, 17.9, 18.7, 21.3, 22.6,
23.1, 26.5, 27.0, 28.2, 30.4, 33.0, 35.5, 39.9, 41.9, 51.2, 56.2, 71.3,
73.1, 79.4, 170.7; HRMS (EI) calcd for C20H36O4 (M+): 340.2613,
found: 340.2613.
[˛]2D3 − 5.7 (c 1.85, CHCl3); IR (film) 1716, 1467, 1252 cm−1
;
1H
NMR ı: 0.04 (3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.63
(3H, s), 0.85 (9H, s), 0.88 (9H, s), 0.94 (3H, d, J = 6.3 Hz), 1.10 (3H,
s), 1.19 (3H, s), 1.20–2.28 (16H, m), 2.43 (1H, dd, J = 11.1, 7.5 Hz),
3.18 (1H, dd, J = 7.5, 2.4 Hz); 13C NMR ı: −3.7, −3.1, −1.9, 12.5,
18.8, 19.2, 23.1, 24.2, 25.9, 26.2, 27.8, 28.9, 29.7, 34.0, 36.2, 39.1,
41.1, 50.0, 62.1, 81.3, 212.3; HRMS (EI) calcd for C30H60O3Si2 (M+):
524.4081, found: 524.4066.
2.28. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4S)-4,5-
di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-
7a-methyloctahydro-1H-indene (42)
2.31. (1R,3aR,7aR)-4-[(E)-Bromomethylene]-1-((1R,4S)-
4,5-di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-
7a-methyl-octahydro-1H-inden (45)
To a solution of 41 (60 mg, 0.176 mmol) in CH2Cl2 (2.0 ml), 2,6-
lutidine (0.13 ml, 1.10 mmol) and tert-butyldimethylsilyl tri-
fluoromethanesulfonate (TBSOTf) (0.17 ml, 0.74 mmol) were
added at 0 ◦C and the mixture was stirred at 0 ◦C for 1.5 h.
To the mixture, saturated NaHCO3 was added and the mix-
ture was extracted with Et2O. The extract was washed with
saturated NH4Cl, dried, and evaporated. The residue was
chromatographed on column (hexane/AcOEt = 50/1) to give
42 (95 mg, 95%) as a colorless oil; [˛]2D1 + 6.3 (c 1.07, CHCl3);
To
a degassed solution of (bromomethyl)triphenylphos-
phonium bromide (Ph3PCH2Br2) (400 mg, 0.917 mmol) in THF
(2.2 ml), sodium hexamethyldisilazide (NaHMDS) (1.0 M in
THF; 0.9 ml, 0.9 mmol) was added at −60 ◦C and the mix-
ture was stirred at −60 ◦C for 1 h. To the mixture, a degassed
solution of 44 (55.9 mg, 0.106 mmol) in THF (0.7 ml) was
added at −60 ◦C. The mixture was warmed to room tem-
perature gradually and stirred at room temperature for 1 h.
To the mixture, saturated NH4Cl was added at 0 ◦C and the
mixture was extracted with AcOEt. The extract was dried
and evaporated. The residue was chromatographed on col-
umn (hexane/benzene = 100/1 to 10/1) to give 45 (35 mg, 55%)
as a yellow oil; [˛]2D3 + 50.1 (c 0.83, CHCl3); IR (film) 1466,
IR (film) 1735 cm−1 1H NMR ı: 0.04 (3H, s), 0.06 (3H, s), 0.07
;
(3H, s), 0.08 (3H, s), 0.85 (9H, s), 0.88 (9H, s), 1.10 (3H, s),
1.20 (3H, s), 2.04 (3H, s), 3.18 (1H, dd, J = 7.5, 2.4 Hz), 5.15 (1H,
brs); 13C NMR ı: 5.8, 6.3, 6.6, 6.9, 7.0, 7.0, 7.3, 13.1, 18.1, 18.7,
21.5, 22.9, 23.6, 27.3, 28.8, 29.0, 30.7, 34.1, 36.1, 40.2, 42.1, 51.5,
56.6, 71.6, 76.3, 81.7, 170.8; MS (EI (m/z)): 553 [(M − Me)+], 173
(100%).
1252 cm−1 1H NMR ı: 0.04 (3H, s), 0.06 (3H, s), 0.07 (3H, s),
;
2.29. (1R,3aR,4S,7aR)-1-[(1R,4S)-4,5-di(tert-
butyldimethylsilyloxy)-1,5-dimethylhexyl]-7a-
methyloctahydro-1H-inden-4-ol (43)
0.08 (3H, s), 0.56 (3H, s), 0.85 (9H, s), 0.88 (9H, s), 0.92 (3H, d,
J = 6.0 Hz), 1.10 (3H, s), 1.18 (3H, s), 1.22–1.36 (5H, m), 1.4–1.74
(7H, m), 1.86–2.4 (4H, m), 2.86 (1H, m), 3.17 (1H, dd, J = 7.5,
2.4 Hz), 5.64 (1H, s); 13C NMR ı: −3.7, −3.1, −1.9, −1.8, 11.9,
18.2, 18.3, 18.9, 22.1, 22.7, 23.2, 25.9, 26.2, 27.9, 29.0, 29.7,
31.2, 34.2, 36.7, 40.0, 45.6, 55.9, 56.0, 76.4, 81.2, 97.4, 145.3;
HRMS (EI) calcd for C30H58O2Si2Br [(M − Me)+]: 587.3128, found:
587.3140.
To a solution of 42 (260 mg, 0.458 mmol) in THF (5.0 ml),
LiAlH4 (26.1 mg, 0.687 mmol) was added at 0 ◦C and the mix-
ture was stirred at 0 ◦C for 1.5 h. To the mixture, 1N NaOH
was added and the mixture was extracted with AcOEt. The
extract was washed with saturated NH4Cl, dried, and evap-
orated. The residue was chromatographed on column (hex-
ane/AcOEt = 40/1) to give 43 (241 mg, 100%) as a colorless oil;
2.32. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4R)-4,5-
dihydroxy-1,5-dimethylhexyl]-7a-methyloctahydro-
1H-indene (46)
[˛]2D1 + 6.3 (c 1.07, CHCl3); IR (film) 3430 cm−1
;
1H NMR ı: 0.04
(3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.85 (9H, s), 0.89
(9H, s), 0.92 (3H, d, J = 6.3 Hz), 1.11 (3H, s), 1.19 (3H, s), 3.18
(1H, dd, J = 7.6, 2.3 Hz), 4.00 (1H, brs); 13C NMR ı: 5.8, 6.3, 6.6,
6.9, 7.9, 7.0, 7.3, 13.1, 18.1, 18.7, 21.5, 22.9, 23.6, 27.3, 28.8,
29.0, 30.7, 34.1, 36.1, 40.2, 42.1, 51.5, 56.6, 71.6, 76.3, 81.7,
170.8; HRMS (EI) calcd for C30H62O3Si2 (M+): 526.4237, found:
526.4247.
To a solution of 40 (50 mg, 0.164 mmol) in 50% tert-BuOH
(2.0 ml) solution, MeSO2NH2 (15.5 mg, 0.164 mmol) and AD-
mix-␤ (Aldrich, 0.5% OsO4; 167 mg, 0.0032 mmol) were added
at 0 ◦C and the mixture was stirred at 2 ◦C for 3 days.
To the mixture, saturated Na2S2O3 was added and the
mixture was extracted with AcOEt. The extract was dried
and evaporated. The residue was chromatographed on col-
umn (CHCl3/MeOH = 20/1). The resultant was dissolved in
EtOH (5.0 ml) and hydrogenated with 10% Pd/C (15 mg)
at room temperature for 10 h. The mixture was filtrated
through Celite® and evaporated to give 26 (53 mg, 95%); IR
2.30. (1R,3aR,7aR)-1-[(1R,4S)-4,5-Di(tert-
butyldimethylsilanyloxy)-1,5-dimethylhexyl]-7a-
methyl-octahydro-1H-inden-4-one (44)
To a solution of 43 (59.0 mg, 0.112 mmol) in CH2Cl2 (7.5 ml), N-
methylmorpholine-N-oxide (NMO) (23.6 mg, 0.201 mmol) and
(film) 3455, 1735 cm−1 1H NMR ı: 0.89 (3H, s), 0.92 (3H, d,
;
J = 6.6 Hz), 1.16 (3H, s), 1.21 (3H, s), 2.04 (3H, s), 3.32 (1H, t,
J = 5.9 Hz), 5.14 (1H, brs); MS (EI (m/z)): 280 [(M − Ac-H2O)+],
59 (100%).
˚
4 A molecular sieves (50 mg) were added at room temperature
and the mixture was stirred at room temperature for 1 h. To the

536
steroids 7 1 ( 2 0 0 6 ) 529–540
2.33. (1R,3aR,4S,7aR)-4-Acetoxy-1-[(1R,4R)-4,5-
di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-
7a-methyloctahydro-1H-indene (47)
2.37. [(R)-6-{(E,3R,3aR)-7-(Bromomethylene)-
octahydro-3a-methyl-1H-inden-3-yl}-2-methylheptan-
2-yloxy]trimethylsilane (52)
Following the same experimental procedure as for 42, 47
(130 mg, 97%) was obtained as a colorless oil from 46 (80 mg,
Following the same experimental procedure as for 50, 52
(12 mg, 25%) was obtained as a colorless oil from 51 (40 mg,
0.235 mmol); [˛]2D1 + 6.3 (c 1.07, CHCl3); IR (film) 1745 cm−1
;
1H
0.11 mmol); IR (neat) 2950 (br), 1247, 1043, 839 cm−1 1H NMR
;
NMR ı: 0.04 (3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.86 (9H,
s), 0.89 (9H, s), 1.10 (3H, s), 1.19 (3H, s), 2.04 (3H, s), 3.22 (1H, brs),
5.15 (1H, brs); 13C NMR ı: 5.8, 6.3, 6.6, 6.9, 7.0, 7.0, 7.3, 13.1, 18.1,
18.7, 21.5, 22.9, 23.6, 27.3, 28.8, 29.0, 30.7, 34.1, 36.1, 40.2, 42.1,
51.5, 56.6, 71.6, 76.3, 81.7, 170.8; MS (EI (m/z)): 553 [(M − Me)+],
173 (100%).
ı: 0.10 (9H, s), 0.56 (3H, s), 0.93 (3H, d, J = 6.3 Hz), 1.20 (6H, s),
1.21–1.70 (14H, m), 1.83–2.05 (4H, m), 2.83–2.89 (1H, m), 5.64
(1H, s); MS (EI (m/z)) 429(M+).
2.38. 1˛,2ˇ,3ˇ-Tris(triethylsilyloxy)-24S,25-di(tert-
butyldimethylsilyloxy)-9,10-secochoresta-5,7,10(19)-
triene (53)
2.34. (1R,3aR,4S,7aR)-1-[(1R,4R)-4,5-di(tert-
butyldimethylsilyloxy)-1,5-dimethylhexyl]-
7a-methyloctahydro-1H-inden-4-ol (48)
To a solution of 33 (14.8 mg, 0.025 mmol) and 45 (9.0 mg,
0.018 mmol) in toluene (0.4 ml) and NEt3 (0.25 ml),
tetrakis(triphenylphosphine)palladium (Pd(PPh3)4) (5.7 mg,
0.005 mmol) was added at room temperature and the mixture
was stirred at room temperature for 15 min and then refluxed
for 1 h. After cooling to room temperature, the mixture was
diluted with Et2O, passed through small amount of silica
gel, and evaporated. The residue was chromatographed on
preparative TLC (hexane/benzene = 20/1) to give 53 (4.9 mg,
27%) as a yellow oil; [˛]2D2 + 44.3 (c 1.28, CHCl3); IR (neat) 3831,
Following the same experimental procedure as for 43, 48
(120 mg, 93%) was obtained from 47 (140 mg, 0.246 mmol);
[˛]2D1 + 6.3 (c 1.07, CHCl3); IR (film) 3425, cm−1
;
1H NMR ı: 0.03
(3H, s), 0.06 (3H, s), 0.07 (3H, s), 0.08 (3H, s), 0.85 (9H, s),
0.89 (9H, s), 1.11 (3H, s), 1.18 (3H, s), 3.22 (1H, brs), 4.08 (1H,
brs); 13C NMR ı: 5.8, 6.3, 6.6, 6.9, 7.9, 7.0, 7.3, 13.1, 18.1, 18.7,
21.5, 22.9, 23.6, 27.3, 28.8, 29.0, 30.7, 34.1, 36.1, 40.2, 42.1, 51.5,
56.6, 71.6, 76.3, 81.7, 170.8; MS (EI (m/z)): 511 [(M − Me)+], 173
(100%).
3737, 1687, 1525, 1461, 1255, 1097 cm−1 1H NMR ı: 0.53–0.67
;
(15H, m), 0.86–0.99 (36H, m) 1.11 (3H, s), 1.19 (3H, s), 2.15 (1H,
dd, J = 9.6, 3.3 Hz), 2.62 (1H, m), 2.84 (1H, d, J = 8.7 Hz), 3.18 (1H,
dd, J = 5.7, 1.8 Hz), 3.75 (1H, brs), 4.02 (1H, d, J = 3.3 Hz), 4.11 (1H,
m), 4.99 (1H, brs), 5.12 (1H, brs), 6.04 (1H, d, J = 8.7 Hz), 6.26 (1H,
d, J = 6.3 Hz); 13C NMR ı: −3.7, −3.1, −1.9, 4.9, 5.0, 5.2, 5.9, 6.8,
6.9, 7.0, 7.0, 12.0, 18.3, 18.9,22.2, 23.2, 23.7, 25.9, 26.2, 28.0, 29.0,
29.7, 34.2, 36.8, 40.4, 40.8, 45.9, 56.5, 56.7, 69.5, 76.5, 81.3, 116.5,
118.1, 123.3, 134.7, 141.3.
2.35. (1R,3aR,7aR)-1-[(1R,4R)-4,5-Di(tert-
butyldimethylsilanyloxy)-1,5-dimethylhexyl]-7a-
methyl-octahydro-1H-inden-4-one (49)
Following the same experimental procedure as for 44, 49
(273 mg, 100%) was obtained as a colorless oil from 48 (274 mg,
0.521 mmol); [˛]2D5 + 16.2 (c 1.02, CHCl3); IR (film) 1716, 1460,
1252 cm−1
;
1H NMR ı: 0.03 (3H, s), 0.06 (3H, s), 0.07 (3H, s),
2.39. 1˛,2ˇ,3ˇ-Tris(triethylsilyloxy)-24R,25-di(tert-
butyldimethylsilyloxy)-9,10-secochoresta-5,7,10(19)-
triene (54)
0.08 (3H, s), 0.63 (3H, s), 0.85 (9H, s), 0.88 (9H, s), 0.94 (3H,
d, J = 6.0 Hz), 1.11 (3H, s), 1.18 (3H,s), 1.20–2.28 (16H, m), 2.43
(1H, dd, J = 7.5, 11.1 Hz), 3.20 (1H, m); 13C NMR ı: −3.7, −3.1,
−1.9, −1.8, 12.5, 18.8, 18.3, 18.9, 19.2, 23.1, 24.2, 25.9, 26.2,
27.8, 28.9, 29.7, 34.0, 36.2, 39.1, 41.1, 50.0, 62.1, 81.3, 212.2;
HRMS (EI) calcd for C30H60O3Si2 [(M − Me)+]: 509.8846, found:
509.8848.
Following the same experimental procedure as for 53, 54
(25.2 mg, 23%) was obtained as a yellow oil from 33 (96.0 mg,
0.159 mmol) and 50 (55.0 mg, 0.110 mmol); [˛]2D1 + 81.2 (c 1.26,
CHCl3); IR (film) 1685, 1461, 1373, 1247, 1093, 1008 cm−1 1H
;
NMR ı: 0.53–0.65 (15H, m), 0.85–0.99 (36H, m) 1.11 (3H, s), 1.18
(3H, s), 2.15 (1H, dd, J = 9.6, 3.3 Hz), 2.62 (1H, m), 2.84 (1H, d,
J = 11.1 Hz), 3.22 (1H, brs), 3.75 (1H, brs), 4.03 (1H, d, J = 4.2 Hz),
4.12 (1H, m), 4.99 (1H, brs), 5.12 (1H, brs), 6.05 (1H, d, J = 11.4 Hz),
6.27 (1H, d, J = 11.4 Hz); 13C NMR ı: −3.9, −3.1, −1.9, −1.9, 4.9,
5.0, 5.2, 6.9, 7.0, 7.0, 12.0, 18.2, 18.3, 19.1, 22.2, 23.6, 26.0, 26.2,
27.9, 29.0, 29.2, 29.6, 34.0, 36.9, 40.3, 40.7, 45.9, 56.5, 56.6, 69.5,
76.3, 76.4, 81.2, 118.1, 123.3, 134.7, 141.3, 144.9.
2.36. (1R,3aR,7aR)-4-[(E)-Bromomethylene]-1-((1R,4R)-
4,5-di(tert-butyldimethylsilyloxy)-1,5-dimethylhexyl]-
7a-methyl-octahydro-1H-inden (50)
Following the same experimental procedure as for 45, 50
(96.1 mg, 34%) was obtained as
a colorless oil from 49
(250 mg, 0.476 mmol); [˛]2D3 + 65.7 (c 1.06, CHCl3); IR (film) 1467,
1253 cm−1 1H NMR ı: 0.04 (3H, s), 0.06 (3H, s), 0.07 (3H,
;
s), 0.08 (3H, s), 0.56 (3H, s), 0.85 (9H, s), 0.89 (9H, s), 0.92
(3H, d, J = 6.0 Hz), 1.11 (3H, s), 1.18 (3H, s), 1.22–1.36 (5H, m),
1.40–1.74 (7H, m), 1.86–2.40 (4H, m), 2.86 (1H, m), 3.21 (1H,
m), 5.64 (1H, s); 13C NMR ı: −3.9, −3.1, −1.9, −1.8, 11.9,
18.3, 19.0, 22.1, 22.7, 23.6, 26.0, 26.2, 27.7, 29.0, 29.6, 31.2,
33.9, 36.8, 40.0, 45.6, 55.9, 56.0, 76.4, 81.1, 97.4, 145.3; HRMS
(EI) calcd for C27H52O2Si2Br [(M − C4H9)+]: 543.2690, found:
543.2692.
2.40. 1˛,2ˇ,3ˇ-Tris(tert-butyldimethylsilyloxy)-
25(trimethylsilyloxy)-9,10-secochoresta-5,7,10(19)-
triene (55)
Following the same experimental procedure as for 53, 55
(3.5 mg, 17%) was obtained as a colorless oil from 33 (12.0 mg,
0.024 mmol) and 52 (12 mg, 0.024 mmol); IR (neat): 2980 (br),
1080 cm−1 1H NMR ı: 0.10 (9H, s), 0.50–0.68 (30H, m), 0.91–1.00
;

steroids 7 1 ( 2 0 0 6 ) 529–540
537
(21H, m), 1.20 (6H, s), 2.57–2.66 (1H, m), 2.79–2.88 (1H, m),
3.
Results and discussion
Synthesis of the oxidized metabolite, methyl ester
3.70–3.78 (1H, m), 4.02 (1H, d, J = 4.4 Hz), 4.08–4.15 (1H, m),
4.99 (1H, s), 5.12 (1H, s), 6.03 (1H, d, J = 11.5 Hz), 6.25 (1H, d,
J = 11.5 Hz); MS (EI (m/z)): 847 (M+).
3.1.
derivative (6), and the truncated metabolite, tetraol
derivative (7), at the 2ˇ-position
2.41. 1˛,2ˇ,3ˇ,24(S),25-Pentahydroxy-
9,10-secochoresta-5,7,10(19)-triene (8)
First, we undertook the synthesis of methyl ester derivative
(6) as an ester standard of the oxidized metabolite, carboxylic
acid (5), which in turn was derived from the oxidation of the
hydroxypropoxy substituent at the 2␤-position. After several
unsuccessful trials, methyl ester derivative (18) was prepared
from known hydroxyethoxy derivative (10) [4]. The hydroxyl
group in the hydroxyethoxy moiety of 10 was acetylated to
give 11 in 65% yield with recovery of 10 in 34% yield. The rest
of three hydroxyl groups of 11 were triethylsilylated with tri-
ethylsilyl trifluoromethanesulfonate (TESOTf) to afford 12 in
76% yield and the acetyl group in 12 was hydrolized to furnish
alcohol (13) in 56% yield. The acid-delicate 5,7-diene moi-
ety of 13 was protected as 4-phenyl-1,2,4-triazoline-3,5-dione
(PTAD) adduct (14) in 82%. The alcohol (14) was converted to
carboxylic acid methyl ester (17) as follows: (i) iodination of
14 with iodine and triphenyl phosphine (Ph3P) produced 15
in 98% yield, (ii) cyanation of 15 with sodium cyanide (NaCN)
afforded 16 in 87% yield, and (iii) methanolysis of 16 with
methanolic hydrochloric acid (concd. HCl/MeOH) gave 17 in
25% yield. The ester (17) was submitted to retrocycloaddition
in 1,3-dimethyl-2-imidazolidione (DMI) at 140 ◦C to generate
5,7-diene (18) in 59% yield. Finally, irradiation of 18 at 0 ◦C
using high pressure mercury lamp (400 W, Vycor filter), fol-
lowed by thermal isomerization in boiling ethanol gave rise to
methyl ester derivative (6) in 25% yield (Fig. 3). The synthesis
has been also disclosed in our patent [14].
Next, we conducted the synthesis of the truncated metabo-
lite, tetraol (7). Although 7 and its derivatives have been syn-
thesized in a highly effective parallel synthesis on solid phase
[15], we adopted an alternative approach using PTAD adduct of
␣-epoxide (19), a key intermediate in a large synthesis of ED-
71 [1]. After acetylation of the 3␤-hydroxyl group of ␣-epoxide
(19), the ␣-epoxy ring in 20 was cleaved in the presence of
boron triflioride diethyl etherate (BF3·OEt2) in THF at 60 ◦C to
give 1,2-diol (21) which was converted to 5,7-diene (22) by ther-
mal retrocycloaddition in a 44% overall yield from 20. After
deacetylation of 22, 5,7-diene (23) was irradiated and ther-
mally isomerized to the tetraol (7) in 5% yield. Tetraol (7)
obtained in the abovementioned linear method (Fig. 4) was
To a solution of 53 (12.5 mg, 0.020 mmol) in toluene (1 ml) and
THF (1.1 ml), tetrabutylammonium fluoride (TBAF) (1.0 M in
THF; 0.12 ml, 0.12 mmol) was added at room temperature and
the mixture was stirred at room temperature for 17 h, at 50 ◦C
for 7 h, and at 70 ◦C for 5 h. The mixture was evaporated. The
residue was chromatographed on reverse-phase preparative
TLC (CH3CN/H2O = 3/1) and the resultant was purified by HPLC
(ODS-M80, 5.0 ml/s, UV detect; 265 nm, MeCN/H2O = 1/1) to give
8 (3.9 mg, 70%) as a colorless powder; [˛]2D4 − 37.3 (c 0.3, CH3OH);
1H NMR ı: 0.46 (3H, s), 0.86 (3H, d, J = 6.4 Hz), 1.05 (3H, s), 1.10 (3H,
s), 2.38 (1H, brs), 2.75 (1H, d, J = 10.0 Hz), 3.14 (1H, d, J = 10.0 Hz),
3.28 (1H, brs), 3.33 (1H, d, J = 2.8 Hz), 3.40 (2H, d, J = 9.2 Hz), 4.02
(1H, brs), 4.10 (1H, d, J = 8.8 Hz), 4.98 (1H, s), 5.38 (1H, s), 5.95 (1H,
d, J = 11.2 Hz), 6.24 (1H, d, J = 11.2 Hz); UV ꢀmax: 265 nm, ꢀmin
:
229 nm; MS (ESI): 466 ([M + NH4]+), 447 ([M − H]−); HRMS (ESI)
calcd for C27H48NO5 [(M + NH4)+]: 466.3532, found: 466.3510.
2.42. 1˛,2ˇ,3ˇ,24(R),25-Pentahydroxy-
9,10-secochoresta-5,7,10(19)-triene (9)
Following the same experimental procedure as for 8, 9 (5.2 mg,
50%) was obtained as a colorless powder from 54 (24.0 mg,
0.023 mmol); [˛]2D4 − 13.8 (c 0.52, MeOH); IR (film) 3363, 1644,
1124 cm−1; 1H NMR ı: 0.38 (3H, s), 0.76 (3H, d, J = 6.4 Hz), 0.92 (3H,
s), 0.95 (3H, s), 1.12 (6H, m), 1.29 (6H, m), 1.48 (2H, m), 1.70–1.87
(3H, m), 2.18 (1H, m), 2.24 (1H, m), 2.65 (1H, m), 3.00 (1H, d,
J = 8.8 Hz), 3.29 (1H, dd, J = 8.4, 3.2 Hz), 3.83 (1H, m), 3.94 (1H, d,
J = 8.0 Hz), 4.78 (1H, brs), 5.20 (1H, brs), 5.86 (1H, d, J = 11.2 Hz),
6.09 (1H, d, J = 11.2 Hz); UV ꢀmax: 265 nm, ꢀmin: 229; MS (ESI): 471
([M + Na]+), 447 ([M − H]−); HRMS (EI) calcd for C27H44O5 (M+):
448.3189, found: 448.3199.
2.43. 1˛,2ˇ,3ˇ,25-Tetrahydroxy-9,10-secochoresta-
5,7,10(19)-triene (7)
Following the same experimental procedure as for 8, 7 (0.9 mg,
50%) was obtained as a white powder from 55 (3.5 mg,
0.004 mmol).
Fig. 3 – Synthesis of methyl ester derivative (6): (a) Ac2O, pyridine, DMAP; (b) TESOTf, 2,6-lutidine; (c) KOH; (d) PTAD; (e) I2,
imidazole, Ph3P; (f) NaCN; (g) concd. HCl/MeOH; (h) DMI, 140 ◦C; (i) (1) hꢀ, (2) ꢀ.

538
steroids 7 1 ( 2 0 0 6 ) 529–540
Fig. 4 – Synthesis of tetraol derivative (7): (a) Ac2O, pyridine; (b) BF3·OEt2; (c) DMI, 140 ◦C; (d) LiAlH4; (e) (1) hꢀ, (2) ꢀ.
completely identical with material prepared by the convergent
method described below (Fig. 7).
32 by hydrolysis and subsequent triethylsilyl ether formation
in 93% yield.
Next, we performed the synthesis of the C/D-ring frag-
ment (45) from the Inhoffen-Lythgoe diol (34) [22,23]. Selective
tosylation of the primary alcohol gave tosylate (35) in 91%
yield. The tosylate (35) was converted to sulfone (36) in 78
% yield in known procedure [24]. The secondary alchol of 36
was protected by triethylsilyl group to afford 37, quantitatively.
Alkylation of 37 with 4-bromo-2-methyl-2-butene in the pres-
ence of n-BuLi and subsequent desilylation gave 38 in 96%
yield. Reductive desulfonylation with sodium mercury amal-
gam (Na/Hg) produced an inseparable ene/diene mixture (4/1)
(39) in 72% yield [25]. After protection of the hydroxy group
of 39 by acetyl group, the ene/diene mixture (4/1) (40) was
treated with the commercially available reagent for sharpless
asymmetric dihydroxylation, AD-mix-␣ followed by catalytic
hydrogenation to afford diol (41) as a sole product in 96% yield
[26]. Since the asymmetric dihydroxylation occurred predomi-
nantly at the trisubstituted olefin, subsequent catalytic hydro-
genation gave the requisite diol as a sole product. Protection
of two hydroxy groups in 41 by tert-butyldimethylsilyl (TBS)
group gave 42 in 95% yield and deprotection of acetyl group
in 42 with lithium aluminum hydride (LiAlH4) furnished 43
quantitatively. The alcohol (43) was oxidized with tetrapropyl-
ammonium perruthenate (TPAP) and N-methylmorpholine-
N-oxide (NMO) to give ketone (44) in 95% yield. Wittig reac-
tion of 44 with (bromomethyl)triphenylphosphonium bro-
mide (Ph3PCH2Br2) in the presence of sodium hexamethyldi-
silazide (NaHMDS) produced 45 in 55% yield. The similar reac-
tion of 40 with AD-mix-␤ gave rise to the C/D-ring fragment
(50) in the 24(R)-series (Fig. 6). This synthetic route represents
3.2.
Synthesis of pentaols (8 and 9) arising from the
combination metabolism of the side chain and the
2ˇ-position
To synthesize pentaols (8 and 9) in 24(S) and 24(R) forms aris-
ing from the combination metabolism between the side chain
and the 2␤-position, we adopted a convergent approach. In
the convergent synthesis, the A-ring fragment (33) is coupled
with the C/D-ring fragments (45 and 50) for the construction
of the triene system of the vitamin D3 structures. First, we
undertook the synthesis of the A-ring fragment (33) (Fig. 5).
Diol (24), obtained from d-mannitol [16], was protected as
the pivalate ester (25) in 72% yield. This ester was subjected
to hydrolysis with 1N hydrochloric acid in THF at 20 ◦C for
12 hours to give diol (26) in 50% yield accompanied by tetraol
(27) in 34% and recovery of 25 in 16%, which were easily sepa-
rated by silica gel column chromatography. Diol (26) was then
converted to orthoester (28) in 88% yield with triethyl ortho-
formate (HC(OEt)3) and p-toluenesulfonic acid monohydrate
(TsOH·H2O) in CH2Cl2. Upon treatment of orthester (28) with
boiling acetic anhydride for 14 h, terminal olefin formation
took place cleanly to produce olefin (29) in 93% yield [17–21].
Deketalization of 29 afforded diol (30) in 93% yield. Subsequent
Mitsunobu reaction of 30 with diisopropyl azodicarboxylate
(DIAD) and Ph3P in dioxane gave epoxide (31) in 74% yield.
Epoxide (31) was treated with lithio trimethylsilysacetylene in
the presence of BF3·OEt2 at −78 ◦C to provide 1,7-eneyne (32)
in 85% yield. Finally, A-ring fragment (33) was obtained from
Fig. 5 – Synthesis of the A-ring fragment: (a) t-BuCOCl, pyridine, DMAP; (b) 1N HCl; (c) HC(OEt)3, TsOH·H2O; (d) Ac2O; (e) 1N
HCl; (f) Ph3P, DIAD; (g) LiC≡CTMS, BF3·OEt2; (h) (1) 1N NaOH, (2) TESOTf, Et3N.

steroids 7 1 ( 2 0 0 6 ) 529–540
539
Fig. 6 – Synthesis of the C/D-ring fragments: (a) TsCl, pyridine, DMAP; (b) (1) NaI, (2) NaSO2Ph·2H2O; (c) TESCl, imidazole; (d)
(1) n-BuLi, (CH3)2C=CHCH2Br, (2) 3N HCl; (e) Na/Hg; (f) Ac2O, pyridine, DMAP; (g) (1) AD-mix-␣, Me3SO2NH2, (2) Pd-C, H2; (h)
TBSOTf, 2,6-lutidine; (i) LiAlH4; (j) TPAP, NMO; (k) Ph3PCH2Br2, NaHMDS; (l) (1) AD-mix-␤, Me3SO2NH2, (2) Pd-C, H2.
Fig. 7 – Coupling the A-ring fragment with C/D-ring fragments: (a) Pd(PPh3)4, Et3N; (b) TBAF.
a novel versatile approach for preparation of 24-hydroxylated
C/D-ring fragments in 24(S) and 24(R) series in good overall
yields for the synthesis of vitamin D3 analogs.
used as authentic samples for metabolic studies of ED-71 (1).
The detailed metabolic pathway and metabolites of ED-71 (1)
will be reported elsewhere.
Having the A-ring fragment (33) and the C/D-ring frag-
ments (45 and 50), we conducted the fragment coupling reac-
tion in the presence of tetrakis(triphenylphosphine)palladium
(Pd(PPh3)4) following Trost’s methodology [27]. This gave rise
to (53) and (54) in 27% and 23% yields, respectively, which were
then deprotected by tetrabutylammonium fluoride (TBAF) to
afford 24(S)OH-pentaol (8) and 24(R)OH-pentaol (9) in 70% and
50% yields, respectively.
Acknowledgement
We are grateful to Professor David Horne of Oregon State Uni-
versity for helpful discussions.
Finally, tetraol (7) was also prepared by desilylation of 55,
which was also obtained in a convergent manner by coupling
A-ring fragment (33) and C/D-ring fragment (52). This reac-
tion sequence delivered 7 that was identical to authentic 7
produced in the aforementioned linear synthesis (Fig. 7).
r e f e r e n c e s
[1] Miyamoto K, Murayama E, Ochi K, Watanabe H, Kubodera
N. Synthetic studies of vitamin D analogs. XIV. Synthesis
and calcium regulating activity of vitamin D3 analogs
bearing a hydroxyalkoxy group at the 2␤-position. Chem
Pharm Bull 1993;41:1111–3.
4.
Conclusion
[2] Okano T, Tsugawa N, Masuda S, Takeuchi A, Kobayashi T,
Takita Y, et al. Regulatory activities of
Four putative metabolites of ED-71 (1) have been synthesized,
namely methyl ester derivative (6), tetraol derivative (7), 24(S)-
pentaol (8), and 24(R)-pentaol (9). These metabolites are now
2␤-(3-hydroxypropoxy)-1␣,25-dihydroxyvitamin D3, a novel
synthetic vitamin D3 derivative, on calcium metabolism.
Biochem Biophys Res Commun 1989;163:1444–9.

540
steroids 7 1 ( 2 0 0 6 ) 529–540
[3] Kobayashi T, Okano T, Tsugawa N, Murano M, Masuda S,
[15] Hijikura I, Doi T, Takahashi T. Parallel synthesis of a
vitamin D3 library in the solid-phase synthesis. J Am
Chem Soc 2001;123:3716–22.
[16] Heathcock CH, Pirrung MC, Lampe J, Buse CT, Young SD.
Acyclic stereoselection. 12. Double stereodifferentiation
with mutual kinetic resolution. A superior class of
reagents for control of Cram’s rule stereoselection in
synthesis of erythro-␣-alkyl-␤-hydroxy carboxylic acids
from chiral aldehydes. J Org Chem 1981;46:
2290–300.
Takeuchi A, et al.
2␤-(3-Hydroxypropoxy)-1␣,25-dihydroxyvitamin D3 (ED-71),
preventive and therapeutic effects on bone mineral loss in
ovariectomized rats. Bioorg Med Chem Lett 1993;3:1815–9.
[4] Ono Y, Watanabe H, Shiraishi A, Takeda S, Higuchi Y, Sato
K, et al. Synthetic studies of vitamin D analogs. XXIV.
Synthesis of active vitamin D3 analogs substituted at the 2
␤-position and their preventive effects on bone mineral
loss in ovariectomized rats. Chem Pharm Bull
1997;45:1626–30.
[17] Veek APM, Putten FH. Alkyl 1,1-dialkoxy-alkane- and
-arene-carboxylates. Part IV (1). Thermal decomposition
reactions of 2-acetoxy-1,3-dioxolanes. Tetrahedron Lett
1970;11:3951–2.
[5] Ono Y, Kawase A, Watanabe H, Shiraishi A, Takeda S,
Higuchi Y, et al. Syntheses and preventive effects of
analogues related to
1␣,25-dihydroxy-2␤-(3-hydroxypropoxy)vitamin D3 (ED-71)
on bone mineral loss in ovariectomized rats. Bioorg Med
Chem 1998;6:2517–23.
[18] Hanessian S, Bargiotti A, LaRue M. A mild and
stereospecific conversion of vicinal diols into olefins.
Tetrahedron Lett 1978;19:737–40.
[6] Bouillon R, Okamura WH, Norman AW. Structure–function
relationships in the vitamin D endocrine system. Endocr
Rev 1995;16:200–57.
[7] Kubodera N, Tsuji N, Uchiyama Y, Endo K. A new active
vitamin D analog, ED-71, causes increase in bone mass
with preferential effects on bone in osteoporotic patients.
J Cell Biochem 2003;88:286–9.
[8] Takasaki Y, Suda T, Yamada S, Takayama H, Nishii Y.
Isolation, identification, and biological activity of
25-hydroxy-24-oxovitamin D3: a new metabolite of vitamin
D3 generated by in vitro incubations with kidney
homogenates. Biochemistry 1981;20:1681–6.
[9] Yamada S, Ohmori M, Takayama H, Takasaki Y, Suda T.
Isolation and identification of 1␣- and 23-hydroxylated
metabolites of 25-hydroxy-24-oxovitamin D3 from in vitro
incubates of chick kidney homogenates. J Biol Chem
1983;258:457–63.
[19] Camps P, Font J, Ponsati O. A short synthesis of
5-hydroxymethyl-(5)-furan-2-one and derivatives from
ribonolactone. Tetrahedron Lett 1981;22:1471–2.
[20] Camps P, Cardellach J, Font J, Ortuno RM, Ponsati O.
Studies on structurally simple ␣,␤-butenolides II:
(−)-(S)-␥-hydroxymethyl-␣,␤-butenolide and derivatives
from d-ribonolactone efficient synthesis of (−)-ranunculin.
Tetrahedron 1982;38:2395–402.
[21] Block E. Olefin synthesis by deoxygenation of vicinal diols.
In: Dauben WG, editor. Organic reactions, 30. John Wiley &
Sons, Inc.; 1983. p. 481.
[22] Infhoffen HH, Gerhard Q, Siegismund S, Gerhard F, Tober
E. Abbau der vitamin D2 und D3 zum
8-methyl-trans-hydrindanol-(4)-on-(1). Chem Ber
1858;91:781–90.
[23] Lythgoe B, Roberts DA, Waterhouse I. Calciferol and its
relatives. Part 20. A synthesis of Windaus and
Grundmann’s C19 ketone. J Chem Soc Perkin Trans 1
1977;23:2608–12.
[24] Vennstra GE, Zwaneburg B. An improved synthesis of
sulfones using tetrabutyl-ammonium sulfinates. Synthesis
1975:519–20.
[10] Kruse R, Popjak G, Bishop JE, Norman AW.
23,25-Dihydroxy-24-oxovitamin D3: a metabolite of vitamin
D3 made in the kidney. Biochemistry 1983;22:1798–805.
[11] Ikekawa N, Fujimoto Y. Active form of vitamin D. J Synth
Org Chem Jpn 1988;46:455–66.
[12] Ikekawa N. Structures and biological activities of vitamin D
metabolites and their analogs. Med Res Rev 1987;7:333–66.
[13] Hatakeyama S, Kawase A, Uchiyama Y, Maeyama J,
Iwabuchi Y, Kubodera N. Synthesis and biological
characterization of
1␣,24,25-trihydroxy-2␤-(3-hydroxypropoxy)vitamin D3
(24-hydroxylated ED-71). Steroids 2001;66:267–76.
[14] Watanabe H, inventor; Chugai Pharmaceutical Co., Ltd.,
Japan, assignee. Vitamin D derivatives at the 2␤-position.
WO99/52863, October 21, 1999.
[25] Florian D, Johann L, Hans-Gu¨ nther S. An approach to
serrulatane diterpenes via endo-selective conjugate
nucleophilic addition to arene-Cr(CO)3 Complexes. Org Lett
2002;4:3915–8.
[26] Sinha SC, Keinan E. Total synthesis of (+)-aspicilin. The
naked carbon skeleton strategy vs the bioorganic
approach. J Org Chem 1997;62:377–86.
[27] Trost BM, Dumas J. New stragegy for the total synthesis of
1␣-hydroxyvitamin D derivatives. J Am Chem Soc
1992;114:1924–5.