Synthesis and biological activity of the (25R)-cholesten-26-oic acids - Ligands for the hormonal receptor DAF-12 in Caenorhabditis elegans

Article abstract of DOI:10.1039/b817358c

We describe the stereoselective transformation of diosgenin (4a) to (25R)-Δ⁴-dafachronic acid (1a), (25R)-Δ⁷- dafachronic acid (2a), and (25R)-cholestenoic acid (3a), which represent potential ligands for the hormonal receptor DAF-12

Full text of DOI:10.1039/b817358c

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Volume 7 | Number 5 | 7 March 2009 | Pages 817–1028
ISSN 1477-0520
FULL PAPER
Hans-Joachim Knölker et al.
PERSPECTIVE
Synthesis and biological activity of the Teather J. Sundstrom et al.
(25R)-cholesten-26-oic acids—ligands Inhibitors of phosphoinositide-3-
for the hormonal receptor DAF-12 in
Caenorhabditis elegans
kinase: a structure-based approach to
understanding potency and selectivity

PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and biological activity of the (25R)-cholesten-26-oic acids—ligands
for the hormonal receptor DAF-12 in Caenorhabditis elegans†
Rene´ Martin,a Arndt W. Schmidt,a Gabriele Theumer,a Tilo Krause,a Eugeni V. Entchev,b
Teymuras V. Kurzchaliab and Hans-Joachim Kno¨lker*a
Received 6th October 2008, Accepted 27th November 2008
First published as an Advance Article on the web 28th January 2009
DOI: 10.1039/b817358c
We describe the stereoselective transformation of diosgenin (4a) to (25R)-D4-dafachronic acid (1a),
(25R)-D7-dafachronic acid (2a), and (25R)-cholestenoic acid (3a), which represent potential ligands for
the hormonal receptor DAF-12 in Caenorhabditis elegans. Key-steps of our synthetic approach are a
modified Clemmensen reduction of diosgenin (4a) and a double bond shift from the 5,6- to the
7,8-position. In the 25R-series, the D7-dafachronic acid 2a exhibits the highest hormonal activity.
Introduction
The nematode Caenorhabditis elegans has been established as
an important model organism for studying developmental and
metabolic processes and moreover, for the functional characteri-
zation of novel drug targets.1,2 Upon starvation or overcrowding
it enters diapause and forms specialized larvae called dauer
(enduring). The activity of a single nuclear hormone receptor,
DAF-12, plays a major role in the dauer formation process.3,4 In
the absence of ligand, DAF-12 is activated and initiates the dauer
formation program. However, the products of the cytochrome
P450 DAF-9 are ligands for the DAF-12 receptor. In the presence
of these ligand(s), DAF-12 is inactivated and a program for
reproductive development is initiated.3 Recently, Mangelsdorf
et al. identified novel steroidal metabolites which they called
dafachronic acids (1a, 1b, 2a, and 2b; Fig. 1).3d–f These hormones
function as ligands for the hormonal receptor DAF-12. The
dafachronic acids are the D4- and D7- isomers of (25R)- and (25S)-
3-ketocholesten-26-oic acid. However, the 25R-diastereoisomer
1a showed significantly lower activity in comparison to the 25S-
diastereoisomer 1b.3d–f In the same year, Gill et al. reported that
(25S)-cholestenoic acid (3b) also effectively binds to DAF-12.5 In
contrast, (25R)-cholestenoic acid (3a) was not active (Fig. 1).
Corey and Giroux described the first synthesis of (25S)-D7-
dafachronic acid (2b), which they called dafachronic acid A,
from b-stigmasterol using a stereoselective ruthenium-catalyzed
hydrogenation as the key-step.6 In 2008, the same group completed
Fig. 1 (25R)- and (25S)-cholesten-26-oic acids 1a–3b, diosgenin (4a), and
the synthesis of (25R)-D7-dafachronic acid (2a) in 10 steps and
13% overall yield starting from b-ergosterol.7 Previously, Khripach
et al. published the synthesis of both diastereoisomers (1a and 1b)
of D4-dafachronic acid as well as both diastereoisomers (3a and 3b)
yamogenin (4b).
of cholestenoic acid via a stereoselective assembly of the steroid
side chain.8
aDepartment Chemie, Technische Universita¨t Dresden, Bergstrasse 66,
01069 Dresden, Germany. E-mail: hans-joachim.knoelker@tu-dresden.de;
Fax: +49 351-463-37030
Results and discussion
In the course of our studies directed towards the synthesis of
hormonally active steroids,4,9 we became interested in the synthesis
of the three (25R)-cholesten-26-oic acids 1a–3a. The commercially
available sapogenin diosgenin (4a) was used as a perfect starting
material as it provides the 25R-configuration present in the target
compounds.10
bMax Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauer-
strasse 108, 01307 Dresden, Germany
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra of compounds 1a, 2a and 3a. CCDC reference number 697766.
For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/b817358c
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Clemmensen reduction of diosgenin (4a) with zinc dust and con-
centrated hydrochloric acid, according to the procedure described
by Williams,11–13 provided the rearrangement product 5 (9% yield)
as the only compound that could be isolated (Scheme 1).14 The
structural assignment of 5 was unequivocally confirmed by an
X-ray crystal structure determination (Fig. 2).†15
yield.11 However, treatment of 9 with an excess of Jones reagent
did not provide the desired (25R)-D4-dafachronic acid (1a) but the
(25R)-3,6-diketocholest-4-en-26-oic acid (10) in 56% yield. Using
PDC in N,N-dimethylformamide16 instead of Jones reagent, the
diketo acid 10 was obtained in 74% yield. Earlier reports described
that oxidation of 3b-hydroxycholest-5-enes with Jones reagent,17
PCC,18 and TPAP/NMO19 provided the corresponding cholest-4-
ene-3,6-diones. More recently, a similar transformation has been
reported for diosgenin (4a).20 In order to avoid allylic oxidation,
it was necessary to achieve a sequential oxidation of the hydroxy
groups at C-3 and C-26. Therefore, the introduction of orthogonal
protecting groups at these positions was required.
Since a selective monodesilylation of compound 8 proved to be
impossible, a differentiation between all three hydroxy groups of 6
had to be achieved. Selective acetylation of 6 with acetic anhydride
provided the C-26 acetate 11 in remarkable 54% yield (Scheme 3).
The sterically more demanding pivaloyl chloride represents an
excellent reagent for selective acylation of primary alcohols in the
presence of secondary alcohols.21 Williams and Gong reported
a double pivaloylation at C-3b and C-26 of the triol 6.13 Using
1.1 equivalents of pivaloyl chloride we achieved a differentiation
between the primary and the secondary hydroxy groups of the
triol 6, providing the C-26 pivalate 12 in 81% yield. Treatment
of 12 with 1.1 equivalents of tert-butylchlorodimethylsilane and
DBU led to selective silylation at C-3b. Compound 13 represents
an orthogonally diprotected derivative of the triol 6 in which all
three hydroxy groups have been differentiated. Mesylation of the
16b-hydroxy group followed by treatment with lithium aluminium
hydride afforded the alcohol 14 in 89% yield by removal of the
hydroxy group at C-16 with concomitant cleavage of the pivalate
at C-26. Acetylation of 14 and subsequent desilylation of the
acetate 15 provided the 3b-alcohol 16 in 88% yield. In order to
avoid allylic oxidation at C-6 (cf. Scheme 2), we used a classical
Oppenauer oxidation to obtain the cholest-4-en-3-one 17 (70%
yield). Saponification of the acetate 17 to the 26-alcohol 18 (93%
yield) with catalytic amounts of potassium carbonate in methanol
and subsequent Jones oxidation provided (25R)-D4-dafachronic
acid (1a) in 79% yield.
Scheme 1 Clemmensen reduction of diosgenin (4a). Reagents and condi-
tions: (a) 147 equiv. Zn dust, conc. HCl, EtOH, reflux, 4.5 h, 9%.
Fig. 2 Molecular structure of the rearrangement product 5 in the crystal
form.
In an optimization of the Clemmensen reduction of 4a, we
found out that using half-concentrated hydrochloric acid instead
of concentrated hydrochloric acid led to the desired triol 6 in 85%
yield on large scale (Scheme 2). Treatment of the triol 6 with tert-
butylchlorodimethylsilane (TBSCl) and DBU provided the disilyl
ether 7 in 85% yield.11,14 Mesylation at the 16b-hydroxy group
of 7 and subsequent reduction with lithium aluminium hydride
afforded 8 in 89% yield.13 Removal of the silyl groups with TBAF
in THF at reflux provided (25R)-26-hydroxycholesterol (9) in 89%
For the synthesis of (25R)-D7-dafachronic acid (2a) we envisaged
a shift of the 5,6-double bond into the 7,8-position. Treatment of
compound 8 with PDC and tert-butyl hydroperoxide in benzene
according to Chandrasekaran and Chidambaram’s procedure22
Scheme 2 Synthesis of (25R)-3,6-diketocholest-4-en-26-oic acid (10). Reagents and conditions: (a) 125 equiv. Zn dust, 19% HCl, EtOH, reflux, 4 h, 85%;
(b) 5.0 equiv. TBSCl, 3.8 equiv. DBU, THF, 25 ◦C, 16 h, 85%; (c) 3.8 equiv. MsCl, pyridine, 0 ◦C to 25 ◦C, 16 h; (d) 10.0 equiv. LiAlH4, Et2O, reflux, 4 h,
89% for 2 steps; (e) 3.0 equiv. TBAF, THF, reflux, 20 h, 89%; (f) 10.0 equiv. PDC, DMF, 25 ◦C, 18 h, 74%.
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Scheme 3 Synthesis of (25R)-D4-dafachronic acid (1a). Reagents and conditions: (a) 10 mol% DMAP, 1.1 equiv. Ac2O, 2.2 equiv. Et3N, THF, 25 ◦C, 16 h,
54% of 11; 1.1 equiv. PivCl, 2.2 equiv. Et3N, THF, 25 ◦C, 20 h, 81% of 12; (b) 1.1 equiv. TBSCl, 2.2 equiv. DBU, THF, 25 ◦C, 17 h, 79%; (c) 3.8 equiv.
MsCl, pyridine, 0 ◦C to 25 ◦C, 16 h; (d) 5.0 equiv. LiAlH4, Et2O, reflux, 4 h, 89% for 2 steps; (e) 10 mol% DMAP, 2.0 equiv. Ac2O, 4.0 equiv. Et3N, THF,
16 h, 100%; (f) 1.2 equiv. TBAF, THF, reflux, 17 h, 88%; (g) 1.7 equiv. Al(Oi-Pr)3, toluene/acetone (9:1), reflux, 15 h, 70%; (h) 10 mol% K2CO3, MeOH,
25 ◦C, 40 h, 93%; (i) 5.0 equiv. Jones reagent, acetone, 0 ◦C, 1 h, 79%.
afforded the cholest-5-en-7-one 19 in 57% yield (Scheme 4). Allylic
oxidation of 8 with chromium trioxide and 3,5-dimethyl-1H-
pyrazole provided a similar result (56% yield of 19).23 Using
catalytic amounts of manganese(III) acetate in the presence
of tert-butyl hydroperoxide led to 19 in 47% yield.24 Transfer
hydrogenation of 19 with ammonium formate and Pd/C afforded
the ketone 20 in 90% yield.
isopropylmagnesium chloride led diastereoselectively to the 7a-
alcohol 21 in 70% yield along with 10% of recovered starting
material 20. Treatment of the 7a-alcohol 21 with thionyl chloride
in pyridine afforded quantitatively the cholest-7-ene 23.25 Removal
of the silyl protecting groups with TBAF to the diol 24 (93% yield)
followed by Jones oxidation provided (25R)-D7-dafachronic acid
(2a) in 74% yield.
For stereoelectronic reasons we required the 7a-alcohol 21
to introduce the D7-double bond via elimination of the hydroxy
group. Reduction of the ketone 20 using lithium aluminium
hydride at low temperature provided the 7a-alcohol 21 in 59%
yield along with 31% of the undesired 7b-alcohol 22 (Table 1).
The epimeric alcohols 21 and 22 were easily separable using
flash chromatography on silica gel. Grignard reduction with
The alcohol 14 has been readily converted to (25R)-cholestenoic
acid (3a) in four steps (Scheme 5). Oxidation of 14 us-
ing tetrapropylammonium perruthenate in the presence of
N-methylmorpholine N-oxide (TPAP/NMO)26 afforded the alde-
hyde 25 in only moderate yield (54%). However, oxidation of 14
with pyridinium dichromate (PDC)16 led to the aldehyde 25 in
89% yield. Oxidation of the aldehyde 25 to the silyl-protected
(25R)-cholestenoic acid 26 has been achieved with sodium chlorite.
Following the standard protocol (aqueous tert-butanol as the
solvent) provided low yields of 26 due to the poor solubility of our
starting material.27 In contrast, using aqueous tetrahydrofuran as
solvent the acid 26 could be obtained in 92% yield. Cleavage of
the silyl ether with TBAF afforded only impure 3a in moderate
yield. Chromatographic purification of (25R)-cholestenoic acid
Table 1 Reduction of the ketone 20
Reaction conditions
Yield
1.0 equiv. LiAlH4, THF, -78 ◦C to 25 ◦C, 24 h
1.2 equiv. i-PrMgCl, Et2O, 25 ◦C, 1 h
59% of 21, 31% of 22
70% of 21, 10% of 20
Scheme 4 Synthesis of (25R)-D7-dafachronic acid (2a). Reagents and conditions: (a) 4.0 equiv. PDC, 4.0 equiv. t-BuOOH, CeliteꢀR , benzene, 0 ◦C to
25 ◦C, 28 h, 57%; (b) 10% Pd/C, 4.0 equiv. ammonium formate, EtOAc/MeOH (3:4), reflux, 8 h, 90%; (c) 1.4 equiv. i-PrMgCl, Et2O, 25 ◦C, 1 h, 70%;
(d) 3.0 equiv. SOCl2, pyridine, 0 ◦C, 1 h, 100%; (e) 2.2 equiv. TBAF, THF, reflux, 20 h, 93%; (f) 5.0 equiv. Jones reagent, acetone, 0 ◦C, 1 h, 74%.
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present methodology to 4b should provide a stereoselective access
to the (25S)-cholesten-26-oic acids 1b–3b. Synthetic studies in this
direction are currently underway in our laboratories and will be
reported in due course.
In order to study the activity of the (25R)-compounds on dauer
formation in vivo, they have been used for feeding mutant worms
daf-9(dh6) that lack the activity of DAF-9, the cytochrome P450
that is involved in the production of dafachronic acids. Mutant
worms daf-9(dh6) do not produce the ligand of DAF-12 and arrest
as dauer-like larvae (Fig. 3A, B). In addition, daf-9(dh6) can be
distinguished by the absence of green fluorescence which is carried
by the rescuing dhEx24 extrachromosomal array in the parental
strain and siblings daf-9(dh6); dhEx24.3c,d Therefore, appearance
of non-fluorescent worms going over dauer arrest can be used as an
assay for the presence of DAF-9 product activity. We found both
compounds 1a and 2a to show activity towards rescuing dauer
arrest in daf-9(dh6) (Fig. 3C–H). More active was 2a, which at
0.5 mM was sufficient for daf-9(dh6) to develop at the same speed
as control fluorescent worms (Fig. 3G). A similar activity was
observed for compound 1a at 5 mM (Fig. 3E). Compound 1a
at 0.5 mM (Fig. 3D) and compound 2a at 0.05 mM (Fig. 3F)
were sufficient for daf-9(dh6) to pass through the arrest but the
worms developed slower than the control and often migration of
the gonad phenotypes could be seen. Thus, (25R)-D7-dafachronic
acid (2a) is one order of magnitude more active than (25R)-
D4-dafachronic acid (1a). Similar to a previous study,5 little or
no activity was detected with (25R)-cholestenoic acid (3a) at
all concentrations applied (Fig. 3I–L). Taken together, we can
conclude that although the (25R)-diastereoisomers are less active
as compared with the (25S)-diastereoisomers,3d–f,5 they can be used
for the clarification of basic principles of dauer larvae formation
(Fig. 3).
Scheme 5 Synthesis of (25R)-cholestenoic acid (3a). Reagents and
conditions: (a) 2.0 equiv. PDC, MS 4 A, CH2Cl2, 25 ◦C, 3.5 h, 89%;
˚
(b) 10.0 equiv. 2-methyl-2-butene, 1.3 equiv. KH2PO4, 1.5 equiv. NaClO2,
THF/H2O (3:1), 25 ◦C, 16 h, 92%; (c) cat. H2SO4, MeOH, reflux, 16 h,
84%; (d) 3.0 equiv. LiOH, THF/MeOH/H2O (2:1:1), 25 ◦C, 16 h, 97%.
(3a) proved to be difficult. Therefore, the silyl-protected (25R)-
cholestenoic acid 26 was converted to the methyl ester 27 with
concomitant cleavage of the silyl ether by treatment with catalytic
amounts of concentrated sulfuric acid in methanol at reflux. The
(25R)-cholestenoic acid methyl ester (27) could be easily purified
by column chromatography on silica gel. Finally, saponification of
the methyl ester 27 with lithium hydroxide provided pure (25R)-
cholestenoic acid (3a) in 97% yield.
Experimental
General
All reactions were carried out in oven-dried glassware under an ar-
gon atmosphere. Tetrahydrofuran, ethyl acetate, dichloromethane,
and diethyl ether were dried using a solvent purification system
(MBraun-SPS). Acetone was distilled from phosphorus(V) oxide
Conclusion
˚
and stored over 3 A molecular sieves. Benzene was dried over
We have developed a highly efficient synthesis of the three (25R)-
cholesten-26-oic acids 1a–3a from diosgenin (4a) as a cheap com-
mercially available starting material. The (25R)-D4-dafachronic
acid (1a) was synthesized in 10 steps and 22% overall yield based
on 4a. The perfect differentiation of the three hydroxy groups
present in the triol 6 led to the orthogonally diprotected alcohol
13 and was the key to solve all regioselectivity problems. This
intermediate has been also exploited for the synthesis of (25R)-
cholestenoic acid (3a) (9 steps and 32% overall yield based on 4a).
Chandrasekaran oxidation followed by reduction and elimination
resulted in a shift of the 5,6-double bond to the 7,8-position,
which represents the key-step of the transformation into (25R)-
D7-dafachronic acid (2a) (10 steps and 16% overall yield based
on 4a). The spectroscopic data of all three natural products 1a–3a
were in full agreement with those reported previously.3d,7,8 Another
sapogenin natural product is yamogenin (4b), which represents the
C-25 diastereoisomer of diosgenin (4a). Thus, application of the
sodium. Toluene was purchased from ACROS Organics (water
content less than 50 ppm). Pyridine was obtained from FLUKA
(water content less than 50 ppm). Dry methanol was purchased
from VWR Prolabo (water content less than 20 ppm). All other
chemicals were used as received from commercial sources. Flash
chromatography was performed using silica gel from ACROS
Organics (0.063–0.200 mm). Thin layer chromatography was per-
formed with TLC plates from Merck (60 F254) using anisaldehyde
solution for visualization. Melting points were measured on an
Electrothermal IA9100 and are uncorrected. Infrared spectra
were recorded on a Thermo Nicolet Avatar 360 FT-IR using
the ATR technique (Attenuated Total Reflectance). Mass spectra
were recorded on a Finnigan MAT-95 (electron impact, 70 eV) or
by GC/MS-coupling using an Agilent Technologies 6890 N GC
System equipped with a 5973 Mass Selective Detector (electron
impact EI, 70 eV). ESI-MS spectra were recorded on an Esquire
LC with an ion trap detector from Bruker. Positive and negative
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Fig. 3 Hormonal activity of 1a, 2a and 3a at 0.05 mM, 0.5 mM and 5 mM; asterisks—non fluorescent daf-9(dh6); the sibling daf-9(dh6); dhEx24 which
are wild type for daf-9 gene can be easily distinguished by the green fluorescence.
ions were detected. Elemental analyses were measured on an
EuroVector EuroEA3000. NMR spectra were recorded on a
ether (2 ¥ 300 mL). The combined organic layers were washed
with a saturated solution of sodium hydrogen carbonate (100 mL)
and brine (100 mL) and then dried over sodium sulfate. Removal
of the solvent and recrystallization of the crude product from
ethanol/water (1:1) provided pure 5, yield: 300 mg (9%). Colorless
solid; mp: 143–145 ◦C; IR (ATR): n = 3289, 2929, 2857, 1463,
1434, 1373, 1331, 1249, 1203, 1134, 1039, 1004, 943, 906, 848,
808 cm-1; 1H NMR (500 MHz, CDCl3): d = 0.69 (d, J = 6.7 Hz,
3 H), 0.90 (d, J = 6.7 Hz, 3 H), 0.97 (s, 3 H), 0.98 (s, 3 H) 1.07–1.62
(m, 14 H), 1.70–1.87 (m, 4 H), 1.91–1.98 (m, 2 H), 2.03–2.24 (m,
5 H), 2.31 (ddd, J = 12.8, 4.8, 2.1 Hz, 1 H), 3.41 (dd, J = 10.4,
6.5 Hz, 1 H), 3.49 (dd, J = 10.4, 6.0 Hz, 1 H), 3.52 (m, 1 H), 5.40
(m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = 14.68
(CH3), 16.48 (CH3), 18.71 (CH3), 22.93 (CH2), 23.22 (CH2), 24.97
(CH3), 25.64 (CH2), 30.94 (CH2), 31.40 (CH2), 31.46 (CH2), 31.62
(2 CH2), 33.05 (CH), 33.44 (CH2), 35.79 (CH), 36.91 (C), 37.02
(CH2), 39.13 (CH), 42.14 (CH2), 49.20 (CH), 52.78 (C), 68.51
(CH2), 72.00 (CH), 121.94 (CH), 136.26 (C), 140.32 (C), 141.34
(C); GC-MS (70 eV): m/z (%) = 271 (100) [(M - C8H17O)+], 253
(17), 175 (6), 157 (9), 147 (7), 133 (11).
1
Bruker DRX 500. Complete assignment of H and 13C signals
was done using HSQC experiments. Chemical shifts are reported
in ppm with the deuterated solvent as internal standard. The
following abbreviations have been used: s: singlet, d: doublet,
dd: doublet of doublets, dt: doublet of triplets, t: triplet, q:
quartet, m: multiplet, br: broad. X-ray analyses: Bruker-Nonius
Kappa CCD with Oxford Cryosystems and STOE IPDS 2 image
plate. Software: Collect (Nonius BV, 1999), Dirax/lsq (Duisen-
berg, 1992), SHELXS-97 (G. M. Sheldrick, 1990), EvalCCD
(Duisenberg et al., 2003), SADABS version 2.10 (G. M. Sheldrick,
Bruker AXS Inc., 2002), SHELXL-97 (G. M. Sheldrick, 1997),
Schakal-99 (E. Keller, 1999).
(3S,8R,9S,10R,17S)-2,3,4,7,8,9,10,11,12,15,16,17-Dodeca-
hydro-17-((2R,6R)-7-hydroxy-6-methylhept-2-yl)-10,17-dimethyl-
1H-cyclopenta[a]phenanthren-3-ol (5). Zinc dust (80.0 g,
1.23 mol) and diosgenin (4a) (3.47 g, 8.37 mmol) were mixed
in ethanol (250 mL) and concentrated aqueous hydrochloric acid
(800 mL) was added under vigorous stirring over a period of
4 h. The resulting mixture was then heated under reflux for
further 30 min. Unreacted zinc was removed by filtration of the
hot reaction mixture. After cooling to room temperature, water
(500 mL) and then diethyl ether (500 mL) were added until the
layers separated. The aqueous layer was extracted with diethyl
Crystallographic data for 5
C27H44O2, crystal size: 0.87 ¥ 0.24 ¥ 0.17 mm3, M = 400.62 g mol-1,
monoclinic, space group P21, a = 6.6550(10), b = 21.3180(11),
◦
3
˚
˚
c = 17.247(2) A, b = 91.568(11) , V = 2445.9(5) A , Z = 4,
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-3
-1
˚
42.19 (C), 42.78 (CH2), 50.17 (CH), 54.53 (CH), 61.38 (CH), 68.49
(CH2), 72.47 (CH), 72.59 (CH), 120.91 (CH), 141.66 (C).
rcalcd = 1.088 g cm , m = 0.066 mm , l = 0.71073 A, T = 198(2) K,
q range = 3.04–25.39◦, reflections collected: 27537, independent:
4577 (Rint = 0.0349), 535 parameters. The structure was solved
by direct methods and refined by full-matrix least-squares on F2;
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)cholest-5-ene
(8).
Methanesulfonyl chloride (1.1 mL, 1.56 g, 13.58 mmol) was
added dropwise at 0 ◦C to a solution of 7 (2.38 g, 3.59 mmol)
in pyridine (20 mL). Stirring was continued for 16 h and the
reaction mixture was allowed to warm to room temperature. The
resulting orange solution was carefully quenched with ice-water
(50 mL) and extracted with diethyl ether (3 ¥ 100 mL). The
combined organic layers were washed with ice-cold 1 N HCl
(50 mL), water (50 mL), a saturated solution of sodium hydrogen
carbonate (100 mL), and brine (100 mL). After drying with
magnesium sulfate and removal of the solvent the crude mesylate
was obtained in quantitative yield (2.60 g). The mesylate (500 mg,
680 mmol) was dissolved in diethyl ether (25 mL) and carefully
treated with lithium aluminium hydride (260 mg, 6.8 mmol)
at 0 ◦C. The resulting suspension was refluxed for 4 h. After
cooling to room temperature, the reaction mixture was carefully
quenched by addition of water (10 mL) and ethyl acetate (10 mL).
An ice-cold 10% aqueous solution of sulfuric acid (20 mL) was
slowly added until the precipitate disappeared. The aqueous
layer was extracted with diethyl ether (3 ¥ 100 mL) and the
combined organic layers were washed with a saturated solution
of sodium hydrogen carbonate (100 mL), water (50 mL), and
brine (50 mL). Drying with magnesium sulfate and evaporation
of the solvent gave the crude product which was purified by flash
chromatography on silica gel (petroleum ether/diethyl ether, 40:1)
final R indices [I > 2s(I)]: R1 = 0.0490; wR2 = 0.1236; maximal
-3
˚
residual electron density: 0.538 e A . CCDC 697766.
(25R)-Cholest-5-ene-3b,16b,26-triol (6). Zinc dust (90.0 g,
1.376 mol) and diosgenin (4a) (4.4 g, 11.0 mmol) were mixed
in ethanol (1 L) and the mixture was heated at reflux. Half-
concentrated aqueous hydrochloric acid (800 mL) was added to the
reaction mixture over a period of 1 h and heating was continued
for a further 30 min. Unreacted zinc was removed by filtration
of the hot reaction mixture. After cooling to room temperature,
water (600 mL) and then diethyl ether (approximately 500 to
1000 mL) were added until the layers separated. The two layers
were separated and the aqueous layer was extracted with diethyl
ether (2 ¥ 300 mL). The combined organic layers were washed with
a saturated solution of sodium hydrogen carbonate (100 mL) and
brine (100 mL) and then dried over magnesium sulfate. Removal
of the solvent and recrystallization of the crude product from
ethanol/water (1:1) provided pure 6, yield: 3.9 g (85%). Colorless
◦
1
solid; mp: 170–174 C; H NMR (500 MHz, pyridine-d5): d =
0.84–0.91 (m, 1 H), 0.97–1.19 (m, 4 H), 1.05 (d, J = 6.7 Hz, 3 H),
1.06 (s, 3 H), 1.09 (d, J = 6.7 Hz, 3 H), 1.13 (s, 3 H), 1.21–1.33 (m,
2 H), 1.44–1.70 (m, 8 H), 1.76–1.86 (m, 3 H), 1.97 (m, 2 H), 2.08
(m, 2 H), 2.27–2.36 (m, 2 H), 2.62 (m, 2 H), 3.65 (dd, J = 10.3,
6.5 Hz, 1 H), 3.74 (dd, J = 10.3, 5.8 Hz, 1 H), 3.85 (m, 1 H), 4.55
(m, 1 H), 5.41 (d, J = 4.7 Hz, 1 H), 5.68 (br s, 1 H), 5.93 (br s, 1 H),
6.20 (br s, 1 H); 13C NMR and DEPT (125 MHz, pyridine-d5): d =
13.43 (CH3), 17.32 (CH3), 18.58 (CH3), 19.63 (CH3), 21.15 (CH2),
24.59 (CH2), 30.39 (CH), 31.95 (CH), 32.27 (CH2), 32.64 (CH2),
34.35 (CH2), 36.71 (CH2), 36.75 (CH), 36.96 (C), 37.80 (CH2),
38.02 (CH2), 40.37 (CH2), 42.59 (C), 43.51 (CH2), 50.60 (CH),
54.92 (CH), 62.12 (CH), 67.75 (CH2), 71.07 (CH), 71.30 (CH),
121.22 (CH), 142.03 (C).
◦
to obtain 8, yield: 382 mg (89%). Colorless solid; mp: 92–93 C;
IR (ATR): n = 2930, 2897, 2856, 1471, 1383, 1368, 1249, 1079,
1023, 1006, 957, 938, 885, 869, 833, 773 cm-1; 1H NMR (500 MHz,
CDCl3): d = 0.02 (s, 6 H), 0.04 (s, 6 H), 0.66 (s, 3 H), 0.84 (d, J =
6.7 Hz, 3 H), 0.876 (s, 9 H), 0.881 (s, 9 H), 0.90 (d, J = 6.5 Hz,
3 H), 0.92 (m, 1 H), 0.97–1.37 (m, 13 H), 0.98 (s, 3 H), 1.42–1.55
(m, 7 H), 1.70 (m, 1 H), 1.77–1.85 (m, 2 H), 1.94–2.00 (m, 2 H),
2.13–2.17 (m, 1 H), 2.26 (m, 1 H), 3.34 (dd, J = 9.7, 6.6 Hz, 1 H),
3.42 (dd, J = 9.7, 5.9 Hz, 1 H), 3.47 (m, 1 H), 5.30 (m, 1 H); 13
C
NMR and DEPT (125 MHz, CDCl3): d = -5.34 (2 CH3), -4.60
(2 CH3), 11.83 (CH3), 16.65 (CH3), 18.27 (C), 18.36 (C), 18.67
(CH3), 19.42 (CH3), 21.04 (CH2), 23.33 (CH2), 24.27 (CH2), 25.93
(3 CH3), 25.95 (3 CH3), 28.23 (CH2), 31.88 (CH), 31.92 (CH2),
32.07 (CH2), 33.56 (CH2), 35.69 (CH), 35.73 (CH), 36.16 (CH2),
36.57 (C), 37.36 (CH2), 39.78 (CH2), 42.30 (C), 42.80 (CH2),
50.18 (CH), 56.11 (CH), 56.79 (CH), 68.56 (CH2), 72.64 (CH),
121.16 (CH), 141.56 (C); MS (70 eV): m/z (%) = 616 (3) [(M -
Me)+], 574 (100) [(M - tert-Bu)+], 572 (10), 442 (10); HRMS: m/z
calc. for C35H65O2Si2 [(M - tert-Bu)+]: 573.4518, found: 573.4532;
Anal. calc. for C39H74O2Si2: C 74.21, H 11.82, found: C 74.25, H
11.92%.
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)cholest-5-en-16b-ol
(7). DBU (700 mL, 4.6 mmol) was added dropwise to a stirred
solution of TBSCl (900 mg, 6.0 mmol) and 6 (500 mg, 1.2 mmol)
in THF (20 mL). After stirring for 16 h at room temperature, the
mixture was quenched with water (50 mL) and extracted with ethyl
acetate (3 ¥ 100 mL). The combined organic layers were dried
with magnesium sulfate and the solvent was removed in vacuo.
Purification of the residue by flash chromatography on silica gel
(petroleum ether/diethyl ether, 10:1) furnished 7, yield: 660 mg
(85%). Colorless solid; mp: 122–124 C; H NMR (500 MHz,
CDCl3): d = 0.02 (s, 6 H), 0.04 (s, 6 H), 0.85 (d, J = 6.7 Hz, 3 H),
0.87 (s, 3 H), 0.875 (s, 9 H), 0.879 (s, 9 H), 0.90–1.59 (m, 17 H),
0.97 (d, J = 6.7 Hz, 3 H), 0.99 (s, 3 H), 1.17 (dd, J = 13.3, 4.5 Hz,
1 H), 1.70 (m, 1 H), 1.79 (dt, J = 13.3, 3.3 Hz, 1 H), 1.82–1.84
(m, 1 H), 1.95–2.01 (m, 2 H), 2.13–2.26 (m, 3 H), 3.35 (dd, J =
9.7, 6.5 Hz, 1 H), 3.42 (dd, J = 9.7, 5.9 Hz, 1 H), 3.46 (m, 1 H),
4.33 (m, 1 H), 5.30 (d, J = 5.2 Hz, 1 H); 13C NMR and DEPT
(125 MHz, CDCl3): d = -5.34 (2 CH3), -4.60 (2 CH3), 13.01 (CH3),
16.68 (CH3), 18.18 (CH3), 18.25 (C), 18.37 (C), 19.41 (CH3), 20.67
(CH2), 23.75 (CH2), 25.93 (3 CH3), 25.96 (3 CH3), 29.75 (CH),
31.48 (CH), 31.83 (CH2), 32.04 (CH2), 33.60 (CH2), 35.79 (CH),
36.29 (CH2), 36.49 (CH2), 36.58 (C), 37.31 (CH2), 39.87 (CH2),
◦
1
(25R)-26-Hydroxycholesterol (9). A 1.0 M solution of TBAF
in THF (2.3 mL, 2.3 mmol) was added to a solution of the disilyl
ether 8 (484 mg, 0.77 mmol) in THF (40 mL). The resulting mixture
was heated at reflux for 20 h. After cooling to room temperature,
water (100 mL) was added and the reaction mixture was extracted
with diethyl ether (3 ¥ 100 mL). The combined organic layers were
dried with magnesium sulfate and the solvent was evaporated.
Purification of the residue by flash chromatography on silica
gel (petroleum ether/ethyl acetate, 2:1) furnished the known
(25R)-26-hydroxycholesterol (9), yield: 275 mg (89%). Colorless
914 | Org. Biomol. Chem., 2009, 7, 909–920
This journal is
The Royal Society of Chemistry 2009
©

solid; mp: 172–173 ◦C; IR (ATR): n = 3292, 2959, 2932, 2897,
2850, 1463, 1435, 1374, 1354, 1254, 1228, 1194, 1126, 1057, 1042,
1023, 990, 955, 841, 798, 737 cm-1; 1H NMR (500 MHz, CDCl3):
d = 0.66 (s, 3 H), 0.90 (d, J = 6.7 Hz, 6 H), 0.99 (s, 3 H), 1.03–1.60
(m, 21 H), 1.80–1.85 (m, 3 H), 1.94–2.02 (m, 2 H), 2.20–2.30 (m,
2 H), 3.41 (dd, J = 10.5, 6.4 Hz, 1 H), 3.49 (dd, J = 10.5, 5.8 Hz,
1 H), 3.51 (m, 1 H), 5.34 (m, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = 11.85 (CH3), 16.49 (CH3), 18.65 (CH3), 19.39 (CH3),
21.06 (CH2), 23.38 (CH2), 24.27 (CH2), 28.24 (CH2), 31.63 (CH2),
31.87 (CH), 31.88 (CH2), 33.50 (CH2), 35.70 (CH), 35.78 (CH),
36.13 (CH2), 36.48 (C), 37.22 (CH2), 39.75 (CH2), 42.27 (CH2),
42.30 (C), 50.08 (CH), 56.09 (CH), 56.73 (CH), 68.53 (CH2), 71.78
(CH), 121.70 (CH), 140.74 (C); MS (70 eV): m/z (%) = 402 (93)
[M+], 400 (16), 387 (34), 385 (100), 369 (33), 317 (36), 300 (23), 299
(35), 291 (62), 273 (22), 271 (90); HRMS: m/z calc. for C27H46O2
[M+]: 402.3498, found: 402.3505; Anal. calc. for C27H46O2: C 80.54,
H 11.51, found: C 80.61, H 11.65%.
(s, 3 H), 2.19–2.29 (m, 3 H), 3.50 (m, 1 H), 3.79 (dd, J = 10.7,
7.1 Hz, 1 H), 3.97 (dd, J = 10.7, 5.9 Hz, 1 H), 4.33 (dt, J = 4.5,
7.6 Hz, 1 H), 5.33 (m, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = 13.01 (CH3), 16.86 (CH3), 18.14 (CH3), 19.36 (CH3),
20.66 (CH2), 20.99 (CH3), 23.55 (CH2), 29.75 (CH), 31.43 (CH),
31.57 (CH2), 31.76 (CH2), 32.34 (CH), 33.59 (CH2), 35.91 (CH2),
36.47 (C), 36.65 (CH2), 37.14 (CH2), 39.80 (CH2), 42.16 (C), 42.22
(CH2), 50.03 (CH), 54.44 (CH), 61.35 (CH), 69.53 (CH2), 71.70
=
(CH), 72.35 (CH), 121.41 (CH), 140.84 (C), 171.40 (C O); MS
(70 eV): m/z (%) = 460 (7) [M+], 442 (97), 427 (24), 424 (79), 409
(50), 383 (23), 331 (17), 271 (100), 253 (54). HRMS: m/z calc. for
C29H46O3 [(M - H2O)+]: 442.3447, found: 442.3454; Anal. calc.
for C29H48O4: C 75.61, H 10.50, found: C 75.15, H 10.34%.
(25R)-26-Pivaloyloxycholest-5-ene-3b,16b-diol (12). Triethyl-
amine (80 mL, 572 mmol), pivaloyl chloride (35 mL, 286 mmol), and
DMAP (3 mg, 26 mmol) were added to a solution of 6 (107 mg,
260 mmol) in THF (10 mL). The resulting solution was stirred
at room temperature for 20 h and then water was added. The
mixture was extracted with diethyl ether three times and dried with
magnesium sulfate. Evaporation of the solvent and purification
of the residue by flash chromatography on silica gel (petroleum
ether/diethyl ether, 1:2) provided the pivalate 12, yield: 106 mg
(81%). Colorless solid; mp: 119–121 ◦C; IR (ATR): n = 3395,
2928, 2850, 1722, 1632, 1479, 1462, 1397, 1375, 1364, 1336, 1282,
(25R)-3,6-Diketocholest-4-en-26-oic acid (10). PDC (700 mg,
1.86 mmol) was added to a solution of (25R)-26-hydroxy-
cholesterol (9) (75 mg, 0.186 mmol) in DMF (20 mL) and the
resulting mixture was stirred for 18 h at room temperature. Water
(100 mL) was added and the mixture was extracted with ethyl
acetate (3 ¥ 100 mL). The combined organic layers were dried
with magnesium sulfate. The solvent was removed in vacuo and
the residue was purified by flash chromatography (petroleum
ether/ethyl acetate, 2:1) to provide compound 10, yield: 59 mg
(74%). Colorless solid; mp: 105–110 ◦C; IR (ATR): n = 2940,
2868, 1699, 1462, 1415, 1379, 1221, 1024, 922, 868 cm-1; 1H NMR
(500 MHz, CDCl3): d = 0.70 (s, 3 H), 0.91 (d, J = 6.5 Hz, 3 H),
1.15 (s, 3 H), 1.17 (d, J = 7.0 Hz, 3 H), 1.19–1.51 (m, 12 H),
1.61 (m, 3 H), 1.80–1.92 (m, 4 H), 2.02 (dd, J = 16.0, 12.4 Hz,
1 H), 2.08 (dt, J = 13.0, 3.3 Hz, 1 H), 2.13 (ddd, J = 13.4, 5.1,
2.6 Hz, 1 H), 2.43–2.53 (m, 3 H), 2.66 (dd, J = 16.0, 4.1 Hz,
1 H), 6.16 (s, 1 H); 13C NMR and DEPT (125 MHz, CDCl3):
d = 11.87 (CH3), 16.76 (CH3), 17.49 (CH3), 18.55 (CH3), 20.84
(CH2), 23.59 (CH2), 23.93 (CH2), 27.97 (CH2), 33.83 (CH2), 33.94
(CH2), 34.16 (CH), 35.50 (CH, CH2), 35.60 (CH2), 39.08 (CH2),
39.21 (CH), 39.78 (C), 42.52 (C), 46.77 (CH2), 50.91 (CH), 55.83
1
1164, 1138, 1045, 1020, 983, 954, 936, 815, 770 cm-1; H NMR
(500 MHz, CDCl3): d = 0.82–0.86 (m, 1 H), 0.87 (s, 3 H), 0.91
(d, J = 6.7 Hz, 3 H), 0.94–0.99 (m, 2 H), 0.96 (d, J = 6.7 Hz,
3 H), 1.00 (s, 3 H), 1.02–1.17 (m, 5 H), 1.19 (s, 9 H), 1.25–1.32
(m, 3 H), 1.45–1.55 (m, 6 H), 1.77–1.85 (m, 4 H), 1.96 (m, 1 H),
1.99 (dt, J = 12.6, 3.5 Hz, 1 H), 2.20–2.30 (m, 3 H), 3.51 (m,
1 H), 3.79 (dd, J = 10.7, 7.0 Hz, 1 H), 3.97 (dd, J = 10.7, 5.6 Hz,
1 H), 4.33 (dt, J = 4.2, 7.6 Hz, 1 H), 5.34 (m, 1 H); 13C NMR and
DEPT (125 MHz, CDCl3): d = 13.02 (CH3), 16.94 (CH3), 18.17
(CH3), 19.37 (CH3), 20.67 (CH2), 23.55 (CH2), 27.21 (3 CH3),
29.78 (CH), 31.45 (CH), 31.58 (CH2), 31.78 (CH2), 32.52 (CH),
33.68 (CH2), 36.03 (CH2), 36.49 (C), 36.66 (CH2), 37.15 (CH2),
38.84 (C), 39.81 (CH2), 42.17 (C), 42.22 (CH2), 50.04 (CH), 54.46
(CH), 61.32 (CH), 69.20 (CH2), 71.74 (CH), 72.38 (CH), 121.45
=
=
(CH), 56.48 (CH), 125.45 (CH), 161.04 (C), 182.27 (C O), 199.55
(CH), 140.84 (C), 178.71 (C O); MS (70 eV): m/z (%) = 502 (2)
+
(C O), 202.34 (C O); MS (70 eV): m/z (%) = 428 (100) [M ], 410
(11), 400 (19), 386 (7), 277 (10), 243 (18); HRMS: m/z calc. for
C27H40O4 [M+]: 428.2927, found: 428.2914.
[M+], 484 (33), 469 (10), 466 (68), 451 (17), 271 (100); HRMS: m/z
calc. for C32H52O3 [(M - H2O)+]: 484.3916, found: 484.3907.
=
=
(25R)-3b-(tert-Butyldimethylsilyloxy)-26-pivaloyloxy-cholest-5-
en-16b-ol (13). tert-Butylchlorodimethylsilane (885 mg,
5.874 mmol) and DBU (1.76 mL, 11.748 mmol) were added to
a solution of 12 (2.687 g, 5.34 mmol) in THF (50 mL). The
mixture was stirred at room temperature for 17 h and then water
(100 mL) was added. The resulting solution was extracted with
diethyl ether (3 ¥ 100 mL) and the combined organic layers were
washed with brine (50 mL) and dried with magnesium sulfate.
Evaporation of the solvent and purification of the residue by
flash chromatography on silica gel (petroleum ether/diethyl ether,
20:1) provided ◦the silyl ether 13, yield: 2.618 g (79%). Colorless
solid; mp: 108 C; IR (ATR): n = 3547, 2931, 2852, 1722, 1464,
1397, 1382, 1367, 1281, 1251, 1167, 1077, 1022, 985, 959, 938,
886, 870, 838, 777 cm-1; 1H NMR (500 MHz, CDCl3): d = 0.04 (s,
6 H), 0.81–0.86 (m, 1 H), 0.869 (s, 3 H), 0.874 (s, 9 H), 0.89–0.98
(m, 2 H), 0.91 (d, J = 6.7 Hz, 3 H), 0.96 (d, J= 6.7 Hz, 3 H),
0.99 (s, 3 H), 1.01–1.16 (m, 5 H), 1.19 (s, 9 H), 1.21–1.34 (m,
(25R)-26-Acetoxycholest-5-ene-3b,16b-diol
(11). Triethyl-
amine (230 mL, 1.65 mmol), acetic anhydride (78 mL, 825 mmol),
and DMAP (9 mg, 76 mmol) were added to a solution of 6
(316 mg, 760 mmol) in THF (30 mL). The resulting mixture was
stirred at room temperature for 16 h, subsequently quenched by
addition of water (50 mL), and the aqueous layer was extracted
with diethyl ether (3 ¥ 50 mL). The combined organic layers were
washed with brine (50 mL) and dried with magnesium sulfate.
Removal of the solvent and purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 1:2)
provided the acetate 11, yield: 188 mg (54%). Colorless solid; mp:
89–90 ◦C; IR (ATR): n = 3381, 2932, 2859, 1739, 1628, 1465,
1437, 1375, 1363, 1336, 1231, 1152, 1043, 985, 954 cm-1; 1H NMR
(500 MHz, CDCl3): d = 0.81–0.99 (m, 3 H), 0.87 (s, 3 H), 0.90
(d, J = 6.7 Hz, 3 H), 0.96 (d, J = 6.7 Hz, 3 H), 1.00 (s, 3 H),
1.02–1.54 (m, 14 H), 1.75–1.84 (m, 4 H), 1.95–2.01 (m, 2 H), 2.04
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 909–920 | 915
©

2 H), 1.38–1.57 (m, 7 H), 1.68–1.72 (m, 1 H), 1.77–1.87 (m,
3 H), 1.95–2.00 (m, 2 H), 2.15 (ddd, J = 13.4, 4.9, 2.1 Hz, 1 H),
2.20–2.28 (m, 2 H), 3.46 (m, 1 H), 3.79 (dd, J = 10.7, 6.9 Hz, 1 H),
tert-Bu)+]: 459.3658, found: 459.3650; Anal. calc. for C33H60O2Si:
C 76.68, H 11.70, found: C 76.85, H 11.79%.
3.97 (dd, J = 10.7, 5.6 Hz, 1 H), 4.33 (m, 1 H), 5.30 (m, 1 H); 13
C
(25R)-26-Acetoxy-3b-(tert-butyldimethylsilyloxy)cholest-5-ene
(15). Triethylamine (863 mL, 6.2 mmol), acetic anhydride
(292 mL, 3.1 mmol), and DMAP (19 mg, 0.155 mmol) were added
to a solution of the alcohol 14 (800 mg, 1.55 mmol) in THF
(30 mL). The resulting mixture was stirred at room temperature
for 16 h and then water (100 mL) was added. The solution
was extracted with diethyl ether (3 ¥ 100 mL), the combined
organic layers were washed with brine (50 mL) and dried with
magnesium sulfate. Evaporation of the solvent and purification
of the residue by flash chromatography on silica gel (petroleum
ether/diethyl ether, 20:1) provided the acetate 15, yield: 866 mg
(100%). Colorless solid; mp: 105–106 ◦C; IR (ATR): n = 2933,
2893, 2857, 1742, 1471, 1362, 1240, 1195, 1092, 1031, 988, 963,
NMR and DEPT (125 MHz, CDCl3): d = -4.60 (2 CH3), 13.02
(CH3), 16.94 (CH3), 18.17 (CH3), 18.26 (C), 19.41 (CH3), 20.66
(CH2), 23.55 (CH2), 25.92 (3 CH3), 27.22 (3 CH3), 29.77 (CH),
31.46 (CH), 31.82 (CH2), 32.03 (CH2), 32.53 (CH), 33.69 (CH2),
36.04 (CH2), 36.57 (C), 36.69 (CH2), 37.29 (CH2), 38.84 (C), 39.85
(CH2), 42.18 (C), 42.76 (CH2), 50.14 (CH), 54.51 (CH), 61.32
(CH), 69.19 (CH2), 72.39 (CH), 72.58 (CH), 120.90 (CH), 141.66
+
=
(C), 178.68 (C O); MS (70 eV): m/z (%) = 598 (2) [(M - H2O) ],
559 (86) [(M - tert-Bu)+], 541 (12), 483 (14), 467 (9), 457 (13), 383
(29), 365 (100), 271 (9); HRMS: m/z calc. for C34H59O4Si [(M -
tert-Bu)+]: 559.4183, found: 559.4182; Anal. calc. for C38H68O4Si:
C 73.97, H 11.11, found: C 74.01, H 11.33%.
1
927, 890, 870, 836, 804, 774 cm-1; H NMR (500 MHz, CDCl3):
(25R)-3b-(tert-Butyldimethylsilyloxy)cholest-5-en-26-ol
(14).
d = 0.04 (s, 6 H), 0.66 (s, 3 H), 0.87 (s, 9 H), 0.900 (d, J = 6.4 Hz,
3 H), 0.903 (d, J = 6.7 Hz, 3 H), 0.98 (s, 3 H), 1.00–1.55 (m,
20 H), 1.69–1.82 (m, 4 H), 1.93–2.01 (m, 2 H), 2.04 (s, 3 H), 2.15
(ddd, J = 13.3, 4.9, 2.2 Hz, 1 H), 2.25 (m, 1 H), 3.47 (m, 1 H),
3.83 (dd, J = 10.7, 7.0 Hz, 1 H), 3.93 (dd, J = 10.7, 6.0 Hz,
1 H), 5.30 (m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3):
d = -4.60 (2 CH3), 11.84 (CH3), 16.78 (CH3), 18.27 (C), 18.65
(CH3), 19.42 (CH3), 21.00 (CH3), 21.04 (CH2), 23.20 (CH2), 24.27
(CH2), 25.93 (3 CH3), 28.24 (CH2), 31.88 (CH), 31.92 (CH2), 32.07
(CH2), 32.48 (CH), 33.73 (CH2), 35.66 (CH), 36.02 (CH2), 36.56
(C), 37.36 (CH2), 39.77 (CH2), 42.31 (C), 42.80 (CH2), 50.17 (CH),
56.05 (CH), 56.77 (CH), 69.61 (CH2), 72.63 (CH), 121.14 (CH),
Methanesulfonyl chloride (1.02 mL, 13.22 mmol) was added
dropwise to a solution of 13 (2.15 g, 3.48 mmol) in pyridine
(30 mL) at 0 ◦C. Stirring was continued for 16 h and the reaction
mixture was allowed to warm to room temperature. The resulting
orange solution was carefully quenched with ice-water (50 mL)
and extracted with diethyl ether (3 ¥ 100 mL). The combined
organic layers were washed with ice-cold 1 N HCl (50 mL), water
(50 mL), a saturated solution of sodium hydrogen carbonate
(50 mL), and brine (50 mL). After drying with magnesium sulfate
and removal of the solvent the crude mesylate was obtained in
quantitative yield (2.42 g). The mesylate (2.42 g, 3.48 mmol)
was dissolved in diethyl ether (40 mL) and carefully treated with
lithium aluminium hydride (660 mg, 17.4 mmol) at 0 ◦C. The
resulting suspension was heated at reflux for 4 h. After cooling to
room temperature, the reaction mixture was carefully quenched
by addition of water (10 mL) and ethyl acetate (10 mL). An
ice-cold 10% aqueous solution of sulfuric acid (20 mL) was
slowly added until the precipitate disappeared. The aqueous
layer was extracted with diethyl ether (3 ¥ 100 mL) and the
combined organic layers were washed with a saturated solution
of sodium hydrogen carbonate (50 mL), water (50 mL), and brine
(50 mL). Drying with magnesium sulfate and evaporation of
the solvent gave the crude product, which was purified by flash
chromatography on silica gel (petroleum ether/diethyl ether, 5:1)
to afford◦the alcohol 14, yield: 1.601 g (89%). Colorless solid; mp:
163–164 C; IR (ATR): n = 3306, 2929, 2901, 2857, 1462, 1381,
1252, 1196, 1086, 1039, 1006, 989, 959, 938, 887, 869, 835, 802,
=
141.56 (C), 171.33 (C O); GC-MS (70 eV): m/z (%) = 501 (64)
[(M - tert-Bu)+], 441 (14), 367 (100).
(25R)-26-Acetoxycholest-5-en-3b-ol (16). A 1 M solution of
TBAF in THF (1.74 mL, 1.74 mmol) was added to a solution of the
silyl ether 15 (811 mg, 1.45 mmol) in THF (25 mL) and the mixture
was heated at reflux for 17 h. After cooling to room temperature,
water (100 mL) was added and the mixture was extracted with
diethyl ether (3 ¥ 100 mL). The combined organic layers were
washed with brine (50 mL) and dried with magnesium sulfate.
Evaporation of the solvent and purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 3:2)
provided the alcohol 16, yield: 563 mg (88%). Light yellow oil,
which slowly solidified on cooling to give a colorless solid; mp:
74 ◦C; IR (ATR): n = 3264, 2931, 2901, 2864, 1739, 1463, 1365,
1233, 1195, 1108, 1058, 1036, 986, 954, 926, 841, 800, 740 cm-1; 1H
NMR (500 MHz, CDCl3): d = 0.66 (s, 3 H), 0.90 (d, J = 6.7 Hz,
6 H), 0.92–1.62 (m, 20 H), 0.99 (s, 3 H), 1.73–1.85 (m, 4 H), 1.93–
2.06 (m, 2 H), 2.04 (s, 3 H), 2.22–2.30 (m, 2 H), 3.51 (m, 1 H),
3.82 (dd, J = 10.7, 7.0 Hz, 1 H), 3.93 (dd, J = 10.7, 6.0 Hz, 1 H),
5.34 (m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = 11.84
(CH3), 16.78 (CH3), 18.64 (CH3), 19.38 (CH3), 21.00 (CH3), 21.05
(CH2), 23.21 (CH2), 24.26 (CH2), 28.23 (CH2), 31.63 (CH2), 31.87
(CH, CH2), 32.47 (CH), 33.72 (CH2), 35.67 (CH), 36.01 (CH2),
36.48 (C), 37.22 (CH2), 39.74 (CH2), 42.27 (CH2), 42.30 (C), 50.08
(CH), 56.05 (CH), 56.72 (CH), 69.61 (CH2), 71.77 (CH), 121.68
1
773 cm-1; H NMR (500 MHz, CDCl3): d = 0.04 (s, 6 H), 0.66
(s, 3 H), 0.88 (s, 9 H), 0.90 (d, J = 6.7 Hz, 6 H), 0.91–1.63 (m,
20 H), 0.98 (s, 3 H), 1.70 (m, 2 H), 1.77–1.83 (m, 2 H), 1.93–2.01
(m, 2 H), 2.16 (ddd, J = 13.3, 4.9, 2.2 Hz, 1 H), 2.25 (m, 1 H),
3.41 (dd, J = 10.5, 6.5 Hz. 1 H), 3.44–3.51 (m, 1 H), 3.48 (dd, J =
10.5, 4.6 Hz, 1 H), 5.31 (m, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = -4.60 (2 CH3), 11.84 (CH3), 16.49 (CH3), 18.27
(C), 18.66 (CH3), 19.42 (CH3), 21.04 (CH2), 23.37 (CH2), 24.27
(CH2), 25.93 (3 CH3), 28.25 (CH2), 31.88 (CH), 31.92 (CH2),
32.06 (CH2), 33.51 (CH2), 35.69 (CH), 35.79 (CH), 36.13 (CH2),
36.56 (C), 37.36 (CH2), 39.78 (CH2), 42.31 (C), 42.80 (CH2), 50.17
(CH), 56.09 (CH), 56.78 (CH), 68.54 (CH2), 72.63 (CH), 121.15
(CH), 141.56 (C); MS (70 eV): m/z (%) = 516 (0.4) [M+], 501
(2), 459 (100), 457 (38); HRMS: m/z calc. for C29H51O2Si [(M -
=
(CH), 140.74 (C), 171.35 (C O); GC-MS (70 eV): m/z (%) = 444
(15) [M+], 429 (16), 426 (100), 411 (27), 384 (16), 359 (27), 333 (47),
271 (12).
916 | Org. Biomol. Chem., 2009, 7, 909–920
This journal is
The Royal Society of Chemistry 2009
©

(25R)-26-Acetoxycholest-4-en-3-one (17). Aluminium iso-
propoxide (232 mg, 1.14 mmol) was added to a solution of the
alcohol 16 (303 mg, 680 mmol) in a mixture of acetone (1 mL) and
toluene (9 mL). The reaction mixture was heated at reflux for 15 h
and after cooling to room temperature, treated with dilute HCl
(20 mL). The two layers were separated and the aqueous layer was
extracted with diethyl ether (2 ¥ 100 mL). The combined organic
layers were dried with magnesium sulfate and the solvent was
evaporated. Purification of the residue by flash chromatography on
silica gel (petroleum ether/diethyl ether, 4:1) furnished the ketone
17, yield: 210 mg (70%). Yellow oil; IR (ATR): n = 2934, 2868,
1737, 1674, 1616, 1465, 1375, 1330, 1230, 1187, 1034, 986, 933,
with ethyl acetate (3 ¥ 50 mL). The combined organic layers were
washed with brine (50 mL), dried with magnesium sulfate and
the solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/ethyl acetate, 60:40
+1% AcOH) provided pure (25R)-D4-dafachronic acid (1a), yield:
158 mg (79%). Colorless solid; mp: 148–150 ◦C; [a]D = +66.0 (c =
20
1.0, CHCl3); IR (ATR): n = 2934, 2865, 2849, 1734, 1703, 1673,
1638, 1614, 1465, 1445, 1435, 1374, 1331, 1269, 1233, 1194, 1161,
1
1141, 1115, 1029, 933, 866, 781, 734 cm-1; H NMR (500 MHz,
CDCl3): d = 0.69 (s, 3 H), 0.88–0.93 (m, 1 H), 0.89 (d, J = 6.5 Hz,
3 H), 0.96–1.63 (m, 17 H), 1.163 (d, J = 6.9 Hz, 3 H), 1.164 (s, 3 H),
1.67 (dt, J = 4.9, 14.0 Hz, 1 H), 1.78–1.86 (m, 2 H), 1.98–2.02 (m,
2 H), 2.25 (ddd, J = 14.5, 4.0, 2.4 Hz, 1 H), 2.30–2.47 (m, 4 H),
5.72 (s, 1 H), 11.15 (br s, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = 11.93 (CH3), 16.74 (CH3), 17.35 (CH3), 18.55 (CH3),
20.99 (CH2), 23.60 (CH2), 24.14 (CH2), 28.15 (CH2), 32.00 (CH2),
32.93 (CH2), 33.86 (CH2), 33.94 (CH2), 35.57 (2 CH), 35.64 (2
CH2), 38.58 (C), 39.26 (CH), 39.57 (CH2), 42.37 (C), 53.75 (CH),
1
865, 778 cm-1; H NMR (500 MHz, CDCl3): d = 0.69 (s, 3 H),
0.88–0.93 (m, 1 H), 0.897 (d, J = 6.6 Hz, 3 H), 0.904 (d, J =
6.7 Hz, 3 H), 0.96–1.15 (m, 7 H), 1.17 (s, 3 H), 1.18–1.84 (m,
14 H), 1.98–2.03 (m, 2 H), 2.04 (s, 3 H), 2.23–2.44 (m, 4 H), 3.82
(dd, J = 10.7, 7.0 Hz, 1 H), 3.93 (dd, J = 10.7, 6.0 Hz, 1 H), 5.71 (s,
1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = 11.93 (CH3),
16.77 (CH3), 17.36 (CH3), 18.57 (CH3), 20.99 (CH3, CH2), 23.20
(CH2), 24.14 (CH2), 28.17 (CH2), 32.00 (CH2), 32.47 (CH), 32.93
(CH2), 33.70 (CH2), 33.97 (CH2), 35.58 (CH), 35.63 (CH), 35.66
(CH2), 35.94 (CH2), 38.58 (C), 39.59 (CH2), 42.37 (C), 53.76 (CH),
=
55.81 (CH), 55.99 (CH), 123.71 (CH), 171.85 (C), 182.48 (C O),
+
=
199.83 (C O); ESI-MS: m/z = 415 [(M + H) ]; Anal. calc. for
C27H42O3: C 78.21, H 10.21, found: C 78.03, H 10.46%.
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)cholest-5-en-7-one
(19). A 5.5 M solution of tert-butyl hydroperoxide in decane
(500 mL, 2.54 mmol) was added to a mixture of the disilyl ether
=
55.83 (CH), 56.01 (CH), 69.59 (CH2), 123.72 (CH), 171.34 (C O),
+
=
171.73 (C), 199.71 (C O); MS (70 eV): m/z (%) = 442 (100) [M ],
440 (8), 427 (7), 425 (15), 400 (23), 319 (15), 271 (11), 269 (19);
R
8 (400 mg. 630 mmol), Celiteꢀ (760 mg) and PDC (950 mg,
HRMS: m/z calc. for C29H46O3 [M+]: 442.3447, found: 442.3433.
2.54 mmol) in benzene (20 mL) at 0 ◦C. The ice-bath was removed
and stirring was continued at room temperature for further 24 h.
A second portion of tert-butylhydroperoxide (500 mL, 2.54 mmol)
was added and stirring was continued for 4 h. The crude mixture
was filtered through a short pad of silica gel (elution with diethyl
ether) and the solvent was removed. Purification of the residue
by flash chromatography on silica gel (petroleum ether/diethyl
ether, 20:1) provided the enone 19, yield: 231 mg (57%). Colorless
solid; mp: 147–148 ◦C; IR (ATR): n = 2930, 2896, 2857, 1669,
1633, 1472, 1376, 1360, 1317, 1283, 1248, 1189, 1081, 1059, 1006,
952, 940, 889, 876, 864, 836, 803, 772 cm-1; 1H NMR (500 MHz,
CDCl3): d = 0.02 (s, 6 H), 0.05 (s, 6 H), 0.66 (s, 3 H), 0.84 (d, J =
6.7 Hz, 3 H), 0.875 (s, 9 H), 0.879 (s, 9 H), 0.90 (d, J = 6.5 Hz, 3 H),
0.96–1.38 (m, 13 H), 1.17 (s, 3 H), 1.44–1.66 (m, 5 H), 1.78–1.91
(m, 3 H), 2.01 (dt, J = 12.6, 3.2 Hz, 1 H), 2.22 (dd, J = 12.1,
11.1 Hz, 1 H), 2.36–2.40 (m, 3 H), 3.33 (dd, J = 9.7, 6.7 Hz, 1 H),
3.42 (dd, J = 9.7, 5.9 Hz, 1 H), 3.58 (m, 1 H), 5.65 (d, J = 1.1 Hz,
1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = -5.34 (2 CH3),
-4.70 (CH3), -4.67 (CH3), 11.94 (CH3), 16.65 (CH3), 17.28 (CH3),
18.14 (C), 18.35 (C), 18.83 (CH3), 21.17 (CH2), 23.35 (CH2), 25.82
(3 CH3), 25.95 (3 CH3), 26.29 (CH2), 28.55 (CH2), 31.71 (CH2),
33.52 (CH2), 35.62 (CH), 35.71 (CH), 36.16 (CH2), 36.39 (CH2),
38.33 (C), 38.69 (CH2), 42.51 (CH2), 43.06 (C), 45.38 (CH), 49.92
(CH), 49.97 (CH), 54.76 (CH), 68.54 (CH2), 71.31 (CH), 125.79
(25R)-26-Hydroxycholest-4-en-3-one (18). Potassium carbon-
ate (6 mg, 45 mmol) was added to a solution of the acetate 17
(200 mg, 450 mmol) in methanol (20 mL) and the reaction mixture
was stirred at room temperature for 20 h. A second portion of
potassium carbonate (6 mg, 45 mmol) was added and stirring
was continued for further 20 h. Water (50 mL) was added and
the solution was extracted with ethyl acetate (3 ¥ 50 mL). The
combined organic layers were dried with magnesium sulfate and
the solvent was evaporated. Purification of the residue by flash
chromatography on silica gel (petroleum ether/diethyl ether, 1:8)
provided◦the alcohol 18, yield: 167 mg (93%). Colorless crystals;
mp: 125 C; IR (ATR): n = 3415, 2928, 2860, 1660, 1615, 1447,
1434, 1376, 1332, 1276, 1231, 1188, 1112, 1038, 958, 932, 861, 779,
1
740 cm-1; H NMR (500 MHz, CDCl3): d = 0.69 (s, 3 H), 0.88–
1.15 (m, 8 H), 0.90 (d, J = 6.7 Hz, 6 H), 1.17 (s, 3 H), 1.21–1.63
(m, 11 H), 1.68 (dt, J = 4.6, 14.0 Hz, 1 H), 1.79–1.88 (m, 2 H),
1.98–2.06 (m, 2 H), 2.23–2.44 (m, 4 H), 3.41 (dd, J = 10.5, 6.4 Hz,
1 H), 3.49 (dd, J = 10.5, 5.8 Hz, 1 H), 5.71 (s, 1 H); 13C NMR
and DEPT (125 MHz, CDCl3): d = 11.94 (CH3), 16.48 (CH3),
17.36 (CH3), 18.57 (CH3), 20.99 (CH2), 23.37 (CH2), 24.15 (CH2),
28.18 (CH2), 32.01 (CH2), 32.93 (CH2), 33.48 (CH2), 33.97 (CH2),
35.58 (CH), 35.65 (CH, CH2), 35.77 (CH), 36.05 (CH2), 38.58
(C), 39.60 (CH2), 42.37 (C), 53.77 (CH), 55.84 (CH), 56.05 (CH),
=
=
68.51 (CH2), 123.72 (CH), 171.72 (C), 199.69 (C O). For further
spectroscopic data see ref. 8.
(CH), 165.88 (C), 202.49 (C O); MS (70 eV): m/z (%) = 644 (0.4)
[M+], 629 (3), 587 (100), 585 (8), 455 (6), 441 (3), 265 (5); HRMS:
m/z calc. for [(M - tert-Bu)+]: 587.4310, found: 587.4313; Anal.
calc. for C39H72O3Si2: C 72.61, H 11.25, found: C 72.83, H 11.52%.
(25R)-3-Ketocholest-4-en-26-oic acid – (25R)-D4-dafachronic
acid (1a). Freshly prepared Jones reagent, consisting of CrO3
(238 mg, 2.4 mmol in 400 mL water) and concentrated sulfuric
acid (209 mL, 3.77 mmol in 500 mL water), was added to a
solution of the diol 18 (193 mg, 480 mmol) in acetone (30 mL)
at 0 ◦C. After stirring for 1 h at 0 ◦C, 2-propanol (10 mL) and
brine (10 mL) were added, and the resulting mixture was extracted
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)-5a-cholestan-7-one
(20). Ammonium formate (40 mg, 620 mmol) was added to a
mixture of the enone 19 (100 mg, 155 mmol) and Pd/C (10%,
16 mg, 15 mmol Pd) in methanol (3 mL) and ethyl acetate (4 mL).
The mixture was heated at reflux for 8 h and then cooled to
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 909–920 | 917
©

R
room temperature. Filtration over a short pad of Celiteꢀ (elution
-78 ◦C. The reaction mixture was allowed to warm to room
temperature over a period of 24 h. Diethyl ether (50 mL), water
(10 mL), and 10% sulfuric acid (5 mL) were added. The layers
were separated and the aqueous layer was extracted with diethyl
ether (3 ¥ 50 mL). The combined organic layers were washed
with a saturated solution of sodium hydrogen carbonate (50 mL)
and brine (50 mL), and then dried over magnesium sulfate.
Evaporation of the solvent and purification of the residue by
flash chromatography on silica gel (petroleum ether/diethyl ether,
10:1) provided as the less polar fraction the 7a-alcohol 21 (59 mg,
85 mmol, 59%) (spectral data, see above) and the more polar 7b-
alcohol 22, yield: 29 mg (31%). Colorless solid; mp: 88–94 ◦C; IR
(ATR): n = 3488, 2928, 2856, 1471, 1374, 1249, 1187, 1100, 1074,
with diethyl ether) and removal of the solvent gave the crude
product, which was purified by flash chromatography on silica
gel (petroleum ether/diethyl ether, 40:1 to 20:1) to provide ◦20,
yield: 90 mg (90%). Colorless crystalline solid; mp: 102–103 C;
IR (ATR): n = 2928, 2856, 1706, 1671, 1471, 1376, 1361, 1252,
1174, 1098, 1053, 1006, 941, 909, 872, 835, 774, 735, 667 cm-1; 1H
NMR (500 MHz, CDCl3): d = 0.021 (s, 6 H), 0.025 (s, 6 H), 0.63
(s, 3 H), 0.84 (d, J = 6.7 Hz, 3 H), 0.84–1.10 (m, 5 H), 0.86 (s, 9 H),
0.88 (s, 9 H), 0.89 (d, J = 7.7 Hz, 3 H), 1.06 (s, 3 H), 1.16–1.58
(m, 16 H), 1.71 (m, 2 H), 1.87 (m, 1 H), 1.95–2.00 (m, 2 H), 2.16
(m, 1 H), 2.33 (m, 2 H), 3.33 (dd, J = 9.7, 6.7 Hz, 1 H), 3.42
(dd, J = 9.7, 5.9 Hz, 1 H), 3.53 (m, 1 H); 13C NMR and DEPT
(125 MHz, CDCl3): d = -5.34 (2 CH3), -4.66 (2 CH3), 11.86 (CH3),
12.03 (CH3), 16.64 (CH3), 18.20 (C), 18.35 (C), 18.74 (CH3), 21.84
(CH2), 23.29 (CH2), 24.93 (CH2) 25.87 (3 CH3), 25.95 (3 CH3),
28.40 (CH2), 31.52 (CH2), 33.50 (CH2), 35.55 (CH), 35.70 (CH),
36.01 (C), 36.11 (CH2), 36.21 (CH2), 38.44 (CH2), 38.75 (CH2),
42.47 (C), 46.21 (CH2), 47.13 (CH), 48.83 (CH), 50.01 (CH), 55.01
1
1026, 1005, 943, 870, 833, 771, 667 cm-1; H NMR (500 MHz,
CDCl3): d = 0.02 (s, 6 H), 0.03 (s, 6 H), 0.66 (s, 3 H), 0.81 (s, 3 H),
0.84 (d, J = 6.6 Hz, 3 H), 0.87 (s, 9 H), 0.88 (s, 9 H), 0.89 (d, J =
6.7 Hz, 3 H), 0.97–1.89 (m, 28 H), 1.97 (m, 1 H), 3.31–3.36 (m,
1 H), 3.34 (dd, J = 9.7, 6.6 Hz, 1 H), 3.42 (dd, J = 9.7, 6.0 Hz, 1 H),
3.53 (m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = -5.34
(2 CH3), -4.58 (2 CH3), 12.13 (CH3), 12.47 (CH3), 16.65 (CH3),
18.24 (C), 18.36 (C), 18.75 (CH3), 21.40 (CH2), 23.35 (CH2), 25.92
(3 CH3), 25.95 (3 CH3), 26.90 (CH2), 28.73 (CH2), 31.86 (CH2),
33.54 (CH2), 34.94 (C), 35.59 (CH), 35.72 (CH), 36.17 (CH2),
37.05 (CH2), 38.09 (CH2), 38.17 (CH2), 39.99 (CH2), 42.16 (CH),
43.43 (CH), 43.60 (C), 52.55 (CH), 55.20 (CH), 55.72 (CH), 68.54
(CH2), 71.91 (CH), 75.24 (CH); GC-MS (70 eV): m/z (%) = 591
(90) [(M - tert-Bu)+], 589 (10), 573 (12), 459 (22), 75 (100); Anal.
calc. for C39H76O3Si2: C 72.15, H 11.80, found: C 72.36, H 11.93%.
=
(CH), 55.45 (CH), 68.53 (CH2), 71.51 (CH), 212.44 (C O); MS
(70 eV): m/z (%) = 646 (0.2) [M+], 631 (2), 589 (100), 587 (9), 459
(16), 445 (12); HRMS: m/z calc. for C35H63O3Si2 [(M - tert-Bu)+]:
589.4467, found: 589.4478; Anal. calc. for C39H74O3Si2: C 72.38,
H 11.53, found: C 72.56, H 11.57%.
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)-5a-cholestan-7a-ol
(21). A 2.0 M solution of isopropylmagnesium chloride in diethyl
ether (110 mL, 220 mmol) was further diluted with diethyl ether
(2 mL), and added dropwise over a period of 30 min to a solution
of the ketone 20 (100 mg, 155 mmol) in diethyl ether (8 mL) at
room temperature. After stirring for 1 h at room temperature,
the reaction mixture was quenched by addition of a saturated
solution of ammonium chloride (25 mL) and the layers were
separated. The aqueous layer was extracted with diethyl ether
(2 ¥ 50 mL). The combined organic layers were washed with
water (50 mL) and brine (50 mL), and dried with magnesium
sulfate. Removal of the solvent and purification of the residue by
flash chromatography on silica gel (petroleum ether/diethyl ether,
10:1) provided diastereoselectively the 7a-alcohol 21, yield: 70 mg
(70%). Colorless solid; mp: 95–98 ◦C; IR (ATR): n = 3455, 2928,
2856, 1471, 1376, 1361, 1250, 1098, 1078, 1031, 1006, 948, 834,
772, 667 cm-1; 1H NMR (500 MHz, CDCl3): d = 0.02 (s, 6 H), 0.03
(s, 6 H), 0.64 (s, 3 H), 0.78 (s, 3 H), 0.84 (d, J = 6.7 Hz, 3 H), 0.86 (s,
9 H), 0.88 (s, 9 H), 0.89 (d, J = 8.2 Hz, 3 H), 0.96–1.68 (m, 27 H),
1.83–1.87 (m, 1 H), 1.93 (dt, J = 12.6, 3.2 Hz, 1 H), 3.33 (dd, J =
9.7, 6.7 Hz, 1 H), 3.42 (dd, J = 9.7, 6.0 Hz, 1 H), 3.57 (m, 1 H),
3.81 (m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = -5.33
(2 CH3), -4.58 (2 CH3), 11.27 (CH3), 11.81 (CH3), 16.64 (CH3),
18.25 (C), 18.36 (C), 18.61 (CH3), 20.96 (CH2), 23.26 (CH2), 23.64
(CH2), 25.95 (6 CH3), 28.20 (CH2), 31.85 (CH2), 33.52 (CH2),
35.57 (C), 35.68 (CH), 35.71 (CH), 36.10 (CH2), 36.28 (CH2),
36.90 (CH2), 37.21 (CH), 38.18 (CH2), 39.51 (CH2), 39.54 (CH),
42.66 (C), 45.92 (CH), 50.58 (CH), 56.09 (CH), 68.12 (CH), 68.55
(CH2), 71.97 (CH); GC-MS (70 eV): m/z (%) = 591 (84) [(M -
tert-Bu)+], 573 (25), 459 (17), 75 (100); Anal. calc. for C39H76O3Si2:
C 72.15, H 11.80, found: C 72.16, H 11.91%.
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)-5a-cholest-7-ene
(23). Thionyl chloride (33 mL, 450 mmol) was added to a solution
of the 7a-alcohol 21 (100 mg, 150 mmol) in pyridine (10 mL) at
0
◦C. After stirring for 1 h at 0 ◦C, water (50 mL) was added
and the mixture was extracted with diethyl ether (3 ¥ 50 mL).
The combined organic layers were dried with magnesium sulfate
and the solvent was evaporated. Purification of the residue by
flash chromatography on silica gel (petroleum ether/diethyl ether,
20:1) provided 23, yield: 95 mg (100%). Colorless crystals; mp: 68–
78 ◦C; IR (ATR): n = 2929, 2890, 2855, 1738, 1471, 1375, 1360,
1
1248, 1160, 1099, 1007, 939, 869, 836, 773, 668 cm-1; H NMR
(500 MHz, CDCl3): d = 0.02 (s, 6 H), 0.04 (s, 6 H), 0.51 (s, 3 H),
0.77 (s, 3 H), 0.84 (d, J = 6.7 Hz, 3 H), 0.87 (s, 9 H), 0.88 (s, 9 H),
0.90 (d, J = 6.6 Hz, 3 H), 0.97–1.06 (m, 5 H), 1.17–1.88 (m, 22 H),
2.00 (dt, J = 12.5, 3.4 Hz, 1 H), 3.34 (dd, J = 9.7, 6.7 Hz, 1 H),
3.42 (dd, J = 9.7, 5.9 Hz, 1 H), 3.53 (m, 1 H), 5.14 (m, 1 H); 13
C
NMR and DEPT (125 MHz, CDCl3): d = -5.34 (2 CH3), -4.58
(2 CH3), 11.81 (CH3), 13.08 (CH3), 16.65 (CH3), 18.28 (C), 18.36
(C), 18.80 (CH3), 21.50 (CH2), 22.94 (CH2), 23.41 (CH2), 25.95 (6
CH3), 27.95 (CH2), 29.69 (CH2), 31.86 (CH2), 33.54 (CH2), 34.21
(C), 35.73 (CH), 36.10 (CH, CH2), 37.31 (CH2), 38.43 (CH2), 39.57
(CH2), 40.38 (CH), 43.37 (C), 49.51 (CH), 55.03 (CH), 56.12 (CH),
68.55 (CH2), 71.89 (CH), 117.49 (CH), 139.59 (C); GC-MS (70
eV): m/z (%) = 615 (2) [(M - Me)+], 573 (100), 497 (8), 441 (7),
258 (14); Anal. calc. for C39H74O2Si2: C 74.21, H 11.82, found: C
73.75, H 12.33%.
(25R)-3b-Hydroxy-5a-cholest-7-en-26-ol (24). TBAF (1.0 M
in THF, 330 mL, 330 mmol) and water (18 mL, 990 mmol) were
added to a solution of 23 (94 mg, 150 mmol) in THF (10 mL) and
the resulting mixture was heated at reflux for 20 h. After cooling to
(25R)-3b,26-Bis(tert-butyldimethylsilyloxy)-5a-cholestan-7b-ol
(22). Lithium aluminium hydride (5.5 mg, 144 mmol) was added
to a solution of 20 (93 mg, 144 mmol) in THF (10 mL) at
918 | Org. Biomol. Chem., 2009, 7, 909–920
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The Royal Society of Chemistry 2009
©

◦
room temperature, water (50 mL) was added and the mixture was
extracted with ethyl acetate (3 ¥ 50 mL). The combined organic
layers were washed with brine (50 mL) and dried with magnesium
sulfate. Purification of the residue by flash chromatography on
silica gel (petroleum ether/diethyl ether, 1:10) provided the diol 24,
yield: 87 mg (89%). Colorless solid; mp: 138–141 C; IR (ATR):
n = 2930, 2896, 2856, 1727, 1462, 1369, 1251, 1197, 1087, 1006,
960, 925, 888, 869, 835, 804, 772, 667 cm-1; 1H NMR (500 MHz,
CDCl3): d = 0.04 (s, 6 H), 0.66 (s, 3 H), 0.88 (s, 9 H), 0.90 (d, J =
6.5 Hz, 3 H), 0.98 (s, 3 H), 1.08 (d, J = 7.0 Hz, 3 H), 1.10–1.72
(m, 21 H), 1.76–1.83 (m, 2 H), 1.93–2.00 (m, 2 H), 2.15 (ddd, J =
13.3, 5.0, 2.2 Hz, 1 H), 2.23–2.34 (m, 2 H), 3.47 (m, 1 H), 5.30 (m,
1 H), 9.60 (d, J = 2.0 Hz, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = -4.60 (2 CH3), 11.83 (CH3), 13.27 (CH3), 18.27
(C), 18.63 (CH3), 19.41 (CH3), 21.03 (CH2), 23.36 (CH2), 24.25
(CH2), 25.93 (3 CH3), 28.23 (CH2), 30.91 (CH2), 31.87 (CH),
31.91 (CH2), 32.06 (CH2), 35.59 (CH), 35.87 (CH2), 36.56 (C),
37.36 (CH2), 39.76 (CH2), 42.31 (C), 42.79 (CH2), 46.38 (CH),
50.15 (CH), 55.95 (CH), 56.76 (CH), 72.62 (CH), 121.12 (CH),
141.56 (C), 205.48 (CHO); GC-MS (70 eV): m/z (%) = 514 (0.5)
[M+], 499 (3), 457 (100); Anal. calc. for C33H58O2Si: C 76.98, H
11.35, found: C 76.92, H 11.44%.
◦
yield: 56 mg (93%). Colorless solid; mp: 148–150 C; IR (ATR):
n = 3289, 2925, 2851, 1734, 1464, 1450, 1373, 1344, 1246, 1160,
1
1134, 1097, 1040, 984, 940, 847, 829, 797, 729 cm-1; H NMR
(500 MHz, CDCl3): d = 0.52 (s, 3 H), 0.78 (s, 3 H), 0.90 (d, J =
6.7 Hz, 3 H), 0.91 (d, J = 6.6 Hz, 3 H), 0.99–1.88 (m, 27 H), 2.01
(dt, J = 12.5, 3.3 Hz, 1 H), 3.41 (dd, J = 10.5, 6.5 Hz, 1 H), 3.49
(dd, J = 10.5, 5.9 Hz, 1 H), 3.58 (m, 1 H), 5.15 (dd, J = 4.6,
2.2 Hz, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = 11.84
(CH3), 13.04 (CH3), 16.49 (CH3), 18.78 (CH3), 21.52 (CH2), 22.93
(CH2), 23.47 (CH2), 27.96 (CH2), 29.62 (CH2), 31.45 (CH2), 33.49
(CH2), 34.18 (C), 35.79 (CH), 36.07 (CH2), 36.11 (CH), 37.11
(CH2), 37.96 (CH2), 39.53 (CH2), 40.21 (CH), 43.37 (C), 49.40
(CH), 55.01 (CH), 56.09 (CH), 68.53 (CH2), 71.05 (CH), 117.44
(CH), 139.56 (C); ESI-MS: m/z = 385 [(M - H2O + H)+]; Anal.
calc. for C27H46O2: C 80.54, H 11.51, found: C 80.42, H 11.63%.
(25R)-3b-(tert-Butyldimethylsilyloxy)cholest-5-en-26-oic acid
(26). A solution of sodium chlorite (38 mg, 0.42 mmol) in
water (1 mL) was added to a solution of the aldehyde 25
(144 mg, 0.280 mmol), 2-methyl-2-butene (304 mL, 2.8 mmol)
and potassium dihydrogen phosphate (50 mg, 0.37 mmol) in a
mixture of THF (6 mL) and water (1 mL). The resulting mixture
was stirred at room temperature for 16 h. The solution was
acidified with dilute HCl (20 mL) and extracted with diethyl
ether (3 ¥ 100 mL). The combined organic layers were washed
with a saturated solution of sodium hydrogen carbonate (50 mL)
and brine (50 mL), and then dried with magnesium sulfate.
Evaporation of the solvent and purification of the residue by
flash chromatography on silica gel (petroleum ether/diethyl ether,
4:1 to 2:1) provided the acid 26, yield: 137 mg (92%). Colorless
crystals; mp: 175 ◦C; IR (ATR): n = 2929, 2895, 2854, 1706,
1463, 1427, 1371, 1249, 1198, 1082, 1007, 963, 940, 926, 890,
(25R)-3-Keto-5a-cholest-7-en-26-oic acid – (25R)-D7-Dafachro-
nic acid (2a). Freshly prepared Jones reagent, consisting of CrO3
(103 mg, 1.03 mmol in 100 mL water) and concentrated sulfuric
acid (90 mL, 1.62 mmol in 400 mL water), was added to a solution
of the diol 24 (83 mg, 206 mmol) in acetone (50 mL) at 0 ◦C. After
◦
stirring for 1 h at 0 C, 2-propanol (10 mL) was added and the
resulting mixture was extracted with ethyl acetate (3 ¥ 100 mL).
The combined organic layers were dried with magnesium sulfate
and the solvent was removed in vacuo. Purification of the residue by
flash chromatography on silica gel (petroleum ether/ethyl acetate,
3:1) provided pure (25R)-D7-dafachronic acid (2a), yield: 63 mg
(74%). Colorless solid; mp: 174–175 ◦C; [a]D = +13.3 (c =
20
0.5, CHCl3); IR (ATR): n = 3093, 2955, 2932, 2862, 1731, 1690,
1446, 1414, 1378, 1328, 1301, 1263, 1233, 1210, 1210, 1196, 1170,
1117, 1101, 948, 874, 847, 835, 807, 764, 736, 637 cm-1; 1H NMR
(500 MHz, CDCl3): d = 0.54 (s, 3 H), 0.91 (d, J = 6.5 Hz, 3 H),
1.00 (s, 3 H), 1.01–1.05 (m, 1 H), 1.17 (d, J = 7.0 Hz, 3 H), 1.18–
1.29 (m, 5 H), 1.35–1.65 (m, 10 H), 1.71–1.91 (m, 5 H), 2.03 (dt,
J = 12.6, 3.3 Hz, 1 H), 2.11 (ddd, J = 13.3, 5.9, 2.3 Hz, 1 H),
2.20–2.28 (m, 3 H), 2.40 (dd, J = 14.6, 6.0 Hz, 1 H), 2.45 (dd, J =
13.5, 6.8 Hz, 1 H), 5.17 (m, 1 H); 13C NMR and DEPT (125 MHz,
CDCl3): d = 11.87 (CH3), 12.44 (CH3), 16.72 (CH3), 18.74 (CH3),
21.66 (CH2), 22.91 (CH2), 23.68 (CH2), 27.90 (CH2), 30.02 (CH2),
33.85 (CH2), 34.35 (C), 35.62 (CH2), 36.01 (CH), 38.09 (CH2),
38.73 (CH2), 39.24 (CH), 39.38 (CH2), 42.82 (CH), 43.33 (C),
44.20 (CH2), 48.78 (CH), 54.87 (CH), 56.01 (CH), 116.99 (CH),
870, 835, 804, 772 cm-1; H NMR (500 MHz, CDCl3): d = 0.04
1
(s, 6 H), 0.66 (s, 3 H), 0.88 (s, 9 H), 0.90 (d, J = 6.5 Hz, 3 H),
0.91–0.97 (m, 2 H), 0.98 (s, 3 H), 0.99–1.15 (m, 5 H), 1.17 (d, J =
7.0 Hz, 3 H), 1.18–1.84 (m, 16 H), 1.93–2.01 (m, 2 H), 2.15 (ddd,
J = 13.3, 4.9, 2.1 Hz, 1 H), 2.23–2.28 (m, 1 H), 2.45 (dd, J =
13.7, 6.9 Hz, 1 H), 3.47 (m, 1 H), 5.30 (m, 1 H); 13C NMR and
DEPT (125 MHz, CDCl3): d = -4.60 (2 CH3), 11.84 (CH3), 16.73
(CH3), 18.27 (C), 18.63 (CH3), 19.41 (CH3), 21.04 (CH2), 23.61
(CH2), 24.27 (CH2), 25.93 (3 CH3), 28.22 (CH2), 31.88 (CH),
31.91 (CH2), 32.06 (CH2), 33.90 (CH2), 35.62 (CH), 35.72 (CH2),
36.56 (C), 37.36 (CH2), 39.19 (CH), 39.76 (CH2), 42.31 (C), 42.79
(CH2), 50.16 (CH), 56.05 (CH), 56.75 (CH), 72.64 (CH), 121.14
=
(CH), 141.55 (C), 182.19 (C O); MS (70 eV): m/z (%) = 515 (2)
=
=
139.46 (C), 182.51 (C O), 212.19 (C O); MS (70 eV): m/z (%) =
414 (100) [M+], 399 (27), 397 (7), 381 (8), 368 (7), 277 (5), 271 (54);
HRMS: m/z calc. for C27H42O3 [M+]: 414.3134, found: 414.3142;
Anal. calc. for C27H42O3: C 78.21, H 10.21, found: C 78.42, H
10.41%.
[(M - Me)+], 473 (100), 471 (16), 455 (18); HRMS: m/z calc. for
C29H49O3Si [(M - tert-Bu)+]: 473.3451, found: 473.3418; Anal.
calc. for C33H58O3Si: C 74.66, H 11.01, found: C 74.82, H 11.10%.
(25R)-3b-Hydroxycholest-5-en-26-oic acid methyl ester (27).
One drop of concentrated sulfuric acid was added to a solution
of the acid 26 (134 mg, 0.252 mmol) in methanol (10 mL). The
reaction mixture was heated at reflux for 16 h, then neutralized
with a saturated solution of sodium hydrogen carbonate (30 mL)
and extracted with dichloromethane (3 ¥ 50 mL). The combined
organic layers were washed with brine (50 mL) and dried with
magnesium sulfate. Evaporation of the solvent and purification
of the residue by flash chromatography on silica gel (petroleum
(25R)-3b-(tert-Butyldimethylsilyloxy)cholest-5-en-26-al
(25).
PDC (143 mg, 0.38 mmol) was added to a mixture of the alcohol
˚
14 (100 mg, 0.19 mmol) and 4 A molecular sieves (95 mg) in
dichloromethane (5 mL). The reaction mixture was stirred at
room temperature for 3.5 h and then the solvent was evaporated.
Purification of the residue by flash chromatography on silica gel
(petroleum ether/diethyl ether, 10:1) afforded the aldehyde 25,
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 909–920 | 919
©

ether/ethyl acetate, 6:1) provided the ester 27, yield: 91 mg (84%).
Colorless solid; mp: 65 ◦C; IR (ATR): n = 3416, 2929, 2864,
2846, 1736, 1461, 1435, 1375, 1363, 1254, 1198, 1163, 1108, 1055,
1022, 985, 955, 926, 882, 840, 799, 740 cm-1; 1H NMR (500 MHz,
CDCl3): d = 0.66 (s, 3 H), 0.89 (d, J = 6.5 Hz, 3 H), 0.91–1.20
(m, 7 H), 0.99 (s, 3 H), 1.13 (d, J = 7.0 Hz, 3 H), 1.23–1.63 (m,
13 H), 1.76–1.85 (m, 3 H), 1.94 (m, 1 H), 1.98 (dt, J = 12.6, 3.5 Hz,
1 H), 2.23 (m, 1 H), 2.28 (ddd, J = 13.1, 5.0, 2.0 Hz, 1 H), 2.42
(dd, J = 13.9, 6.9 Hz, 1 H), 3.51 (m, 1 H), 3.66 (s, 3 H), 5.34
(m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3): d = 11.84
(CH3), 16.95 (CH3), 18.61 (CH3), 19.38 (CH3), 21.05 (CH2), 23.67
(CH2), 24.26 (CH2), 28.20 (CH2), 31.62 (CH2), 31.87 (CH), 31.91
(CH2), 34.17 (CH2), 35.61 (CH), 35.72 (CH2), 36.47 (C), 37.22
(CH2), 39.44 (CH), 39.73 (CH2), 42.27 (CH2), 42.30 (C), 50.07
(CH), 51.46 (CH3), 56.03 (CH), 56.71 (CH), 71.77 (CH), 121.67
Notes and references
1 S. Brenner, Genetics, 1974, 77, 71.
2 T. Kaletta and M. O. Hengartner, Nat. Rev. Drug Disc., 2006, 5,
387.
3 (a) A. Antebi, J. G. Culotti and E. M. Hedgecock, Development, 1998,
125, 1191; (b) A. Antebi, W.-H. Yeh, D. Tait, E. M. Hedgecock and
D. L. Riddle, Genes Dev., 2000, 14, 1512; (c) B. Gerisch, C. Weitzel,
C. Kober-Eisermann, V. Rottiers and A. Antebi, Dev. Cell, 2001, 1,
841; (d) D. L. Motola, C. L. Cummins, V. Rottiers, K. V. Sharma, T.
Li, Y. Li, K. Suino-Powell, H. E. Xu, R. J. Auchus, A. Antebi and
D. J. Mangelsdorf, Cell, 2006, 124, 1209; (e) V. Rottiers, D. L. Motola,
B. Gerisch, C. L. Cummins, K. Nishiwaki, D. J. Mangelsdorf and A.
Antebi, Dev. Cell, 2006, 10, 473; (f) B. Gerisch, V. Rottiers, D. Li, D. L.
Motola, C. L. Cummins, H. Lehrach, D. J. Mangelsdorf and A. Antebi,
Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 5014.
4 V. Matyash, E. V. Entchev, F. Mende, M. Wilsch-Bra¨uninger, C. Thiele,
A. W. Schmidt, H.-J. Kno¨lker, S. Ward and T. V. Kurzchalia, PLoS
Biol., 2004, 2, 1561.
5 J. M. Held, M. P. White, A. L. Fisher, B. W. Gibson, G. J. Lithgow and
M. S. Gill, Aging Cell, 2006, 5, 283.
6 S. Giroux and E. J. Corey, J. Am. Chem. Soc., 2007, 129, 9866.
7 S. Giroux and E. J. Corey, Org. Lett., 2008, 10, 801.
8 V. A. Khripach, V. N. Zhabinskii, O. V. Konstantinova, N. B. Khripach,
A. V. Antonchick, A. P. Antonchick and B. Schneider, Steroids, 2005,
70, 551.
9 (a) A. W. Schmidt, T. Doert, S. Goutal, M. Gruner, F. Mende, T. V.
Kurzchalia and H.-J. Kno¨lker, Eur. J. Org. Chem., 2006, 3687; (b) R.
Martin, F. Da¨britz, E. V. Entchev, T. V. Kurzchalia and H.-J. Kno¨lker,
Org. Biomol. Chem., 2008, 6, 4293.
=
(CH), 140.74 (C), 177.45 (C O); MS (70 eV): m/z (%) = 430 (54)
[M+], 415 (26), 412 (42), 397 (24), 345 (22), 319 (51), 271 (100);
HRMS: m/z calc. for C28H46O3 [M+]: 430.3447, found: 430.3444.
(25R)-3b-Hydroxycholest-5-en-26-oic acid – (25R)-Cholestenoic
acid (3a). Lithium hydroxide (16 mg, 654 mmol) was added to
a solution of the methyl ester 27 (91 mg, 211 mmol) in a mixture
of tetrahydrofuran/methanol/water (2:1:1) (4 mL). The reaction
mixture was stirred at room temperature for 16 h. Further water
(10 mL) was added and the reaction mixture was extracted with
dichloromethane (3 ¥ 20 mL) to remove traces of unreacted
starting material. The aqueous layer was acidified with dilute HCl
(20 mL) and then extracted with dichloromethane (3 ¥ 50 mL).
The combined organic layers were dried with magnesium sulfate
and the solvent was evaporated to afford pure (25R)-cholestenoic
acid (3a), yield: 85 mg (97%). An analytically pure sample was
prepared by recrystallization from acetonitrile. Colorless solid;
10 R. Martin, A. W. Schmidt, G. Theumer, T. V. Kurzchalia and H.-J.
Kno¨lker, Synlett, 2008, 1965.
11 J. R. Williams, D. Chai and D. Wright, Steroids, 2002, 67,
1041.
12 J. R. Williams, D. Chai, J. D. Bloxton, H. Gong and W. R. Solvibile,
Tetrahedron, 2003, 59, 3183.
13 H. Gong and J. R. Williams, Org. Lett., 2006, 8, 2253.
14 Y. Ni, H.-S. Kim, W. K. Wilson, A. Kisic and G. J. Schroepfer,
Tetrahedron Lett., 1993, 34, 3687.
15 CCDC-697766 contains the supplementary crystallographic data for
structure 5. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/
data_request/cif.
mp: 168–170 ◦C (MeCN); [a]D = -30.0 (c = 0.1, CHCl3); IR
20
(ATR): n = 3399, 2934, 2899, 2862, 1702, 1460, 1377, 1333, 1178,
1106, 1052, 1022, 987, 955, 911, 881, 840, 804, 735 cm-1; 1H NMR
(500 MHz, CDCl3): d = 0.66 (s, 3 H), 0.90 (d, J = 6.5 Hz, 3 H),
0.92–1.14 (m, 7 H), 0.99 (s, 3 H), 1.16 (d, J = 6.9 Hz, 3 H), 1.20–
1.27 (m, 2 H), 1.36–1.63 (m, 11 H), 1.77–1.84 (m, 3 H), 1.93–2.01
(m, 2 H), 2.22–2.30 (m, 2 H), 2.45 (dd, J = 13.7, 6.9 Hz, 1 H), 3.52
(m, 1 H), 5.34 (m, 1 H); 13C NMR and DEPT (125 MHz, CDCl3):
d = 11.85 (CH3), 16.74 (CH3), 18.63 (CH3), 19.38 (CH3), 21.05
(CH2), 23.62 (CH2), 24.27 (CH2), 28.21 (CH2), 31.58 (CH2), 31.87
(CH, CH2), 33.91 (CH2), 35.62 (CH), 35.72 (CH2), 36.47 (C),
37.22 (CH2), 39.27 (CH), 39.73 (CH2), 42.21 (CH2), 42.30 (C),
16 E. J. Corey and G. Schmidt, Tetrahedron Lett., 1979, 399.
ˇ
17 B. A. Solaja, D. R. Milic´ and L. I. Dosˇen-Mic´ovic´, Steroids, 1994, 59,
330; H. Chodounska´, V. Pouzar, M. Budeˇsˇ´ınsky´, B. Slav´ıkova´ and L.
Kohout, Steroids, 2004, 69, 605.
18 J. M. Brunel, C. Loncle, N. Vidal, M. Dherbomez and Y. Letourneux,
Steroids, 2005, 70, 907.
19 (a) M. J. S. M. Moreno, M. L. Sa´ e Melo and A. S. Campos Neves,
Tetrahedron Lett., 1991, 32, 3201; (b) C. K. Acosta, P. N. Rao and H. K.
Kim, Steroids, 1993, 58, 205.
20 K. Q. Shawakfeh, N. H. Al-Said and R. M. Al-Zoubi, Steroids, 2008,
73, 579.
21 (a) H. Nagaoka, W. Rutsch, G. Schmid, H. Ilio, M. R. Johnson and
Y. K i s h i , J. Am. Chem. Soc., 1980, 102, 7965; (b) D. Boschelli, T.
Takemasa, Y. Nishitani and S. Masamune, Tetrahedron Lett., 1985,
26, 5239; (c) K. C. Nicolaou and S. E. Weber, Synthesis, 1986,
453.
50.07 (CH), 56.05 (CH), 56.70 (CH), 71.82 (CH), 121.71 (CH),
+
=
140.69 (C), 182.46 (C O); MS (70 eV): m/z (%) = 416 (100) [M ],
401 (36), 399 (73), 398 (76), 383 (52), 331 (44), 305 (99), 299
(24), 277 (22), 271 (43), 255 (33), 231 (18), 213 (43); HRMS: m/z
calc. for C27H44O3 [M+]: 416.3290, found: 416.3297; Anal. calc. for
C27H44O3: C 77.83, H 10.64, found: C 77.26, H 10.33%.
22 N. Chidambaram and S. Chandrasekaran, J. Org. Chem., 1987, 52,
5048.
23 W. G. Salmond, M. A. Barta and J. L. Havens, J. Org. Chem., 1978, 43,
2057.
24 T. K. M. Shing, Y.-Y. Yeung and P. L. Su, Org. Lett., 2006, 8,
3149.
Acknowledgements
25 H.-J. Kno¨lker, A. Ecker, P. Struwe, A. Steinmeyer, G. Mu¨ller and G.
Neef, Tetrahedron, 1997, 53, 91.
26 S. V. Ley, J. Norman, W. P. Griffith and S. P. Marsden, Synthesis, 1994,
639.
We thank Dr. Margit Gruner for extensive 2D NMR experiments.
We thank Prof. Adam Antebi for daf-9(dh6); dhEx24 strain. We
are grateful to JADO Technologies, Dresden, for their support.
27 B. O. Lindgren and T. Nilsson, Acta Chem. Scand., 1973, 27, 888.
920 | Org. Biomol. Chem., 2009, 7, 909–920
This journal is
The Royal Society of Chemistry 2009
©