A new highly stereoselective synthesis of cerebrosterol, an agonist of the nuclear receptor LXRs

Article abstract of DOI:10.1016/S0040-4020(01)00812-2

The title compound, cerebrosterol 1, was synthesized stereoselectively (97% d.e.) in 41% overall yield from methyl hyodeoxycholanate 2 in 10 steps with desmosterol acetate 8 as the key intermediate and the modified Sharpless asymmetric dihydroxylation as the key step.

Full text of DOI:10.1016/S0040-4020(01)00812-2

TETRAHEDRON
Pergamon
Tetrahedron 57 )2001) 8291±8296
A new highly stereoselective synthesis of cerebrosterol, an agonist
of the nuclear receptor LXRs
²
Xiang-Dong Zhou and Wei-Shan Zhoup
Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China
Received 8 May 2001; revised 10 July 2001; accepted 2 August 2001
AbstractÐThe title compound, cerebrosterol 1, was synthesized stereoselectively )97% d.e.) in 41% overall yield from methyl hyodeoxy-
cholanate 2 in 10 steps with desmosterol acetate 8 as the key intermediate and the modi®ed Sharpless asymmetric dihydroxylation as the key
step. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
binaphthyl lithium aluminum hydride reagent, which gave
the 24S-alcohol with 94% d.e.16 Furthermore, addition of
diisopropylzinc to steroidal 24-aldehyde with a chiral
b-amino alcohol as ligand also gave 24S-hydroxycholes-
terol in good yield with high diastereoselectivity )d.e.
87%).17 Corey and Grogan18 disclosed a successful
Sharpless dihydroxylation of desmosterol )8, C3±OH) in
82% yield with 92% d.e. However, this method still required
a long reaction time. This led us to develop an ef®cient and
highly stereoselective method using readily available
starting materials, such as methyl hyodeoxycholanate
)Me-HDCA, 2), which is also a ligand of LXRa.19 Here
we report a new method for highly stereoselective synthesis
of the target molecule 1 using Me-HDCA 2 as the starting
material.
Cerebrosterol )24S-hydroxycholesterol, 1) is presentin
small amounts in human and animal brain,1 adrenals,2 and
liver.3 It was reported that the liver X receptors, LXRa4 and
LXRb,5 are the speci®c biological sensors controlling the
metabolism and reverse transportation of cholesterol in the
body. This operates by means of formation of a permissive
heterodimer with the 9-cis-retinoic acid receptor RXR,6
which regulates the transcriptional expression of their target
genes, such as CYP7A,7 CETP,7,8 ABC-1,9 and ApoE,10 etc.
Subsequently, LXRs have become a promising targets for
the development of drugs for the treatment for athero-
sclerosis.10,11 Thus, as one of the most potent agonists of
LXRs,7a,12 cerebrosterol can be a potential drug for
treatment for atherosclerosis. Moreover, cerebrosterol is
involved in cholesterol homeostasis and in some diseases
of the brain such as Alzheimer's.13 Hence itmightalso be
used as an early biochemical marker for diagnosis of
Alzheimer's and vascular demented diseases.13f
2. Results and discussion
Me-HDCA, which is very readily available in China, is
selected as the starting material because the 3a and 6a-dihy-
droxy group can be easily converted to the D5-3b-ol
moiety20. The AB-ring of Me-HDCA itself is essential for
subtype selective ligation of LXRa.19 The 24-carboxylic
acid sidechain can also be transformed to other desired
ones,21 such as that in desmosterol, which is a common
precursor to many LXR ligands, including 24S,25-epoxy-
cholesterol; all these will expand the scope of the ligands of
LXRs.
The early syntheses of cerebrosterol were performed by
benzoylation of a mixture of 24R- and 24S-hydroxy-
cholesterol, followed by either resolution via recrystalli-
zation or chromatography and hydrolysis.14 Recently
several stereoselective methods have been reported for
introducing a 24S-hydroxy group into the sidechain. One
of them is the coupling of a C22-sulfone derived from
stigmasterol with a chiral C5 epoxide or a chiral C5 iodide
to give a 24S-hydroxysteroid.15 A second method is the
reduction of a cholest-25-en-24-one system with 3 molar
equivalents of Noyori's )S)-)2)-2,20-dihydroxy-1,10-
Our synthesis of cerebrosterol is divided into two stages. In
the ®rst stage, the desmosterol acetate 8, an important inter-
mediate of cerebrosterol, was synthesized as depicted in
Scheme 1. Thus, the 3a,6a-dihydroxy groups in 2 were
protected with dimethoxymethane to afford the 3a,6a-
bismethoxy methyl ether 3 in 91% yield. Then, 3 was
subjected to reduction with lithium aluminum hydride in
dry tetrahydrofuran to afford 24-alcohol 4 in high yield
Keywords: cerebrosterol; Sharpless AD reaction; stereoselective.
Corresponding author. Tel.: 186-21-6416-3300; fax: 186-21-6416-
6128; e-mail: zhws@pub.sioc.ac.cn
Present address: Department of Chemistry, the Third Military Medical
University, Chongqing 400038, China
p
²
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020)01)00812-2

8292
X.-D. Zhou, W.-S. Zhou / Tetrahedron 57 +2001) 8291±8296
Scheme 1. Reagents and conditions: )a) CH3OCH2OCH3, P2O5, CHCl3, rt91%; )b) LiAlH 4, THF, rt94%; )c) )COCl) 2, DMSO, Et3N, CH2Cl2, 2788C, 100%;
)d) BuLi, Ph3P1CH)CH3)2I2, THF, rt, 97%; )e) PPTS, t-Butanol, re¯ux, 89%; )f) TsCl, Py, 08C, then KOAc, DMF±H2O, 1058C, then Ac2O, Py, rt, 87%;
)g) )DHQ)2PHAL, K2OsO2)OH)4, K3Fe)CN)6, K2CO3, CH3SO2NH2, t-Butanol±H2O±Methyl Butyl Ether )2.5:2.5:3), rt 98.6% d.e., 83%; )h) Ac2O, Py, rt,
95%; )i) CH3SO2Cl, DMAP, Et3N, CH2Cl2, 08C rt91%; )j) NH 2OH, EtOAc, DMF, 958C, then KOH, MeOH, rt, 90%.
)94%). Swern oxidation of 4 provided the 24-aldehyde 5 in
quantitative yield, which was treated with the THF solution
of n-butyl lithium and isopropyltriphenylphosphonium
iodide atrtto give D24-compound 6 in 97% yield. Removal
of 3a,6a-dihydroxy protective groups by re¯ux with PPTS
in t-butanol gave D24-5b-3a,6a-dihydroxycholestene 7 in
89% yield. Ditosylation of 7 with p-toluenesulfonyl chloride
in pyridine at0 8C, followed by treatment of the resulting
crude mixture with potassium acetate in DMF±water at
1058C20 and acetylation with acetic anhydride and pyri-
dine22 gave desmosterol acetate 8 in 87% yield. Thus the
important intermediate was synthesized from Me-HDCA 2
in 6 steps in 64% overall yield on a multigram scale. The
desmosterol could also be used for syntheses of another
oxysterol 24S,25-epoxycholesterol18,23 and some other
cytotoxic oxydesmosterols.24
and K2OsO2)OH)4 in t-BuOH±H2O±methyl t-butyl ether
)2.5:2.5:3) within 20 h to give 3b-acetoxy-24S,25-di-
hydroxycholesterol 9 in 98.6% d.e. and 83% yield. The
result is much better than the usual AD-reaction procedure
in terms of d.e. value and reaction time.18,23 Selective
acetylation of the 24S-hydroxy of 9 with acetic anhydride
and pyridine gave the diacetate 10 in 95% yield. Dehydra-
tion of 10 with methanesulfonyl chloride, triethylamine and
DMAP provided D5,25-3b,24S-dihydroxycholestadiene±
diacetate 11 in 91% yield.27 Finally, diimide reduction28
of 11 with high regioselectivity followed by hydrolysis
with potassium hydroxide in methanol gave the target
molecule, cerebrosterol 1 in 90% yield with 97% d.e.
3. Conclusion
With the key intermediate in hand, our attention turned to
the second stage of stereoselective synthesis of cerebro-
sterol from desmosterol acetate. In this stage, the key step
is the stereoselective introduction of 24S,25-dihydroxy
groups into the sidechain via Sharpless catalytic asymmetric
dihydroxylation.25 In the course of the application of
Sharpless catalytic asymmetric dihydroxylation on the
unsaturated steroidal sidechain, we found that the low
solubility of steroids in t-butanol±water solvent system
led to the lower value of %d.e and the longer reaction
time.18,23 Fortunately, we found that addition of methyl
t-butyl ether as an auxiliary solvent to the Sharpless
dihydroxylation solvent system increased the solubility of
the steroid, and consequently gave better results.26 Thus,
using an improved Sharpless catalytic asymmetric di-
hydroxylation, 8 was dihydroxylated with )DHQ)2PHAL
The main advantages of the present synthesis of cerebro-
sterol include the easy availability of starting material, a
high yield in the preparation of the key intermediate
desmosterol acetate, high stereoselectivity for introduction
of the 24S-hydroxy group and high regioselectivity for the
diimide reduction of the double bonds. Furthermore, this
method could be used to prepare various analogs of cerebro-
sterol. Extension of the improved Sharpless AD reaction to
the unsaturated sidechain of steroid is in progress.
4. Experimental
4.1. General
All melting points are uncorrected. IR spectra were recorded

X.-D. Zhou, W.-S. Zhou / Tetrahedron 57 +2001) 8291±8296
8293
with FT-IR apparatus. 1H- and 13C-NMR spectra were
recorded at300 and 75 MHz in CDCl . Chemical shifts
The stirring was continued for another 35 min. Triethyl-
amine )17 mL) was added dropwise to the reaction mixture
and a large amount of white solid appeared. After stirring
for 10 min, the reaction mixture was allowed to warm to
room temperature and washed in turn with sat. NH4Cl
)3£50 mL), sat. NaHCO3)3£50 mL), brine )3£50 mL) and
dried over sodium sulfate. Removal of the solvent in vacuo
and puri®cation by ¯ash chromatography )Pet. Ether/ethyl
acetate 20:1) afforded pure compound 5 )colorless oil
3
are reported in ppm relative to TMS as internal standard.
Mass spectra were recorded by EI methods. Flash column
chromatography was carried out with silica gel )300±
400 mesh). Solvent THF was distilled over sodium, with
dichloromethane being distilled over CaH2. The d.e. value
was determined by HPLC analysis on Inersil ODS-3 column
with CH3CN±H2O as an eluent.
20
9.427 g, 100%). [a]D ˆ110.6 )c, 0.34, CHCl3); MS-
4.1.1. Methyl 3,6-dimethoxymethyl-3a,6a-dihydroxy-
5b-cholanate .3). To a solution of methyl hyodeoxy-
cholanate 2 )5.00 g, 12.3 mmol) in dry chloroform
)75 mL) and dimethoxymethane )16.30 mL, 184 mmol)
was added phosphorus pentoxide )5.5 g, 36.9 mmol) with
stirring at room temperature. After being stirred for 8 h,
the mixture was ®ltered through a pad of silica gel, washed
with chloroform )3£30 mL). Removal of the solvent in
vacuo afforded a lightyellow oil )7.565 g), which was
puri®ed by ¯ash chromatography )Pet ether/ethyl acetate
25:l) to afford pure 3 )5.562 g, 91%). Recrystallization
from methanol/water gave colorless needles: mp 78±798C;
EI)m/z): 464 )M1), 400 )M122MeOH), 340 )M12
1
2CH3OCH2OH); IR )cm21): 1726 )±CHO); H NMR d:
0.64 )3H, s, 18-CH3), 0.91 )3H, s, 21-CH3), 0.92 )3H, s,
19-CH3), 3.36 )3H, s, OCH3), 3.37 )3H, s, OCH3), 3.50
)1H, m, 3b-H), 3.91 )1H, m, 6b-H), 4.63 and 4.65 )2H,
each d, J1ˆ6.7 Hz, J2ˆ6.1 Hz, ±OCH2O±), 4.67 and 4.71
)2H, each d, J1ˆ6.80 Hz, J2ˆ6.85 Hz, ±OCH2O±), 9.77
)1H, t, Jˆ1.6 Hz, 24-CHO); Anal. Calcd for C28H48O5´1/
3H2O: C, 71.45%; H, 10.42%; Found. C, 71.52%; H,
10.58%.
4.1.4. D24-3,6-Dimethoxymethyl-3a,6a-dihydroxy-5b-
cholestene .6). n-Butyl lithium )2.6 M, 0.70 mL) was
added to a suspension of isopropyltriphenylphosphonium
iodide )684 mg, 1.58 mmol) in dry tetrahydrofuran, giving
a deep red solution. Then 5 )367 mg, 0.79 mmol) in dry
tetrahydrofuran )8 mL) was added. After stirring for 4.5 h,
TLC showed disappearance of starting material. Sat. NH4Cl
)2 mL) was added to the reaction mixture, and a large
amount of white solid appeared. The mixture was ®ltered
through a pad of Celite and washed with ethyl acetate
)2£20 mL). The ®ltrate was then washed with 5% HCl
)3£10 mL), sat. NaHCO3 )3£10 mL) and brine )3£10 mL)
and dried over sodium sulfate. Removal of the solvent in
vacuo and puri®cation by ¯ash chromatography )Pet. Ether/
acetone 30:1) afforded 6 as a colorless oil )375 mg, 97%):
[a]D ˆ111.9 )c, 0.3, CHCl3) )lit.29 mp 79±79.58C;
16
[a]D ˆ114.2 )c, 0.72, CHCl3)); MS-EI)m/z): 426 )M12
25
CH3OH), 430 )M122CH3OH), 370 )M122HOCH2OCH3);
1
IR )cm21): 1743 )COOCH3); H NMR d 0.62 )3H, s,18-
CH3), 0.90 )3H, s, 21-CH3), 0.89 )3H, s, 18-CH3), 2.22
)2H, m, 23-CH2), 3.35 )3H, s, OCH3), 3.36 )3H, s, OCH3),
3.50 )1H, m, 3b-H), 3.65 )3H, s, COOCH3), 3.89 )1H, m,
6b-H), 4.62 and 4.64 )2H, each d, J1ˆ6.8 Hz, J2ˆ6.5 Hz,
±OCH2O±), 4.65 and 4.70 )2H, each d, J1ˆ6.6 Hz, J2ˆ
6.7 Hz, OCH2O)
4.1.2.
3,6-Dimethoxymethyl-3a,6a,24-trihydroxy-5b-
cholane .4). A solution of 3 )5.038 g, 10.20 mmol) in dry
tetrahydrofuran )50 mL) was added to a suspension of
lithium aluminum hydride )572 mg, 15.0 mmol) in dry
tetrahydrofuran )50 mL) under argon over 30 min. The
reaction mixture was stirred for another hour before being
quenched with Na2SO4´10H2O, ®ltered through a pad of
Celite, and washed with ethyl acetate. Removal of the
solventin vacuo and puri®caiton by ¯ash chromaot graphy
)Pet. ether/ethyl acetate 10:1) afforded pure 4 )4.483 g,
[a]D ˆ114.4 )c, 0.17, CHCl3); MS-EI )m/z): 491 )M111),
22
430 )M1112OCH2OCH3), 414 )M1112OCH2OCH3±
Me), 397 )M11122HOCH3±2Me), 381 )M1112
2HOCH3±MeO±Me), 367 )M11122HOCH2OCH3), 351
)M122HOCH2OCH3±Me); 1H NMR d: 0.62 )3H, s,
18-CH3), 0.90 )3H, s, 19-CH3), 0.92 )3H, d, Jˆ6.8 Hz,
21-CH3), 1.59 )3H, s, 26-CH3), 1.63 )3H, s, 27-CH3), 3.35
and 3.36 )3H, s, OCH3), 3.49 )1H, m, 3b-H), 3.89 )1H, m,
6b-H), 4.62 )2H, s, ±OCH2O), 4.66 and 4.70 )2H, each d,
Jˆ7.1 Hz, ±OCH2O), 5.08 )1H, t, Jˆ7.1 Hz, 24-H); Anal.
Calcd for C31H54O4´1/4 H2O: C, 75.17%; H, 11.09%; Found:
C, 74.99%; H, 11.25%.
23
94%): mp 74.5±758C; [a]D ˆ120.6 )c, 0.5, CHCl3);
MS-EI )m/z): 404 )M12HOCH2OCH3), 401 )M12
2HOCH3±H2O), 371 )M122HOCH32HCHO), 342 )M12
1
2HOCH2OCH3); IR )cm21): 3486 )OH); H NMR d 0.62
)3H, s, 18-CH3), 0.91 )3H, d, Jˆ8.2 Hz, 21-CH3), 0.97 )3H,
s, 19-CH3), 3.35 )3H, s, OCH3), 3.36 )3H, s, OCH3), 3.50
)1H, m, 3b-H), 3.60 )2H, t, Jˆ6.3 Hz, 24-CH2), 3.90 )1H,
m, 6b-H), 4.6 and 4.63 )2H, each d, J1ˆ6.4 Hz, J2ˆ6.8 Hz,
±OCH2O), 6.66 and 6.70 )2H, each d, J1ˆ6.6 Hz, J2ˆ
6.7 Hz, ±OCH2O); Anal. Calcd for C28H50O5: C, 72.04%;
H, 10.80%; Found. C, 72.19%; H, 10.88%.
4.1.5. D24-3a,6a-Dihydroxy-5b-cholestene .7).
A
mixture of 6 )150 mg, 0.306 mmol) and PPTS )335 mg,
1.34 mmol) in t-butanol )15 mL) was re¯uxed for 10 h.
After removal of the solvent in vacuo, the residue was
dissolved in ethyl acetate )50 mL) and washed with 10%
HCl )3£10 mL), sat. NaHCO3 )3£10 mL) and brine
)3£5 mL) and dried over sodium sulfate. Removal of the
solventin vacuo and puri®caiton by ¯ash chromaot graphy
)Pet. ether/acetone 4:1) afforded pure 7 )granular crystal,
4.1.3.
3,6-Dimethoxymethyl-3a,6a-dihydroxy-5b-
cholane-24-al .5). A solution of dimethyl sulfoxide
)3.25 mL, 45.85 mmol) in dry dichloromethane )10 mL)
was added to a solution of oxalyl chloride )2 mL,
22.93 mmol) in dry dichloromethane )50 mL) stirred in a
dry ice±acetone bath under argon over 10 min. After stirring
for another 10 min, a solution of 4 )9.437 g, 20.33 mmol) in
dichloromethane )35 mL) was added dropwise over 15 min.
20
140 mg, 89%): mp 168±1698C; [a]D ˆ110.6 )c, 0.6,
CHCl3); MS-EI )m/z): 402 )M1), 384 )M12H2O), 369
)M12H2O±Me), 351 )M122H2O±Me); IR )cm21): 3369
)OH); 1H NMR d 0.64 )3H, s, 18-CH3), 0.89 )3H, s,
19-CH3), 0.92 )3H, d, Jˆ7.5 Hz, 21-CH3), 3.61 )1H, m,

8294
X.-D. Zhou, W.-S. Zhou / Tetrahedron 57 +2001) 8291±8296
3b-H), 4.05 )1H, m, 6b-H), 5.09 )1H, t, Jˆ6.6 Hz, 24-H);
13C NMR d 130.9, 125.2, 71.6, 68.1, 56.3, 56.2, 48.5, 42.9,
40.0, 39.9, 36.1, 36.0, 35.6, 35.0, 34.9, 30.2, 29.3, 28.2,
25.7, 24.8, 24.3, 23.5, 20.8, 18.6, 17.6, 12.0; Anal. Calcd
for C27H46O2´1.5H2O: C, 75.47%; H, 11.49%; Found. C,
75.29%; H, 11.33%.
chromatography )Pet. ether/acetone 4:1) to afford pure 9
)1.258 g, 98.6% d.e., 83%): mp 135±1368C, [a]D
23
ˆ
227.2 )c, 0.9, CHCl3); MS-EI )m/z): 400 )M12HOAc),
382 )M12HOAc±H2O); IR )cm21): 3445 )OH), 1735
)CH3CO2); H NMR d 0.68 )3H, s, 18-CH3), 0.94 )3H, d,
1
Jˆ6.5 Hz, 21-CH3), 1.02 )3H, s, 19-CH3), 1.18 )3H, s,
26-CH3), 1.22 )3H, s, 27-CH3), 2.04 )3H, s, CH3CO2),
2.32 )2H, d, Jˆ8.2 Hz, 7-H), 3.28 )1H, m, 24b-H), 4.61
)1H, m, 3a-H), 5.38 )1H, d, Jˆ4.7 Hz, 6-H); 13C NMR d
170.6, 139.7, 122.6, 79.6, 74.0, 73.2, 56.7, 56.0, 50.0, 42.4,
39.7, 38.1, 37.0, 36.6, 36.0, 33.3, 31.9, 28.3, 28.2, 27.8,
26.5, 24.3, 23.2, 21.4, 21.0, 19.3, 18.8, 11.9; Anal. Calcd
for C29H48O4´1/4 H2O: C, 74.88%; H, 10.51%; Found: C,
75.08%; H, 10.70%.
4.1.6. D5,24-3b-Hydroxy-cholestadiene-acetate .desmos-
terol acetate) .8). A solution of 7 )4.020 g, 10 mmol) in
pyridine )10 mL) was cooled to 08C and p-toluenesulfonyl
chloride )4.736 g, 25 mmol) was added. The resulting
mixture was stirred for two days. The reaction mixture
was poured into a ice-cooled dilute HCl solution )150 mL)
and a large amount of precipitate appeared. After stirring for
2 h, the mixture was extracted with ethyl acetate
)3£50 mL). The combined organic layer was washed with
sat. NaHCO3 )3£150 mL), brine )3£150 mL), dried over
sodium sulfate and concentrated. The resulting residue
was dissolved in a mixture of DMF )40 mL) and water
)4.3 mL). Potassium acetate )10.03 g) was added. The
mixture was heated to 1058C and stirred for 5 h. The reac-
tion mixture was cooled to room temperature and poured
into ice-cooled dilute HCl solution. A large amount of pre-
cipitate was produced, which was ®ltered off and washed
with a large amount of water until neutral, and dried to give
a lightyellow solid residue )4.08 g). TLC showed it
contained the 3b-OH compound. The residue was dissolved
in a mixture of acetic anhydride )40 mL) and pyridine
)40 mL) and stirred at room temperature for 25 h. The reac-
tion mixture was taken up in ethyl acetate, washed with
water )5£100 mL), 10% HCl )3£100 mL), sat. NaHCO3
)3£100 mL), brine )3£100 mL) and dried over sodium
sulfate. Removal of the solvent in vacuo and puri®cation
by ¯ash chromatography )Pet. ether/acetate 9:1) afforded
pure 8 )3.705 g, 87%): mp 90.5±918C )lit.23 mp 91±
4.1.8.
D5-3b,24S,25-Trihydroxycholestene-3,24-di-
acetate .10). A solution of 9 )336 mg, 0.730 mmol) in acetic
anhydride )4 mL) and pyridine )4 mL) was stirred at room
temperature for 9 h. The reaction mixture was partitioned
between ethyl acetate )50 mL) and water. The organic layer
was washed with 5% HCl )3£10 mL), sat. NaHCO3
)3£10 mL), brine )3£10 mL), dried over sodium sulfate,
evaporated and puri®ed by ¯ash chromatography )Pet.
ether/ acetone 4:1) to afford pure compound 10 )378 mg,
22
95%): mp 175±1768C; [a]D ˆ231.1 )c, 0.5, CHCl3); MS-
EI )m/z): 485 )M1112H2O), 442 )M12CH3COOH), 425
)M1112CH3COOH±H2O); IR )cm21): 3470 )OH), 1734
)CH3CO2), 1713 )CH3CO2); 1H NMR d 0.67 )3H, s,
18-CH3), 0.93 )3H, d, Jˆ6.5 Hz, 21-CH3), 1.02 )3H, s,
19-CH3), 1.20 )3H, s, 26-CH3), 1.20 )3H, s, 27-CH3), 2.03
)3H, s, 3b-CH3CO2), 2.11 )3H, s, 24a-CH3CO2), 4.60 )1H,
m, 3a-H), 4.72 and 4.76 )1H, d, d, J1ˆ2.4 Hz, J2ˆ10.1 Hz,
24b-H), 5.37 )1H, d, Jˆ4.8 Hz, 6-H); 13C NMR d 171.3,
170.5, 139.7, 122.6, 80.8, 74.0, 72.5, 56.6, 55.7, 50.0, 42.3,
38.1, 37.0, 36.6, 35.8, 32.5, 31.9, 28.1, 27.8, 26.8, 26.0,
25.0, 24.2, 21.4, 21.1, 21.0, 19.3, 18.8, 11.9; Anal. Calcd
for C31H50O5: C, 74.06%; H, 10.02%; Found. C, 74.14%; H,
10.06%.
22
938C); [a]D ˆ240.8 )c, 0.3, CHCl3); MS-EI )m/z): 411
)M12Me), 366 )M12HOAc), 351 )M12Me±HOAc); IR
)cm21): 1732 )COO); 1H NMR d 0.71 )3H, s, 18-CH3), 0.94
)3H, d, Jˆ6.5 Hz, 21-CH3), 1.04 )3H, s, 19-CH3), 1.61 )3H,
s, 26-CH3), 1.69 )3H, s, 27-CH3), 2.03 )3H, s, CH3CO), 2.32
)1H, m, 7-H), 4.61 )1H, m, 3a-H), 5.10 )1H, m, 24-H), 5.38
)1H, d, Jˆ4.7 Hz, 6-H); 13C NMR d 170.6, 139.7, 130.9,
125.2, 122.6, 74.0, 56.7, 56.1, 50.1, 50.0, 42.4, 39.7, 38.1,
37.0, 36.6, 36.1, 35.6, 31.9, 31.8, 28.2, 27.8, 25.7, 24.7,
24.3, 21.4, 21.0, 19.3, 18.6, 11.9; Anal. Calcd for
C29H46O2: C, 81.63%; H, 10.87%; Found: C, 81.64%; H,
11.16%.
4.1.9.
D5,25-3b,24S-Dihydroxycholestadiene-diacetate
.11). To a solution of 10 )110 mg, 0.219 mmol), DMAP
)1.075 mg, 5 mol%) and triethylamine )0.09 mL, 0.65
mmol) in dichloromethane )2 mL) stirred at 08C under
argon was added dropwise methanesulfonyl chloride
)0.0256 mL, 0.33 mmol). After 2 h, the reaction mixture
was diluted with ethyl acetate )20 mL), washed with brine
)3£20 mL), dried over sodium sulfate, concentrated and
puri®ed by ¯ash chromatography )Pet. Ether/acetone 4:1)
to afford pure 11 )64 mg, 91%, recovered 10 37 mg): mp
4.1.7. D5-3b,24S,25-Trihydroxycholestene-3-acetate .9).
A solution of K3Fe)CN)6 )3.257 g, 9.87 mmol), K2CO3
)1.362 g, 9.87 mmol), CH3SO2NH2 )313 mg, 3.29 mmol),
)DHQ)2PHAL )128 mg, 5 mol%), and K2OsO2)OH)4
)12 mg, 1 mol%) in t-butanol±water )1:1, 33 mL) was
cooled to 08C. A solution of 8 )1.400 g, 3.29 mmol) in
methyl t-butyl ether )20 mL) was added dropwise. The
resulting mixture was stirred vigorously for 20 h. The
reaction was quenched at 08C with sodium sul®te
)4.930 g). Stirring was continued for another hour. The
aqueous layer was extracted with ethyl acetate )3£25 mL).
The combined organic layers were washed with 2 M KOH
)3£25 mL), 10% HCl )3£10 mL), sat. NaHCO3 )3£10 mL),
brine )3£10 mL), dried over sodium sulfate and evaporated
to give the crude product, which was puri®ed by ¯ash
24
95±968C; [a]D ˆ243.3 )c, 0.13, CHCl3); MS-EI )m/z):
424 )M12CH3COOH), 364 )M122CH3COOH), 349
)M11122CH3COOH±H2O); IR )cm21): 3080 )CvCH2),
1733 )CH3CO2), 1654 )CvCH2), 904 )CvCH2); H NMR
1
d 0.67 )3H, s, 18-CH3), 0.93 )3H, d, Jˆ6.5 Hz, 21-CH3),
1.02 )3H, s, 19-CH3), 1.72 )3H, s, 27-CH3), 2.03 )3H, s,
3b-CH3CO2), 2.06 )3H, s, 24b-CH3CO2), 4.60 )1H, m,
3a-H), 4.89 )1H, t, Jˆ1.4 Hz, 26-H), 4.94 )1H, s, 26-H),
5.12 )1H, t, Jˆ6.8 Hz, 24b-H), 5.38 )1H, d, Jˆ4.9 Hz, 6-H);
Anal. Calcd for C31H48O4: C, 76.82%; H, 9.98%; Found: C,
76.74%; H, 10.01%.
4.1.10. Cerebrosterol .1). To a suspension of NH2OH´HCl
)3.59 g, 51.7 mmol) and DMF )10 mL) stirred at 08C was

X.-D. Zhou, W.-S. Zhou / Tetrahedron 57 +2001) 8291±8296
8295
)d) Teboul, M.; Enmark, E.; Li, Q.; Wikstrom, A. C.; Pelto-
Huikko, M.; Gustafsson, J. A. Proc. Natl. Acad. Sci. USA
1995, 92, 2096±2100.
added KOH )85%, 3.41 g, 51.7 mmol). After being stirred
for 30 min, the mixture was ®ltered and the solid was
washed with DMF )2±3 mL). The combined ®ltrate and
washings were cooled to 08C, and then ethyl acetate
)2.22 mL, 22.6 mmol) was added dropwise. After stirring
for another 30 min, the solution was added dropwise to
another ¯ask containing 11 )35 mg, 0.064 mmol) stirred at
90±958C. After stirring for 5 h, the mixture was cooled to
room temperature, water )15 mL) was added. The mixture
was then extracted with ethyl acetate )3£25 mL). The
combined organic layers were washed with water )3£
50 mL), 5% HCl )3£50 mL), sat. NaHCO3 )3£50 mL),
brine )3£50 mL), dried over sodium sulfate and evaporated
to afford the crude product which was dissolved in methanol
)5 mL) containing water )0.3 mL) and KOH )30 mg) and
re¯uxed for 5 h. After removal of solvents in vacuo, the
residue was dissolved in ethyl acetate )50 mL), washed
with water )3£10 mL), brine )3£10 mL), dried over sodium
sulfate, evaporated and puri®ed by ¯ash chromatography
)Pet. Ether/ethyl acetate 6:1) to afford pure cerebrosterol
1 )26 mg, 90%, 97% d.e.): mp 181±1828C )lit.14a 175±
6. Willy, P. J.; Mangelsdorf, D. J. Genes Dev. 1997, 11, 289±
298.
7. )a) Lehmann, J. M.; Kliewer, S. A.; Moore, L. B.; Smith-
Oliver, T. A.; Oliver, B. B.; Su, J.-L.; Sundseth, S. S.;
Winegar, D. A.; Blanchard, D. E.; Spencer, T. A.; Willson,
T. M. J. Biol. Chem. 1997, 272, 3137±3140. )b) Peet, D. J.;
Turley, D.; Ma, W.; Janowski, B. A.; Lobaccaro, J. M.;
Mangelsdorf, D. J. Cell 1998, 93, 693±704.
8. Luo, Y.; Tall, A. R. J. Clin. Invest. 2000, 105, 513±520.
9. )a) Repa, J. J.; Turley, S. D.; Lobaccaro, J.-M. A.; Medina, J.;
Li, L.; Lustig, K.; Shan, B.; Heyman, R. A.; Dietschy, J. M.;
Mangelsdorf, D. J. Science 2001, 289, 1524±1529.
)b) Venkateswaran, A.; Laf®tte, B. A.; Joseph, S. B.; Mak,
P. A.; Wilpitz, D. C.; Edwards, P. A.; Tantonoz, P. Proc.
Natl. Acad. Sci. USA 2000, 97, 12097±12102. )c) Costet, P.;
Luo, Y.; Wang, N.; Tall, A. R. J. Biol. Chem. 2000, 276,
28240±28245.
10. )a) Laf®tte, B. A.; Repa, J. J.; Joseph, S. B.; Wilpitz, D. C.;
Mangelsdorf, D. J.; Tantonoz, P. Proc. Natl. Acad. Sci. USA
2001, 98, 507±509. )b) Claudel, T.; Leibowitz, M. D.; Fievet,
C.; Taileux, A.; Wagner, B.; Repa, J. J.; Torpier, G.;
Lobaccaro, J.-M.; Paterniti, J. R.; Mangelsdorf, D. J.;
Heyman, R. A.; Auwerx, J. Proc. Natl. Acad. Sci. USA
2001, 98, 2610±2616.
24
1768C; lit.30 mp 181±182.58C); [a]D ˆ249.5 )c, 0.028,
CHCl3) )lit.30 [a]Dˆ248.3, CHCl3); MS-EI )m/z): 402
)M1), 384 )M12H2O), 369 )M12H2O±Me), 351 )M1
1
2H2O±Me); IR )cm21): 3369 )OH); H NMR d 0.68 )3H,
S, 18-CH3), 0.89 )3H, d, Jˆ6.8 Hz, 21-CH3), 1.01 )3H, s,
19-CH3), 3.30 )1H, m, 24-H), 3.53 )1H, m, 3a-H), 5.35 )1H,
d,Jˆ5.1 Hz, 6-H); 13C NMR dppm: 140.8 )5-C), 121.8 )6-C),
77.5 )24S-C), 77.3 )24R-C), 71.9 )3-C), 56.8 )14-C), 56.0
)17-C), 50.2 )9-C), 42.4 )13-C), 42.4 )4-C), 39.9 )12-C),
37.3 )1-C), 36.6 )10-C), 36.0 )20-C), 33.2 )25-C), 32.3
)22-C), 32.1 )2-C), 32.0 )8-C), 31.8 )7-C), 30.8 )23-C),
28.3 )16-C), 24.4 )15-C), 21.2 )11-C), 19.5 )19-C), 19.2
)21-C), 18.9 )27-C), 16.8 )26-C), 12.0 )18-C); Anal. Calcd
for C27H46O2´1/2 H2O: C, 78.78%, H, 11.51%; Found: C,
78.58%, H, 11.67%.
11. )a) Chawla, A.; Saez, E.; Evans, R. M. Cell 2000, 103, 1±4.
)b) Ferber, D. Science 2000, 289, 1446.
12. )a) Janowski, B. A.; Willy, P. J.; Devi, T. R.; Falck, J. R.;
Mangelsdorf, D. J. Nature 1996, 383, 728±731. )b) Janowski,
B. A.; Grogan, M. J.; Jones, S. A.; Wisely, G. B.; Kliewez,
S. A.; Corey, E. J.; Mangelsdorf, D. J. Proc. Natl. Acad. Sci.
USA 1999, 96, 266±271.
13. )a) LuÈtjohann, D.; Breuer, O.; Ahlborg, G.; Nennesmo, I.;
Siden, A.; Diczfalusy, U.; Bjorkhem, I. Proc. Natl. Acad.
È
È
Sci. USA 1996, 93, 9799. )b) Bjorkhem, I.; Lutjohann, D.;
Breuer, O.; Sakinis, A.; Wennmalm, A. J. Biol. Chem. 1997,
272, 30178±30184. )c) Zhang, J.; Akwa, Y.; El-Etr, M.;
Baulieu, E.-E.; Sjovall, J. Biochem. J. 1997, 322, 175±184.
Acknowledgements
È
È
È
)d) Bjorkhem, I.; Lutjohann, D.; Diczfalusy, U.; Stahle, L.;
Ahlborg, G.; Wahren, J. J. Lipid Res. 1998, 39, 1594±1600.
)e) Kolsch, H.; Lutjohann, D.; Tulke, A.; Bjorkhem, I.; Rao,
M. L. Brain Res. 1999, 818, 171±175. )f) Lutjohann, D.;
Papassotiropoulos, A.; Bjorkhem, I.; Locatelli, S.; Bagli, M.;
Oehring, R. D.; Schlegel, U.; Jessen, F.; Rao, M. L.; von
Bergmann, K.; Heun, R. J. Lipid Res. 2000, 41, 195±198.
14. )a) Ercoli, A.; DeRuggieri, P. J. Am. Chem. Soc. 1953, 75,
3284. )b) Ikekawa, N.; Morisaki, M.; Koizumi, N.; Sawamura,
M.; Tanaka, Y.; Deluca, H. F. Biochem. Biophys. Res.
Commun. 1975, 62, 485±491.
We thank the National Natural Science Foundation of China
for ®nancial support)GrantNo.20072047). We also ht ank
Professor Li-Jun Xia and Ms Jin-Hua Wang for performing
the HPLC analysis.
References
1. Ercoli, A.; DeRuggieri, P. Gazz, Chim. Ital. 1953, 83, 78±86.
2. Prasad, K. V. V.; Ponticorvo, L.; Lieberman, S. J. Steroid
Biochem. 1984, 21, 733±736.
15. )a) Koch, P.; Nakatani, Y.; Luu, B.; Ourisson, G. Bull. Soc.
Chim. Fr. 1983, II, 189±194. )b) Moriarty, R. M.; Enache,
L. A.; Kinney, W. A.; Allen, C. S.; Canary, J. W.; Tuladhar,
S. M.; Guo, L. Tetrahedron Lett. 1995, 36, 5139±5142.
16. Koizumi, N.; Ishiguro, M.; Yasuda, M.; Ikekawa, N. J. Chem.
Soc., Perkin Trans. 1 1983, 1401±1410.
3. Saucier, S. E.; Kandutsch, A. A.; Gayen, A. K.; Swahn, D. K.;
Spencer, T. A. J. Biol. Chem. 1989, 264, 6863±6869.
4. )a) Apfel, R.; Benbrook, D.; Lernhardt, E.; Ortiz, M. A.;
Salbert, G.; Pfahl, M. Mol. Cell. Biol. 1994, 14, 7025±7035.
)b) Willy, P. J.; Umesono, K.; Ong, E. S.; Evans, R. M.;
Heyman, R. A.; Mangelsdorf, D. J. Genes Dev. 1995, 9,
1033±1045.
17. Okamoto, M.; Tabe, M.; Fujii, T.; Tanaka, T. Tetrahedron:
Asymmetry 1995, 6, 767±778.
5. )a) Song, C.; Kokontis, J. M.; Hiipakka, R. A.; Liao, S. Proc.
Natl. Acad. Sci. USA 1994, 91, 10809±10813. )b) Shinar,
D. M.; Endo, N.; Rutledge, S. J.; Vogel, R.; Roda, G. A.;
Schmidt, A. Gene 1994, 147, 273±276. )c) Seol, W.; Choi,
H. S.; Moore, D. D. Mol. Endocrinol. 1995, 9, 72±85.
18. Corey, E. J.; Grogan, M. J. Tetrahedron Lett. 1998, 39, 9351±
9354.
19. Song, C.; Hiipakka, R. A.; Liao, S.-T. Steroids 2000, 65, 423±
427.

8296
X.-D. Zhou, W.-S. Zhou / Tetrahedron 57 +2001) 8291±8296
20. Bharucha, K. R.; Buckley, G. C.; Cross, C. K.; Rubin, L. J.;
Ziegler, P.; Can J. Chem. 1956, 34, 982±990.
24. )a) Sheu, J.-H.; Huang, S.-Y.; Wang, G.-H.; Duh, C.-Y. J. Nat.
Prod. 1997, 60, 900±903. )b) Sheu, J.-H.; Huang, S.-Y.; Duh,
C.-Y. J. Nat. Prod. 1996, 59, 23±26.
21. Jiang, B.; Huang, L. F.; Tian, W. S.; Zhou, W. S. Method for
Construction of Sidechain of Brassinolide and Application to
syntheses of Brassinolides in Studies in Natural Products;
Atta-ur-Rahman, A., Ed.; Elsevier: New York, 1997; Vol.
19, pp. 245±287.
25. Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483±2547.
26. Zhou, X.-D., Zhou, W.-S., Chinese Patent ZL99124007.3.
27. Yadav, J. S.; Mysorekar, S. V. Synth. Commun. 1989, 19,
1057±1060.
22. Because the hydrolytic product is partially produced under
this reaction condition, the reaction mixture was further
acetylized with acetic anhydride and pyridine for completion
of the reaction.
28. Wade, P. A.; Amin, N. V. Synth. Commun. 1982, 12, 287±291.
29. Wang, Z.-Q.; Ruan, B.-F. Acta Chim. Sinica 1994, 52, 403±
411.
30. Saucier, S. E.; Kandutsch, A. A.; Clark, D. S.; Spencer, T. A.
Biochem. Biophys. Acta 1993, 1166, 115±123.
23. Tomkinson, N. C. O.; Willson, T. M.; Russel, J. S.; Spencer,
T. A. J. Org. Chem. 1998, 63, 9919±9923.