A concise synthesis of ent-cholesterol

Article abstract of DOI:10.1021/jo702694g

(Chemical Equation Presented) ent-Cholesterol was synthesized in 16 steps from commercially available (S)-citronellol. The overall yield for the synthesis was 2.0%. This route is amenable to gram-scale preparation of ent-cholesterol. Isotopic incorporatio

Full text of DOI:10.1021/jo702694g

A Concise Synthesis of ent-Cholesterol
Jitendra D. Belani and Scott D. Rychnovsky*
Department of Chemistry, 1102 Natural Sciences II, UniVersity of California, IrVine,
IrVine, California 92697
srychnoV@uci.edu
ReceiVed December 18, 2007
ent-Cholesterol was synthesized in 16 steps from commercially available (S)-citronellol. The overall yield
for the synthesis was 2.0%. This route is amenable to gram-scale preparation of ent-cholesterol. Isotopic
incorporation near the end of the synthesis was achieved using labeled methyl iodide. This synthesis is
the most practical to date and will make ent-cholesterol more readily available to use as a probe of the
function and metabolism of cholesterol.
Introduction
Cholesterol is a steroidal biomolecule that plays a vital role
in cellular functions. Molecular mechanisms underlying these
cellular functions of cholesterol are still largely unclear. The
unnatural enantiomer of cholesterol (Figure 1) can serve as a
valuable biological probe as its chemical composition, bonding
pattern, and relative configuration are the same as the natural
cholesterol. Since the enantiomer of cholesterol is not found in
nature, it must be prepared by chemical synthesis. Our group
reported the first synthesis of ent-cholesterol in 1992.1 It was
then used as a probe to study the interactions of cholesterol
with amphotericin B and its role in the formation of ion
channels.2 ent-Cholesterol has subsequently been synthesized
using several different procedures.3 The most effective route
involved the preparation of a D-ring synthon with an intact side
chain and subsequent elaboration of the CBA rings. Herein we
report a new and more efficient synthesis of ent-cholesterol.
Cholesterol plays a central role in cell membranes and is the
biogenetic precursor to a number steroid hormones. Its enan-
tiomer, ent-cholesterol has been used to probe the role of
cholesterol and specifically to differentiate the general role of
cholesterol in membranes with stereospecific interactions with
proteins and other chiral molecules. Covey and co-workers have
led the way in using ent-cholesterol to probe the function of
FIGURE 1. Natural cholesterol and ent-cholesterol.
cholesterol.4 Westover and Covey studied the effects of
cholesterol on epidermal growth factor (EGF) and found that
the effects are not enantioselective.5 More recently, Cohen and
Rychnovsky have conducted experiments to define the structural
features of sterols required for mammalian growth. The results
indicated that sterols fulfill two roles in mammalian cells: a
bulk membrane requirement in which phytosterols can substitute
for cholesterol and other processes that specifically require small
amounts of cholesterol but are not completely enantioselective.6
Epand and Rychnovsky have shown that specific peptide
chirality is not required for cholesterol containing membranes.
However, a specific chirality of membrane lipids is required
for peptide-induced formation of cholesterol-rich domains.7
(4) (a) Luker, G. D.; Pica, C. M.; Kumar, A.S.; Covey, D. F. Biochemistry
2000, 39, 39:7651-7661. (b) Crowder, C. M.; Westover, E. J.; Kumar, A.
S.; Ostlund, R. E., Jr.; Covey, D. F. J. Biol. Chem. 2001, 276, 44369-
44372.
(5) Westover, W. J.; Covey. D. F.; Brockman. H. L.; Brown, R. E.; Pike,
L. J. J. Biol. Chem. 2003, 278, 51125-51133.
(6) Xu, F.; Rychnovsky, S.; Belani, J. D.; Hobbs, H. H.; Cohen, J. C.;
Rawson, R. B. P. Natl. Acad. Sci. U.S.A. 2005, 102, 14551-14556.
(1) Rychnovsky, S. D.; Mickus, D. E. J. Org. Chem. 1992, 57, 2732-
2736.
(2) Rychnovsky, S. D.; Levitt, D. G.; Mickus, D. E. J. Am. Chem. Soc.
1992, 114, 359-360.
(3) Jiang, X. J.; Covey D. F. J. Org. Chem. 2002, 67, 4893-4900.
10.1021/jo702694g CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/13/2008
2768
J. Org. Chem. 2008, 73, 2768-2773

Synthesis of ent-cholesterol
SCHEME 1. Synthesis of the C-H Insertion Product
FIGURE 2. Retrosynthetic analysis of ent-cholesterol.
These and other studies have provided new insights into the
mechanisms of cellular processes involving cholesterol; how-
ever, much remains to be learned.
One approach to probing cholesterol metabolism would be
to study ent-cholesterol distribution in whole animals. Such
studies would require significant quantities of ent-cholesterol,
and none of the syntheses reported thus far are easily adaptable
to preparation of ent-cholesterol on a gram scale. Our current
efforts were aimed at the development of a practical, scalable
route to this useful probe molecule. An ideal synthesis would
be short, proceed in high yield, and allow for easy incorporation
of heavier isotopes of carbon (13C, 14C) and hydrogen (2H, 3H)
to facilitate distribution and metabolism analysis in vivo. We
have developed a new route that achieves these goals. The
retrosynthetic analysis of our new route is shown in the Figure
2. The synthesis is based on a ring D to C to B to A approach
and incorporates the cholesterol side chain early in the synthesis.
The synthesis of the D-ring intermediate was inspired by Taber’s
elegant synthesis of (-)-astrogorgiadiol, in which he prepared
the intermediate 2 using CH insertion methodology.8
into R-diazo-â-keto ester 3 using p-acetamidobenzenesulfonic
azide (p-ABSA) in 96% yield.11 Taber reported use of Hash-
imoto’s PTPA catalyst12 for the diastereoselective C-H insertion
reaction to provide a ratio of 2.6:1 in favor of the desired
diastereomer. This key CH insertion reaction is pivotal to the
success of the route, and we hoped to improve this ratio by
catalyst optimization. Hence, the precursor for the C-H
insertion was subjected to a series of chiral Rh catalysts. Both
phthalate and naphthalate imides were investigated, with the
smaller phthalates showing consistently higher selectivity (see
Supporting Information). The valine-derived phthalate ligand
gave the best ratio (3.6:1) and was more selective than ligands
with either more or less bulky substituents. The keto ester 2
was crystalline and could be easily recrystallized from ethanol
to >99% ee.
The yield on the C-H insertion step is a bit vague in the
literature because the separation of the two diastereomers is
difficult. When the reaction was carried out on a small scale
and the product was purified by chromatography, we isolated
76% of a 3.6:1 mixture of keto ester 2 and its C13, C14
diastereomer 5. When conducted on a large scale, the crude
product was filtered through a plug of silica gel and crystallized
directly to give 29.5% of keto ester 2 with 99% ee and 99%
de. The mother liquors contained a great deal more keto ester
2, accompanied by its diastereomer 5. Taber had shown that
the mother liquor diastereomers (14% de) could be separated
using an enantioselective reaction to reduce the ketone 5
selectively.8 This process resulted in the recovery of 29% of
the crude mass with 99% de after recrystallization. Taber’s total
yield (40% + 16%) is 56%. Our total yield of pure keto ester
2 on large scale is 29.5%, because we have not processed the
mother liquors. The overall yield for the synthesis is calculated
using 29.5% for this step but could be almost twice that based
on precedent from Taber’s group.
Results
Commercially available (S)-citronellol (>97% ee)9 was
converted to the corresponding benzenesulfonate and subse-
quently alkylated with the dianion of methyl acetoacetate (see
Scheme 1).10 The resultant â-keto ester 4 could be easily distilled
on large scale under high vacuum to provide the product in
63% yield over two steps. The â-keto ester 4 was converted
(7) Epand, R. M.; Rychnovsky, S. D.; Belani, J. D.; Epand, R. F.
Biochem. J. 2005, 390, 541-548.
(8) Malcolm, S. C.; Taber, D. F. J. Org. Chem. 2001, 66, 944-953.
(9) The optical purity of (S)-citronellol was found to vary with supplier,
and the optical rotation was an unreliable indicator of optical purity. (S)-
Citronellol was derivatized with (R)-Mosher acid chloride to give the (S)-
Mosher ester. Ozonolysis and NaBH4 reduction gave a primary alcohol.
The optical purity of this alcohol could be assayed using HPLC analysis
on a Chiracel OD-H column, eluting with 3% i-PrOH in hexanes. The optical
purity of (S)-citronellol purchased from TCI was found to be ca. 60% ee,
whereas the more expensive (S)-citronellol from Aldrich was found to be
>97% ee.
The presence of the C24-C25 alkene in keto ester 2 could
be used to prepare functionalized or substituted sterol side
(11) Baum, J. S.; Shook, D. A.; Davies, H. M. L.; Smith, H. D. Synth.
Commun. 1987, 17, 1709-1716.
(12) Hashimoto, S.; Watanabe, N.; Sato, T.; Shiro, M.; Ikegami, S.
Tetrahedron Lett. 1993, 34, 5109-5112.
(10) Huckins, S. N.; Weiler, L. J. Am. Chem. Soc. 1974, 96, 1082-
1087.
J. Org. Chem, Vol. 73, No. 7, 2008 2769

Belani and Rychnovsky
SCHEME 2. Formation of C,D-Ring Enone
SCHEME 3. Synthesis of Thiophenol Adduct
SCHEME 4. Completing the Synthesis of ent-Cholesterol
chains; however, for the synthesis of ent-cholesterol, the alkene
simply needed to be reduced. Hydrogenation of keto ester 2
using Pd/C provided compound 6 with a saturated side chain
(Scheme 2). Methylation of â-keto ester 6 using MeI and K2-
CO3, followed by decarbomethoxylation gave the desired
R-methyl ketone 8 as a single diastereomer in 86% yield over
three steps. Robinson annulation of ketone 8 with methyl vinyl
ketone in the presence of NaOMe proceeded from the more
substituted thermodynamic enolate. Treatment of the Michael
adduct with p-toluenesulfonic acid catalyzed the aldol condensa-
tion and provided the CD enone 1 in 55% yield from ketone 8.
Taber’s C-H insertion strategy drastically shortens the synthesis
of the sterol side chain and allows the C20 stereogenic center
to be introduced from a chiral pool source.
The initial strategy for the conversion of enone 1 to
ent-cholesterol used the same BA double annulation strategy
developed by Hoffmann La Roche chemists that was used in
our first synthesis (Scheme 3).1,13 Thus, treatment of CD-enone
1 with Stile’s reagent14 provided the desired acid 9 in 90% yield.
Purification of the acid was not easy as the hydrophobic side
chain made the acid-base extraction difficult. Hydrogenation
of the crude acid with Pd/BaSO4 at 10 °C was monitored by
TLC and reaction was worked up rapidly. The solvent, methanol,
was removed under high vacuum while maintaining the tem-
perature below 0 °C because the saturated â-keto acid was very
susceptible to decarboxylation.15 The crude saturated acid was
treated with fresh aqueous formaldehyde in the presence of
piperidine, followed by the addition of a solution of thiophenol
in CH2Cl2 and Et3N to mask the newly formed exo-enone as a
thioether. The desired thioether 10 was obtained in 51% overall
yield over four steps from CD enone 1. While this strategy was
successful, it required very careful handling to avoid the
problematic decarboxylation.15
The BA rings were introduced using the â-keto ester 11 as
illustrated in Scheme 4.13 Annulation of the thiophenol adduct
10 with â-keto ester 1113c proceeded as expected to provide
the tricyclic enone 12 in 73% yield. Reduction of the enone
with Li/NH3 followed by in situ alkylation with MeI installed
the C19 methyl group stereoselectively in 80% yield. This step
must be run at moderate dilution to avoid over-alkylation and
(13) (a) Hajos, Z. G.; Parrish, D. R. J. Org. Chem. 1973, 38, 3244-
3249. (b) Micheli, R. A.; Hajos, Z. G.; Cohen, N.; Parrish, D. R.; Portland,
L. A.; Sciamanna, W.; Scott, M. A.; Wehrli, P. A. J. Org. Chem. 1975, 40,
675-681. (c) Poirier, J. M.; Hennequin, L. Tetrahedron 1989, 45, 4191-
4202.
(15) Covey synthesized CD enone 1 by a different route and reported
that its conversion to acid 9 was problematic due to difficulties in the
purification.3 Their attempted conversion of acid 9 to the exo enone
intermediate by treatment with formaldehyde and piperidine led to a
“complex mixture of products.” In our hands the sequence was successful
with very careful handling but was not easily scalable.
(14) Stile, M. J. J. Am. Chem. Soc. 1959, 81, 2598-2599.
2770 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of ent-cholesterol
SCHEME 5. Improved Strategy for Preparing the CD-Ring
trans-Hydrindane
of the product, and added acid minimized the formation of the
over-reduced product 16. In the optimized conditions, a
combination of methanol, acetic acid and dilute HCl (1 N) in a
ratio of 70:30:0.01 gave the trans-hydrindane as the major
product (8:1) in a combined 82% yield. Recrystallization from
isopropanol provided diastereomerically pure sulfone 15 with
trans-ring junction (62%). Purification of the mother liquor
containing about a 1:1 mixture of the two diastereomers using
HPLC provided another 9% of the desired trans-hydrindane
product. A crystal structure of the sulfone 15 confirmed its
configuration (see Supporting Information). A major side
product (8-12%) in hydrogenation reactions was compound
16, which results from elimination of the sulfone followed by
hydrogenation of the resulting exocyclic olefin. This byproduct
was easily removed by column chromatography.
The synthesis was completed by AB ring annulations and
enone reduction as outlined in Scheme 6. Annulation of trans-
sulfone 15 with â-keto ester 11 afforded the desired tricyclic
enone in 71% yield. Tricyclic enone 12 was identical by
spectroscopy and optical rotation to that obtained by annulation
with thiophenyl adduct 10 (Scheme 4). Enone 12 was elaborated
to ent-cholesterol, as described previously, in four steps and
57% overall yield.
other side reactions. Acid-catalyzed deprotection of the ketal
followed by aldol condensation provided the A ring of ent-
cholestenone in 90% yield. The AB ring functionality was
modified by deprotonation using KOtBu followed by kinetic
protonation to provide the deconjugated ketone and diastereo-
selective reduction of the ketone with Li(OtBu)3AlH gave ent-
cholesterol in 80% yield over two steps.1,16
The synthesis described above used a D-C-B-A assembly
of the steroid molecule in a 17 steps linear sequence from
commercially available (S)-citronellol. The synthesis proceeded
with a 1.9% overall yield from (S)-citronellol. However,
conversion of the CD-enone to the thiophenol adduct was a
challenging transformation: the temperature control and short
handling times were difficult to achieve with larger-scale
reactions, and the yields were reduced upon scaling up due to
premature decarboxylation. Furthermore, the workup for acid
9 is difficult due to solubility problems. For these reasons, the
synthetic approach to ent-cholesterol was further refined to avoid
the issues inherent in the Stiles’ carboxylation route. We
explored two separate strategies to replace the Stile’s carboxy-
lation route.
In many cases biochemical studies can be facilitated by
isotopically labeling the unnatural compound to facilitate
subsequent analysis by MS or NMR spectroscopy. There are
several points where one could add an isotopic label in this
improved ent-cholesterol synthesis. The simplest labeling
strategy was to replace the C19 methyl group, which is derived
from methyl iodide, with an isotopic variant. This strategy was
easily achieved by replacing methyl iodide with CD3I in the
SCHEME 6. Improved Synthesis of ent-Cholesterol and
Synthesis of 19d3-ent-Cholesterol
The first alternative involved a selective conjugate reduction
of enone 1 to provide the trans-hydrindane system followed by
in situ trapping of the enolate with an electrophile (see
Supporting Information). The conjugate reduction step was
investigated, and although yields were generally high, the best
selectivity was only 4:1 in favor of the trans ring junction.
Hence, we abandoned further consideration of this approach.
In the second approach, we turned our attention to an addition-
elimination sequence first reported by Sauer for the synthesis
of D-norgestrel.17 The improved route to a CD enone precursor
15 is illustrated in Scheme 5. Treatment of the CD-enone 1
with formaldehyde and benzenesulfinic acid in a mixture of
N,N,N′,N′-tetramethylenediamine and acetic acid provided sul-
fone 14 in 66% yield. Hydrogenation using Pd/C in EtOH with
5% aqueous hydrochloric acid provided the saturated sulfone
in 2:1 (trans:cis) facial selectivity. Optimization of hydrogena-
tion conditions is shown in Supporting Information. More polar
solvents and the addition of acid improved the trans/cis ratio
(16) (a) Wheeler, O. H.; Mateos, J. L. Can. J. Chem. 1958, 36, 1431-
1435.(b) Ringold, H. J; Malhotra, S. K. Tetrahedron Lett. 1962, 669-672.
(c) Dauben, W. G.; Eastham, J. F. J. Am. Chem. Soc. 1951, 73, 4463-
4464.
(17) Sauer, G.; Eder, U.; Haffer, G.; G.; Neef, G.; Wiechert, R. Angew.
Chem., Int. Ed. 1975, 14, 413-414.
J. Org. Chem, Vol. 73, No. 7, 2008 2771

Belani and Rychnovsky
56.0, 45.2, 39.3, 37.2, 35.9, 34.5, 33.7, 29.1, 28.2, 27.0, 23.9, 22.9,
22.7, 18.9, 16.3 IR (film in CDCl3) 2929, 1742, 1463, 1383, 1158
cm-1; HRMS (ESI) m/z calcd for C18H30ONa (M + Na)+ 285.2194,
found 285.2191.
reductive alkylation of enone 12 (Scheme 6). Further elaboration
and modification of ring A provided deutero-ent-cholesterol in
good yields.
Sulfone (14). Enone 1 (1.6 g, 6.15 mmol, 1.0 equiv) was
dissolved in a premixed solution of HOAc (20 mL) and TMEDA
(60 mL), and sodium benzenesulfinic acid (recrystallized from
EtOH, 1.32 g, 8.39 mmol, 1.36 equiv) and paraformaldehyde (0.36
g, 12 mmol, 1.95 equiv) were added. The reaction mixture was
heated to 60 °C for 5 h under inert atmosphere. The reaction was
quenched with H2O (10 mL), and EtOAc (75 mL) was added. The
mixture was then washed with H2O (250 mL), aqueous HCl (10%,
50 mL) and brine (50 mL). The organic layer was dried,
concentrated and passed through a 1-in. silica plug to provide 2.7
g of crude oil. Flash chromatography with 10% EtOAc-hexanes
provided 1.7 g (66%) of desired sulfone 14 as a colorless oil. The
reaction can also be carried out at lower temperature (35 °C);
however reaction times are usually longer (∼36 h) and yields are
very similar. Sulfone 14: [R]24D -67.5, (c ) 2.3, CHCl3); 1H NMR
(500 MHz, CDCl3) δ 7.86 (2 H, d, J ) 7.2 Hz), 7.62 (1 H, t, J )
7.6 Hz), 7.52 (2 H, t, J ) 8 Hz), 4.26 (1 H, d, J ) 13.6 Hz), 4.02
(1 H, d, J ) 13.6 Hz), 2.82 (1 H, dd, J ) 10.1, 20.4 Hz), 2.66 (1
H, m), 2.35 (1 H, m), 2.20 (1 H, m), 2.05 (1 H, m), 1.77 (1 H, td,
J ) 5, 13.9 Hz), 1.40-1.65 (3 H, m), 1.25-1.50 (5 H, m), 1.10-
1.22 (4 H, m), 1.09 (3 H, s), 0.96 (3 H, d, J ) 6.6 Hz), 0.868 (3
H, d, J ) 6.6 Hz), 0.863 (3 H, d, J ) 6.6 Hz); 13C NMR (100
MHz, CDCl3) δ 195.3, 182.5, 139.1, 133.8, 129.0, 128.8, 120.3,
56.0, 53.1, 46.5, 39.6, 36.3, 35.9, 34.2, 32.9, 29.0, 28.2, 27.3, 23.9,
22.9, 22.85, 22.74, 19.0, 16.5, 14.3; IR (film in CDCl3) 2929, 1742,
1463, 1383, 1158 cm-1 HRMS (ESI) m/z calcd for C25H36O3SNa
(M + Na)+ 439.2282, found 439.2289.
trans-Hydrindane (15). Sulfone 14 (0.85 g, 2 mmol) was
dissolved in a mixture of MeOH (14 mL), acetic acid (4 mL), and
aqueous HCl (1 N, 0.2 mL). The 10% Pd/C catalyst (0.80 g) was
added, and the mixture was cooled and stirred at 0 °C under an
atmosphere of hydrogen for 12 h. The mixture was filtered through
Celite to give 0.85 g of crude mixture of two epimers at the ring
junction is a ratio of 8:1 in favor of trans. The ratio of the two
isomers can be easily obtained from GC analysis (column RTX-
1701), cis isomer (16.72 min) and trans isomer (17.44 min).
Recrystallization of the crude mixture provided 0.53 g (62%) of
the desired trans isomer, and this process provided a 1:1 mixture
of isomers in the mother liquor along with variable amounts of
ketone 16. The mother liquor was concentrated and purified by
chromatography to remove ketone 16. The partially purified mixture
was loaded as a solution in 50% IPA-hexanes on a preparative
HPLC system (column 300 × 50 mm, KromaSpher 80, 5 µM silica
column; solvent system 0.5% IPA-hexanes; retention times trans
37.64 min; cis 38.77 min; flow rate 40 mL/min at 41 bar) to provide
77 mg (9%) of desired trans isomer and 93 mg of cis epimer
Conclusion
The total synthesis of ent-cholesterol using C-H insertion
as the key step was achieved in 16 steps with an overall yield
of 2.0%. The route uses commercially available (S)-citronellol
and a diastereoselective C-H insertion reaction to provide the
D-ring synthon with the ent-cholesterol side chain already
attached. Two crystalline intermediates (the CH insertion product
2 and the hydrogenated CD-ring sulfone 15) facilitate purifica-
tion and separation of minor diastereomers. While the reductive
alkylation step must be run at moderate dilution (and thus on a
limited scale), this step occurs near the end of the synthesis
and does not present a practical limitation for material through-
put. Isotopic labels may be introduced late in the synthesis using
labeled methyl iodide, a practical and inexpensive source of
isotopic atoms. This synthetic pathway has a potential to supply
gram quantities of ent-cholesterol for biological studies.
Experimental Section
â-Ketoester (2). To a stirred solution of diazo-â-ketoester 3 (64
g, 0.22 mol) in dry CH2Cl2 (800 mL) cooled in an ice bath was
added Rh2 (R)-(PTV)4 (1.5 g, 0.45 mol%) at 10 °C. Immediate
evolution of N2 gas was observed. Temperature was not allowed
to rise above room temperature. Reaction mixture was stirred until
evolution of the gas ceased (∼2-3 h). Solvent was removed under
vacuum, and the crude material was loaded on a silica plug (∼500
mL) and eluted with 20% ether-hexanes mixture (6 × 1000 mL)
to afford 56.2 g of crude yellow oil that was seeded with 99% ee
crystals to afford 17 g (30%) of the desired diastereomerically pure
CH insertion product 2. Physical and spectroscopic data were
consistent with literature data:8 mp ) 54-55 °C; H NMR (500
1
MHz, CDCl3) δ 5.07 (1H, m), 3.74 (3H, s), 2.96 (1H, d, J ) 11.4
Hz), 2.56 (1H, m), 2.45-2.30 (2H, m), 2.17 (1H, m), 2.05 (1H,
m), 1.92 (1H, m), 1.69 (3H, s), 1.61 (3H, s), 1.59-1.40 (3H, m),
1.15 (1H, m), 0.87 (3H, d, J ) 7.3 Hz); 13C NMR (125 MHz,
CDCl3) δ 212.5, 170.9, 131.9, 124.4, 59.8, 52.6, 47.0, 38.8, 37.0,
34.0, 25.9, 25.5, 25.4, 17.8, 17.1; IR (film in CDCl3) 2956, 1755,
1726, 1435 cm-1 HRMS (ESI) m/z calcd for C15H24O3Na (M +
Na)+ 275.1623, found 275.1623.
Enantiopure CD-Enone (1). To a solution of NaOMe (freshly
prepared from 30 mL MeOH and 0.6 g of Na) was added a solution
of ketone 8 (3.6 g, 17.1 mmol) in MeOH (10 mL) at 0 °C. After
stirring for 5 min at 0 °C, methyl vinyl ketone (5 mL, 61.7 mmol)
was added, and the yellow mixture was stirred at 23 °C for 6 h.
The reaction was quenched with saturated aqueous NH4Cl (20 mL)
and was concentrated. The crude oil was extracted with EtOAc
(3 × 25 mL), concentrated, and then passed through a silica plug
using a mixture of EtOAc, hexanes, and MeOH (10:89:1) to afford
a yellow oil (3.7 g) that was dissolved in toluene (100 mL).
p-Toluenesulfonic acid (250 mg, 1.4 mmol) was added, and the
mixture was heated at reflux temperature for 3 h in a flask attached
to Dean-Stark trap and condenser. The reaction mixture was
washed with saturated NaHCO3, concentrated, and chromatographed
with 10% EtOAc-hexanes to provide 2.45 g (55% over two steps)
of the CD enone 1 as a yellow oil. Physical and spectroscopic data
(10.9%). The combined yield for the trans hydrindane system was
1
71%. trans Isomer: ([R]24 -15.6, c ) 1.45, CHCl3); H NMR
D
(500 MHz, CDCl3) δ 7.90 (2 H, m), 7.62 (1 H, tt, J ) 7.2, 2 Hz),
7.55 (2 H, m), 4.0 (1 H, dd, J ) 6.5, 14.5 Hz), 3.03 (1 H, ddd, J
) 2, 6.5, 11 Hz), 2.87 (1 H, dd, J ) 2.5, 14 Hz), 2.50 (1 H, td, J
) 6.5, 14.5 Hz), 2.30 (1 H, ddd, J ) 14.9, 4.9, 2.0 Hz), 2.17 (1 H,
ddd, J ) 1, 4.5, 11 Hz), 1.94 (1 H, m), 1.65 (2 H, m), 1.30-1.55
(7 H, m), 1.20-1.10 (4 H, m), 1.04 (3 H, s), 0.95 (1 H, m), 0.90
(3 H, J ) 7 Hz), 0.865 (3 H, d, J ) 6.6 Hz), 0.860 (3 H, d, J ) 6.6
Hz); 13C NMR (100 MHz, CDCl3) δ 208.0, 140.1, 133.8, 129.3
(2C), 128.2 (2C), 55.7, 55.1, 53.0, 45.9, 43.5, 39.6, 38.5, 37.5, 35.9,
35.7, 28.8, 25.0, 23.9, 23.0 (2C), 22.7, 18.7, 11.5; IR (film in CDCl3)
2929, 1742, 1463, 1383, 1158 cm-1; HRMS (ESI) m/z calcd for
were consistent with literature data:3 [R]24 -64.3, (c ) 1.0,
D
1
CHCl3); H NMR (500 MHz, CDCl3) δ 5.72 (1 H, s), 2.61 (1 H,
C25H38O3SNa (M + Na)+ 441.2439, found 441.2430. cis Isomer:
1
dd, J ) 10.9, 19.7 Hz), 2.51 (1 H, m), 2.40 (1 H, m), 2.34-2.20
(2 H, m), 2.0 (1 H, m), 1.80 (1 H, td, J ) 4.9, 13.8, 18.6 Hz), 1.53
(3 H, m), 1.30-1.45 (3 H, m), 1.10-1.25 (4 H, m), 1.08 (3 H, s),
0.97 (3 H, d, J ) 6.6 Hz), 0.862 (3 H, d, J ) 6.6 Hz), 0.858 (3 H,
d, J ) 6.6 Hz); 13C NMR (100 MHz, CDCl3) δ 199.5, 180.3, 121.6,
([R]24 +82.6, c ) 0.52, CHCl3); H NMR (500 MHz, CDCl3) δ
D
7.90 (2 H, m), 7.62 (1 H, tt, J ) 7.5, 2 Hz), 7.55 (2 H, m), 4.0 (1
H, dd, J ) 7.4, 14 Hz), 2.98 (1 H, ddd, J ) 1.5, 7.5, 10 Hz), 2.32
(2 H, m), 2.05 (1 H, m), 2.0-1.87 (3 H, m), 1.53-1.26 (9 H, m),
1.17-1.038 (4 H, m), 0.93 (1 H, m), 0.88 (3 H, J ) 6.5 Hz), 0.87
2772 J. Org. Chem., Vol. 73, No. 7, 2008

Synthesis of ent-cholesterol
(3 H, d, J ) 6.6 Hz), 0.865 (3 H, d, J ) 6.6 Hz), 0.841 (3 H, s);
13C NMR (100 MHz, CDCl3) δ 210.0, 139.9, 133.8, 129.3 (2C),
128.2 (2C), 55.4, 54.3, 53.4, 47.6, 43.2, 39.6, 36.3, 35.7, 34.8, 33.6,
30.8, 29.5, 28.2, 24.1, 23.0, 22.7, 22.6, 19.6; IR (film in CDCl3)
2929, 1742, 1463, 1383, 1158 cm-1; HRMS (ESI) m/z calcd for
C25H38O3SNa (M + Na)+ 441.2439, found 441.2441.
refluxed for 12 h. Solid NaHCO3 (1 g) was added carefully, and
the mixture was concentrated. Water (20 mL) was added, the
mixture was extracted with EtOAc (3 × 25 mL), and the organic
layers were combined, dried, and concentrated. Chromatography
of the crude solid using 5% EtOAc-hexanes gave 234 mg (90%)
of desired enone. Physical and spectroscopic data were consistent
1
with literature data:3 ([R]24 -88.3, c ) 0.5, CHCl3); δ H NMR
Tricyclic Enone (12). To a solution of freshly prepared NaOMe
(0.133 g, 5.52 mmol, 2.76 equiv) was added â-keto ester 1113c (0.61
g, 2.5 mmol, 1.25 equiv) as a solution in MeOH (5 mL) at 10 °C,
and the mixture was stirred for 10 min. A solution of sulfone 15
(0.84 g, 2 mmol, 1 equiv) in dry benzene (10 mL) was added, and
the reaction mixture was stirred at room temperature for 12 h. A
solution of NaOH (4 g) in H2O (16 mL) was added to the reaction
mixture. Sufficient MeOH (∼25 mL) was added to cause formation
of one layer. The mixture was stirred for 3-4 h at 23 °C and then
concentrated. Toluene (50 mL) and H2O (50 mL) were added, and
the mixture was cooled to 0 °C. An aqueous solution of HOAc
(50%) was added dropwise till the pH was ∼4. Mixture was
extracted with toluene (2 × 25 mL), the organic layers were mixed,
dried (Na2SO4), and concentrated to provide a yellow oil. The oil
was heated at 80 °C for 3 h under vacuum (0.2 Torr). It was
necessary to stir the oil for efficient decarboxylation. The crude
material was then chromatographed on silica gel with 10% EtOAc-
hexanes to provide 0.61 g of desired tricyclic enone 12. Physical
D
(500 MHz, CDCl3) δ 5.71 (1 H, s), 2.40-2.25 (4 H, m), 2.05-
1.95 (2 H, m), 1.85-1.75 (2 H, m), 1.72 (1 H, td, J ) 4.7, 13.9
Hz), 1.65-1.20 (10 H, m), 1.16 (3 H, s), 1.05-1.15 (6 H, m), 0.94-
1.04 (3 H, m), 0.88 (3 H, d, J ) 6.5 Hz), 0.867 (3 H, d, J ) 6.6
Hz), 0.854 (3 H, d, J ) 6.6 Hz), 0.69 (3 H, s); 13C NMR (125
MHz, CDCl3) δ199.7, 171.7, 123.7, 56.1, 55.9, 53.8, 42.4, 39.6,
39.5, 38.6, 36.1, 35.79, 35.72, 35.6, 34.0, 32.9, 32.0, 28.2, 28.0,
24.2, 23.8, 22.8, 22.6, 21.0, 18.6, 17.4, 11.9; IR (film in CDCl3)
2934, 1676, 1618, 1467 cm-1; HRMS (ESI) m/z calcd for C27H44-
ONa (M + Na)+ 407.3289, found 407.3274.
ent-Cholesterol. To a solution of ent-cholest-4-en-3-one (200
t
mg, 0.52 mmol) in BuOH (10 mL) under inert atmosphere was
added KOtBu (0.35 g, 3.1 mmol, 6 equiv), and the mixture was
stirred for 4 h at 23 °C. An aqueous solution of 50% HOAc (5
mL) was added in one portion. The mixture was extracted with
EtOAc (3 × 20 mL). The organic layer was dried, (Na2SO4),
concentrated, and then azeotroped with benzene (2 × 20 mL) to
give a crude solid that was taken to the next step. To a solution of
the crude solid in THF (10 mL) was added solid Li(OtBu)3Al-H
(0.8 g, 3.1 mmol, 6 equiv). The reaction was stirred for 2 h at 23
°C. TLC analysis showed complete disappearance of the starting
material. The reaction was quenched with 5% HCl (20 mL) and
extracted with ethyl acetate (3 × 25 mL) to provide 159 mg (80%)
yield of the desired ent-cholesterol. A small sample was recrystal-
lized from methanol for analysis. Physical and spectroscopic data
and spectroscopic data were consistent with literature data:3 ([R]24
D
+18.7, c ) 3.6, CHCl3); 1H NMR (500 MHz, CDCl3) δ 3.93 (4H,
m), 2.76 (1 H, m), 2.46 (2 H, m), 2.36 (1 H, td, J ) 5.2, 11.9 Hz),
2.25 (3 H, m), 2.12 (1 H, m), 1.98 (1 H, m), 1.90 (1 H, m), 1.70-
1.45 (5 H, m), 1.42 (2 H, m), 1.36 (3 H, s), 1.34-1.27 (3 H, m),
1.20-1.08 (6 H, m), 1.00 (1 H, m), 0.93 (3 H, d, J ) 6.5 Hz),
0.870 (3 H, d, J ) 6.6 Hz), 0.865 (3 H, d, J ) 6.6 Hz), 0.82 (3 H,
s); 13C NMR (125 MHz, CDCl3) δ198.8, 159.9, 133.6, 110.0, 64.8
(2C), 56.5, 42.1, 39.6, 39.4, 39.1, 38.3, 37.3, 36.2, 35.8, 28.4, 28.1,
27.3, 27.1, 24.6, 23.9, 23.7, 22.9 (2C), 22.7, 20.1, 18.7, 11.4; IR
(film in CDCl3) 2952, 1667, 1466, 1373, 1060 cm-1; HRMS (ESI)
m/z calcd for C28H46O3Na (M + Na)+ 453.3347, found 453.3345.
Tricyclic Ketone (13). To 65 mg of lithium wire (9.3 mmol, 20
equiv) in 100 mL of anhydrous NH3 (distilled from Na/FeNO3,
100 mg) at -78 °C was added 200 mg of enone 12 (0.46 mmol,
1.0 equiv) in 40 mL of THF. The reaction mixture was stirred for
1 h and MeI (1.62 mL, 27.9 mmol, 60 equiv) was added as a
solution in THF (20 mL) over 30 min. The reaction mixture was
stirred for 2-3 h at -33 °C and then the reaction was quenched
with excess solid NH4Cl. After allowing the NH3 to evaporate, water
(25 mL) was added. The layers were separated and aqueous layer
was extracted with ether (3 × 50). The combined organic layers
were dried (Na2SO4), filtered, and concentrated under reduced
pressure. The crude oil was purified by flash chromatography (ethyl
acetate-hexanes 5:95) to give 165 mg (80%, 0.37 mmol) of
were consistent with literature data:1,3 mp ) 145-147 °C; [R]23
D
+38.8 (c ) 0.5, CHCl3); 1H NMR (500 MHz, CDCl3) δ 5.34 (1 H,
m), 3.51 (1 H, t, J ) 6.1 Hz), 2.27 (2 H, m), 2.01 (2H, M), 1.82
(3 H, m), 1.65-1.45 (8 H, m), 1.35-1.32 (2 H, m), 1.28-1.22 (4
H, m), 1.17-1.05 (8 H, m), 1.03 (3 H, s), 0.96 (3 H, d, J ) 6.5
Hz), 0.87 (3 H, d, J ) 6.6 Hz), 0.85 (3 H, d, J ) 6.6 Hz), 0.67 (3
H, s); 13C NMR (125 MHz, CDCl3) δ 140.9, 121.39, 72.0, 56.9,
50.3, 42.5, 40.0, 39.7, 37.4, 36.7, 36.7, 36.4, 36.0, 32.1, 31.8, 29.9,
28.4, 28.2 (2C), 24.51, 24.0, 23.0, 22.7, 21.3, 19.6, 18.9, 12.0; IR
(film in CDCl3) 3347, 2969, 2926, 1429, 754 cm-1
.
Acknowledgment. This work was supported by the National
Cancer Institute (GM-43854) and a generous gift from the
Schering-Plough Research Institute. We would like to thank Dr.
Joseph Ziller (UCI) for the X-ray determination of sulfone 15
and Matthew Morin for technical assistance. The authors would
like to acknowledge Professor Douglass F. Taber (U. Delaware)
for encouraging us to explore this route and for helpful advice
along the way.
colorless oil. Physical and spectroscopic data were consistent with
1
literature data:3 ([R]24 -32.3, c ) 1.3, CHCl3); H NMR (500
D
MHz, CDCl3) δ 3.93 (4 H, m), 2.51 (1 H, td, J ) 6.5, 14.5 Hz),
2.24 (1 H, m), 2.02 (1 H, m), 1.95-1.80 (4 H, m), 1.76-1.38 (8
H, m), 1.35 (3 H, s), 1.32-1.21 (6 H, m), 1.20-1.10 (6 H, m),
1.08 (3 H, s), 1.05-0.95 (1 H, m), 0.91 (3 H, d, J ) 6.5 Hz),
0.867 (3 H, d, J ) 6.6 Hz), 0.854 (3 H, d, J ) 6.6 Hz), 0.71 (3 H,
s); 13C NMR (125 MHz, CDCl3) δ 215.5, 110.5, 64.7, 64.6, 56.2,
56.0, 50.6, 47.5, 42.7, 39.6 (2C), 38.4, 36.3, 35.9, 35.0, 33.2, 31.4,
29.2, 28.29, 28.20, 24.4, 23.6, 23.0 (2C), 22.75, 21.4, 21.2, 18.8,
Supporting Information Available: Experimental procedures
for the preparation of compounds 3, 4, 6, 7, 8, 10, 12, 17, and
19d3-ent-cholesterol are included. X-ray data for sulfone 15 in CIF
format and NMR spectra of the new compounds are provided,
including detailed comparisons between the synthetic and isolated
natural products described herein. This material is available free
of charge via the Internet at http://pubs.acs.org.
12.1; IR (film in CDCl3) 2936, 1707, 1466, 1366 cm-1
.
ent-Cholest-4-ene-3-one. A solution of tricyclic ketone 13 (300
mg, 0.67 mmol) in MeOH (20 mL) and 6 N HCl (3 mL) was
JO702694G
J. Org. Chem, Vol. 73, No. 7, 2008 2773