A Scalable stereoselective synthesis of scymnol

Article abstract of DOI:10.1071/CH04175

A high-yielding, stereoselective synthesis of scymnol 1 has been carried out in five steps starting from commercially available cholic acid 2. The synthesis was designed with the aim of eventual large-scale processing. Triformyloxycholic acid chloride 4 was treated with the magnesium enolate of diethyl malonate to afford the β-keto diester, diethyl 3α,7α, 12α-triformyloxy-24-oxo-5β-cholestane-26,27-dioate 5. The key step in the synthesis was the stereoselective hydroganation of β-keto diester 5 to give the corresponding β-hydroxy diester 6 using a BINAP nithenium(n) catalyst. Subsequent reduction of the diester moiety and deprotection of the hydroxyl groups afforded scymnol 1.

Full text of DOI:10.1071/CH04175

Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2005, 58, 34–38
www.publish.csiro.au/journals/ajc
A Scalable Stereoselective Synthesis of Scymnol
Raju Adhikari,A Darren J. Cundy,A Craig L. Francis,A,B Mariana Gebara-Coghlan,A
Beata Krywult,A Carolyn Lubin,A Gregory W. Simpson,A and QiYangA
A CSIRO Molecular Science, Bag 10, Clayton South VIC 3169, Australia.
B Corresponding author. Email: craig.francis@csiro.au
A high-yielding, stereoselective synthesis of scymnol 1 has been carried out in five steps starting from commercially
available cholic acid 2. The synthesis was designed with the aim of eventual large-scale processing. Triformyloxy-
cholic acid chloride 4 was treated with the magnesium enolate of diethyl malonate to afford the β-keto diester, diethyl
3α,7α,12α-triformyloxy-24-oxo-5β-cholestane-26,27-dioate 5. The key step in the synthesis was the stereoselective
hydrogenation of β-keto diester 5 to give the corresponding β-hydroxy diester 6 using a BINAP ruthenium(ii)
catalyst. Subsequent reduction of the diester moiety and deprotection of the hydroxyl groups afforded scymnol 1.
Manuscript received: 16 July 2004.
Final version: 5 October 2004.
Introduction
aim was large-scale processing. This paper describes such a
synthesis.
Sodium scymnol sulfate was originally isolated by Ham-
marsten from the gall bladder of the shark species Scym-
nus borealis.[1] The structure of the parent compound,
scymnol [(24R)-5β-cholestane-3α-7α-12α-24,26,27-hexol]
1 (Figure 1), w hich was obtained by basic hydrolysis of nat-
ural sodium scymnol sulfate, was determined by Bridgwater
et al.[2] Scymnol 1 has since found application in the treat-
ment of various skin conditions[3] and liver dysfunction.[4,5]
Discovery of the pharmacological activity of scymnol 1
prompted interest in the development of a laboratory synthe-
sis with a view to make larger quantities for further studies
and to ultimately satisfy the commercial market, as the yield
from natural sources is very poor. The isolation and partial
synthesis of scymnol sulfate and scymnol 1 has been inves-
tigated by various groups.[2,4,6,7] More recently, Macrides
et al.[8] published details of a synthesis of 1 starting from
commerciallyavailablecholicacid 2.Thisprocedureafforded
a mixture of scymnol 1 and its C24 epimer.
Results and Discussion
Our synthetic pathway is outlined in Scheme 1. 3α,7α,12α-
Triformyloxycholic acid 3 was prepared in almost quanti-
tative yield by treatment of cholic acid 2 with a catalytic
amountofperchloricacidinformicacidandaceticanhydride.
We chose to synthesize formate esters rather than acetates[8]
in order to simplify processing, as chromatography would
thereby not be required.[8]
The triformyloxy β-keto diester 5 was prepared as illus-
trated in Scheme 1 by condensation of triformyloxycholic
acid chloride 4 (prepared in situ from 3) with the magnesium
enolate of diethyl malonate in accord with modified literature
procedures.[8,9] The use of magnesium chloride and triethy-
lamine as reagents and ethyl acetate as solvent in this step
was more amenable to scale-up than the use of metallic mag-
nesium, ethanol, tetrahydrofuran, and carbon tetrachloride,
which had been described for a similar reaction.[8]
Ouraimwastodevelopastereoselectiveandhigh-yielding
synthesis of scymnol from cholic acid. We decided to use
inexpensive and easily manageable reagents as our long-term
With β-keto diester 5 in hand, we required a stereo-
selective reduction of the C24 carbonyl group to afford the
corresponding (24R)-alcohol.
OH
Since the advent of bis(diarylphosphino)binapthyl
(BINAP) ruthenium(ii)-based catalysts, the asymmetric
hydrogenation of β-keto esters has received considerable
attention.[10–12] However, we were unaware of any report
that described a stereoselective hydrogenation of a β-keto
diester to afford the corresponding β-hydroxy diester. We
developed a procedure for the asymmetric hydrogenation of
β-keto diester 5 that employed [RuCl2(R-BINAP)]2·NEt3,
RuCl2(R-BINAP), or RuBr2(R-BINAP)[10] in either a
21
22
20
26
25
CO2H
HO
OH
24
18
14
OH
23
11
12
27
17
19
OH
13
16
1
8
9
2
15
10
5
3
7
OH
HO
OH
HO
6
4
H
H
1
2
Fig. 1.
© CSIRO 2005
10.1071/CH04175
0004-9425/05/010034

Stereoselective Synthesis of Scymnol
35
O
O
O
O
O
COX
H
H
O
H
H
H
O
H
OEt
OEt
(b)
O
O
5
H
O
O
H
O
O
O
O
3 X ϭ OH
4 X ϭ Cl
(a)
(c)
O
O
O
O
O
O
OH
O
H
O
H
H
H
O
OEt
OEt
OEt
OEt
O
(d)
7
6
O
O
O
HO
O
H
O
H
O
(e)
1
Scheme 1.
(a) (COCl)2 or SOCl2; (b) CH2(CO2Et)2, Et3N, MgCl2; (c) H2, RuBr2(R-BINAP); (d) DHP, PPTS;
(e) LiAlH4 then HCl/MeOH.
O
OH
O
O
stainless steel bomb or a Parr hydrogenator. We preferred
using RuBr2(R-BINAP) (formed in situ from benzene ruthe-
nium(ii) bromide dimer[13] and R-BINAP) in a glass Parr
hydrogenation bottle.
To demonstrate the procedure in a vessel type more
amenable to large scale processing (the Parr apparatus has
obvious limitations of scale), the hydrogenation was carried
out in a stainless steel bomb. Some trials were successful,
but the procedure was not as reliable as when conducted in
the Parr apparatus. Although not explored fully, the effect of
less efficient agitation and/or catalyst poisoning by the metal
surface was suspected to be influential.
The desired (24R)-isomer 6 (concomitant cleavage of the
3-O-formyl group occurred during the reaction) was obtained
in high yield with none of the S isomer being detected by
NMR spectroscopy. For reference purposes, small samples
of β-keto diester 5 were: (a) hydrogenated using S-BINAP to
give (24S)-β-hydroxy diester 8 (Scheme 2); and (b) reduced
with sodium borohydride[8] to provide both C24 hydroxy
epimers. The C24 13C NMR resonances for epimers 6 and
8 were found to be distinct. Furthermore, in the asymmet-
ric reductions, only one C24 resonance was observed in
each case.
To minimize the use of protective groups, we briefly
examined the direct reduction of the diester residue in the
β-hydroxydiester 6 to afford the desired triol moiety. Unfor-
tunately, at the temperatures required to effect reduction, each
of the reducing agents employed (LiAlH4, LiBH4, NaBH4,
NaBH(OMe)3) cleaved the C24–C25 bond and displaced
the diethyl malonate residue presumably by a reverse aldol-
type reaction. These observations were consistent with those
H
H
O
H
OEt
OEt
H2
5
RuBr2(S-BINAP)
8
HO
O
O
Scheme 2.
reported by others[8,14] for similar systems and indicates the
necessity to protect the β-hydroxyl group.
The β-hydroxy diester 6, which was obtained from the
hydrogenation reaction, was converted into the C24,C3-
bis(tetrahydropyranyl) (THP) ether 7 by treatment with
3,4-dihydro-2H-pyran (DHP) in the presence of pyridinium
p-toluenesulfonate (PPTS).
Reduction of the diester moiety in protected 24-O-THP
diethyl ester 7 was achieved by treatment with lithium alu-
minium hydride (LAH) in tetrahydrofuran. Deprotection to
deliver scymnol 1 in an isolated yield of 57% (from 7) was
subsequently effected in the same vessel by using a catalytic
amount of hydrochloric acid in methanol. To verify the iden-
tity of the final product, we compared the NMR spectra of
our synthetic samples of scymnol with the spectra of scym-
nol obtained from hydrolysis of the natural product sodium
scymnol sulfate.The NMR spectra were found to be identical.
We also investigated the purification of crude scymnol
by Soxhlet extraction in an effort to avoid chromatogra-
phy. A methanolic solution of the crude material was mixed

36
R. Adhikari et al.
with silica gel (flash-chromatography type, sample/silica
gel ≈ 1 : 10) and the solvent was subsequently removed under
vacuum. The free-flowing residue was then placed in a Soxh-
let thimble and extracted with diethyl ether over several days.
The more soluble, higher RF impurities were extracted by the
ether and gradually concentrated in the solvent reservoir,
the concentration of which was monitored by HPLC. Once
the concentration of the higher RF materials had stabilized
(usually this coincided with a small amount of scymnol 1
having leached into the reservoir), the solvent in the reservoir
was removed and replaced with ethyl acetate. The extraction
process was then continued for 1–2 days until the relative
concentration of scymnol 1 (as determined by HPLC) was
optimal. The ethyl acetate solution was subsequently cooled
and scymnol 1 was slowly allowed to crystallize (sometimes
the ethyl acetate solution required some concentration before
crystallization occurred). Samples of scymnol 1 crystallized
by this method were typically of >95% purity, and a sec-
ond slow recrystallization from aqueous methanol delivered
material with a purity of about 99%. Although the yield
of material obtained by Soxhlet extraction was higher than
that obtained by chromatography, in order to obtain analyti-
cally pure samples, column chromatography was still usually
required.
quadrupole using electrospray ionization (ESI) in the positive (+) ion
or negative (−) ion mode with 30 eV cone voltage and either 1 : 1 aceto-
nitrile/water or 2 : 1 : 1 methanol/acetonitrile/water as solvent. Samples
wereusually introducedas solutions in methanol/dichloromethane. Fast-
atom bombardment (FAB) mass spectra were recorded using either a
glycerol or poly(ethyleneglycol) matrix in the negative-ion or positive-
ion mode on a Jeol DX-303 spectrometer. Only the major fragments
are given with their relative abundances shown in parentheses. Micro-
analyses were performed at the Campbell Microanalytical Laboratory,
University of Otago, New Zealand.
3α,7α,12α-Tri-O-formylcholic Acid 3
Perchloric acid (1.0 mL) was added to a stirred solution of cholic acid
(100.0 g, 0.247 mol) in formic acid (400 mL, 1.72 mol) at room tem-
perature (20◦C, mild exotherm). The temperature of the mixture was
increased to 45–50◦C for 2 h and was then adjusted to 40◦C. Acetic
anhydride (350 mL) was subsequently added dropwise over 1.5 h and
the mixture was stirred for an additional 30 min. The solution was
allowed to cool to room temperature before being poured into water
(4 L) and extracted with dichloromethane (2 L). The organic layer was
washed with water to neutrality, dried, and evaporated under reduced
pressure to yield the title compound (118.0 g, 98%) as a foamy solid
that was used without further purification. An analytical sample was
recrystallized from EtOAc/light petroleum, mp 200–202◦C, [α]D 91.80
(c 0.5 in EtOH) (Found: C 65.6, H 8.2. C27H40O8 requires C 65.8, H
8.2%). δH 8.15 (1H, s, OC(O)H12), 8.11 (1H, s, OC(O)H7), 8.02 (1H,
s, OC(O)H3), 5.23 (1H, br s, H12), 5.05 (1H, br s, H7), 4.79–4.57 (1H,
m, H3), 2.45–0.59 (complex m), 0.93 (3H, s, CH3), 0.85 (3H, d, J 6.0,
C21H3), 0.77 (3H, s, CH3). δC 180.0 (C24), 160.6 (formate C=O), 75.2
(C12), 73.7 (C3), 70.7 (C7), 47.2 (C17), 45.0 (C13), 42.9 (C14), 40.7
(C5), 37.7 (C8), 34.7 (C20), 34.5 (C1 and C4), 34.2 (C10), 31.3 (C6),
30.9 (C23), 30.4 (C22), 28.5 (C9), 27.1 (C16), 26.5 (C11), 25.5 (C2),
22.7 (C15), 22.3 (C19), 17.4 (C21), 12.1 (C18). m/z (ESI−) 491 (100%,
[M – H]•−).
The overall yield of scymnol 1 from cholic acid 2 over five
steps was 38%, with the synthesis being well suited for scale-
up. However, future improvements such as decreasing the
catalyst-to-substrate ratio in the stereoselective hydrogena-
tion(BINAPRu(ii)catalystsareexpensive), findingasuitable
large-scale reactor for this step, and improving efficiency in
the final purification process are still required.
Diethyl 3α,7α,12α-Triformyloxy-24-oxo-5β-cholestane-
26,27-dioate 5
Reactant A: The Magnesium Enolate of Diethyl Malonate
Experimental
Triethylamine (10.0 mL, 71.7 mmol) was added to a stirred solution
of diethyl malonate (3.25 g, 20.3 mmol) in ethyl acetate (100 mL) at
0◦C under an atmosphere of nitrogen and then anhydrous magnesium
chloride (3.13 g, 32.8 mmol) was added portionwise over 30 min. The
resultant mixture was warmed to 40◦C and stirred for 14 h.
General
Melting points were determined on a Gallenkamp MPD350.BM2.5
apparatus and are uncorrected. Light petroleum refers to the frac-
tion that boils between 40 and 60◦C. All organic extracts were dried
over anhydrous magnesium sulfate unless otherwise stated. Analytical
thin-layer chromatography (TLC) was carried out on Macherey-Nagel
Polygram pre-coated plastic sheets (0.2 mm layer of silica gel with
fluorescent indicator UV254). Developed plates were visualized upon
heating with a solution of 10% (w/v) phosphomolybdic acid in ethanol
or 6% (w/v) p-anisaldehyde in ethanol that contained 2% H2SO4.
Flash chromatography[15] was performed using Merck Silica gel 60,
particle size 0.040–0.063 mm (230–400 mesh ASTM). Analytical high-
performance liquid chromatography (HPLC) was performed with a
Waters Maxima 820 pump control system, a Waters 410 RI detector at
254 nm, andaMerckLichrospher100RP-18250 × 4.5 mmC18 column.
For β-keto diester 5 and β-hydroxy diester 6, an isocratic mobile phase
Reactant B: Tri-O-formylcholic Acid Chloride 4
Oxalyl chloride (2.13 mL, 24.4 mmol) was added dropwise to a
stirred solution of tri-O-formylcholic acid 3 (10.0 g, 20.3 mmol) in dry
dichloromethane (50 mL) at room temperature under an atmosphere of
nitrogen, and after the bubbling had ceased, N,N-dimethylformamide
(0.25 mL) was added dropwise, which caused some further efferves-
cence. The resultant reaction mixture was stirred at room temperature
for 3 h and the solvent was removed under vacuum to yield 4 as a pale
beige foam.
A solution of the acid chloride 4 (reactant B) in dry ethyl acetate
(100 mL) was added dropwise over 30 min to reactant A at 0◦C under
an atmosphere of nitrogen, and after addition was complete, the mix-
ture was stirred at room temperature for 15 h. Hydrochloric acid (13%,
54 mL) was added dropwise, while the temperature of the reaction mix-
ture was maintained below 25◦C. After being stirred for 15 min, the
aqueous layer was extracted with ethyl acetate (3 × 100 mL). The com-
bined organic layers were washed with 13% hydrochloric acid followed
by water to neutrality. The combined extracts were dried and evaporated
under vacuum to afford the title compound (12.30 g, 95%) as a light
brown, foamy solid, which was used without further purification. An
analytical sample was recrystallized from diethyl ether/light petroleum
to give colourless crystals, mp 96–98◦C, [α]D 67.00 (c 0.5 in EtOH)
(Found: C 64.3, H 7.7. C34H50O11 requires C 64.3, H 7.9%). NMR
analysis showed the product to be a mixture of keto (k) and enol (e)
of 75% methanol/25% water was used at a flow rate of 1 mL min−1
,
for diTHP ether 7 an isocratic mobile phase of 75% methanol/10%
acetonitrile/15% water was used at a flow rate of 1 mL min−1, while
for scymnol 1, an isocratic mobile phase of 5 mM ammonium formate
in 70% methanol/30% water was used at a flow rate of 0.9 mL min−1
.
Data were processed using a Waters Maxima 820 integrator system.
Proton nuclear magnetic resonance (1H NMR) and carbon nuclear mag-
netic resonance (13C NMR) spectra were recorded at room temperature
on a Bruker AC-200 or Varian Gemini-200 spectrometer operating at
200 and 50.3 MHz, respectively, using CDCl3 as solvent and an inter-
nal reference unless otherwise specified. Solute concentrations in the
5 mm tubes were approximately 10 mg mL−1. Unless otherwise indi-
cated, mass spectra were recorded on a Fisons Instruments VG Platform

Stereoselective Synthesis of Scymnol
37
tautomers. δH 12.59 (1H, enol H), 8.15 (1H, s, OC(O)H12), 8.08 (1H, s,
OC(O)H7), 8.00 (1H, s, OC(O)H3), 5.17 (1H, br s, H12), 5.01 (1H, br
s, H7), 4.75–4.54 (1H, m, H3), 4.39 (1H, s, H25), 4.28–4.08 (4H, com-
plex m, OCH2), 2.60–0.60 (complex m), 0.91 (3H, s, CH3), 0.80 (3H, m,
C(21)H3), 0.72 (3H, s, CH3). δC 199.1 [C24(k)], 183.2 [C24(e)], 171.1
and 166.1 (C26 and C27), 160.5 (formate C=O), 99.6 [C25(e)], 75.2
(C12), 73.7 (C3), 70.6 (C7), 65.3 [C25(k)], 62.3 [CH2(k)], 61.3 and 61.0
[CH2(e)], 47.3 (C17), 45.0 (C13), 42.9 (C14), 40.8 (C5), 38.8 [C23(k)],
37.7 (C8), 35.1 (C20), 34.5 (C1 and C4), 34.2 (C10), 32.7 [C23(e)], 31.3
(C6), 30.6 [C22(e)], 29.0 [C22(k)], 28.5 (C9), 27.1 (C16), 26.5 (C11),
25.5 (C2), 22.7 (C15), 22.3 (C19), 17.5 (C21), 14.1 and 14.0 (CH3),
12.1 (C18). m/z (ESI−) 633 (100%, [M – H]•−).
Diethyl 3α,24R-Bis(tetrahydropyran-2-yloxy)-7α,12α-diformyloxy-
5β-cholestane-26,27-dioate 7
Freshly distilled DHP (6.91 g, 7.49 mL, 82.25 mmol) was added drop-
wise to a stirred solution of the (24R)-dihydroxy diester 6 (10.00 g,
16.45 mmol)indrydichloromethane(80 mL)atroomtemperatureunder
an atmosphere of nitrogen. PPTS (0.41 g, 1.65 mmol) was added and the
resultant mixture was left to stir at room temperature for 16 h. The mix-
ture was diluted with diethyl ether (180 mL) and sequentially washed
with water (150 mL) and brine (100 mL). The organic phase was dried,
and the solvent was removed under reduced pressure.The residual brown
syrup was triturated three times with cold light petroleum, the solvent
with the unwanted by-products being decanted each time. The residue
was then placed under high vacuum to afford the title compound (9.25 g,
73%) as an amorphous foam, which was used without further purifica-
tion. δH 8.09 (1H, s, OC(O)H12), 8.07 (1H, s, OC(O)H7), 5.22 (1H, br
s, H12), 5.02 (1H, br s, H7), 4.65 (2H, br s, 2 × OCHO), 4.17 (4H, m,
2 × OCH2CH3), 4.10 (1H, m, H3), 3.95–3.30 (5H, m, THP ring OCH2
and H24), 2.30–0.90 (m, ring CH), 1.23 (6H, m, 2 × OCH2CH3), 0.88
(3H, s, CH3), 0.78 (3H, d, J 5.5, C21H3), 0.70 (3H, s, CH3). δC 168.8,
168.1, 167.7, and 167.4 (ester C=O), 160.8 and 160.6 (formyl C=O),
99.8, 97.5, 97.0, and 96.7 (OCHO), 75.9 and 75.6 (C3), 75.3 (C12),
70.8 and 70.7 (C24), 70.5 (C7), 62.9 (OCH2CH3), 61.6 and 61.3 (THP
OCH2), 56.8 and 56.4 (C25), 47.2 (C17), 44.9 (C13), 42.9 (C14), 41.3
(C5), 37.7 (C8), 36.5 (C4), 35.1 (C1), 34.9 (C20), 34.5 (C10), 31.6,
31.4, 31.1, 30.9, 30.7, and 30.1 (C2, C6, C11, C22, C23, and THP
CH2), 28.5 (C9), 27.2 (C16), 25.4 (THP CH2), 22.8 (C15), 22.4 (C19),
20.0 (THP CH2), 17.7 (C21), 14.0 (OCH2CH3), 12.1 (C18). m/z (ESI+)
797 (100%, [M + Na]•+).
On larger scales (up to 100 g), thionyl chloride was success-
fully used instead of oxalyl chloride, but the yield and purity were
lower.
Diethyl 7α,12α-Diformyloxy-3α,24R-dihydroxy-5β-cholestane-
26,27-dioate 6
(R)-(–)-2,2ꢀ-Bis(diphenylphosphino)-1,1ꢀ-binaphthyl(R-BINAP;75 mg,
0.12 mmol) was added in a single portion to a mixture of the tris-
formyl β-keto diester 5 (20.05 g, 31.6 mmol), benzene ruthenium(ii)
bromide dimer[13] (35.6 mg), and dry ethanol (200 mL) in a glass
Parr hydrogenation bottle. The mixture was deoxygenated by five
evacuation/re-pressurization cycles with hydrogen gas, left under 32 psi
of hydrogen, warmed to 40◦C, and shaken for 18 h. The mixture was
then filtered through glass filter paper and the filtrate was evaporated
under reduced pressure to afford the title compound (18.85 g, 98%) as a
tan/grey, foamy solid, which was unstable to chromatography and used
without further purification, mp 55–61◦C, [α]D 59.69 (c 0.5 in EtOH).
δH 8.05 (1H, s, OC(O)H12), 7.99 (1H, s, OC(O)H7), 5.19 (1H, br s,
H12), 4.98 (1H, br s, H7), 4.27–4.06 (4H, m, OCH2CH3), 4.05–3.91
(1H, m, H24), 3.35 (1H, m, H3), 3.23 (1H, d, J 6.0, H25), 2.50–0.50
(m), 1.20 (6H, m, 2 × OCH2CH3), 0.85 (3H, s, CH3), 0.75 (3H, d, J 6.0,
C21H3), 0.70 (3H, s, CH3). δC 168.8 and 168.2 (ester C=O), 160.7 and
160.6 (formyl C=O), 75.3 (C12), 71.4 (C7), 70.8 (C3), 70.4 [C24(R)],
61.6 (OCH2CH3), 57.4 (C25), 47.3 (C17), 44.9 (C13), 42.9 (C14), 40.9
(C5), 37.7 (C8), 34.8 (C20), 34.6 (C1 and C4), 34.2 (C10), 31.5 (C6),
31.1 (C23), 30.3 (C22), 28.5 (C9), 27.2 (C11 and C16), 25.5 (C12),
22.7 (C15), 22.3 (C19), 17.6 (C21), 14.0 (OCH2CH3), 12.1 (C18). m/z
(ESI−) 607 ([M – H]•−).
(24R)-5β-Cholestane-3α,7α,12α,24,26,27-hexol (Scymnol) 1
The bisformyl-(24R)-diTHP diester 7 (55.14 g, 0.071 mol) in dry THF
(350 mL) was added slowly to a stirred suspension of LAH (10.77 g,
0.284 mol) in dryTHF (660 mL) at −5 to −10◦C under an atmosphere of
nitrogen. The reaction mixture was stirred at this temperature for 90 min
and was then slowly allowed to warm to room temperature overnight.
The reaction mixture was cooled once again and cautiously quenched
by the dropwise addition of 12% aqueous hydrochloric acid (to pH 5).
A further volume of 12% hydrochloric acid (40 mL) was then added to
bring the reaction mixture to a pH of about 1 and the resultant mixture
was stirred for 1 h. The THF was removed under vacuum and methanol
(500 mL) was added to the aqueous suspension followed by concen-
trated hydrochloric acid (50 mL). The resultant mixture was stirred at
room temperature overnight (pH still 1) and was then heated at reflux
for 30 min to afford a clear, golden-coloured solution. The solvent was
evaporated under vacuum and the aqueous residue was extracted with
1 : 1 butanol/EtOAc (3 × 300 mL). The combined organic layers were
washed with water (200 mL), brine (100 mL), dried, and evaporated.The
residue was purified by flash chromatography on silica gel. Elution with
15% methanol in chloroform afforded an almost colourless foam, which
was recrystallized from aqueous methanol to give the title compound
(18.90 g, 57%) as a white solid, mp 191.4–192.4◦C (phase change at
130◦C; lit.[2] 120–123◦C, scymnol dihydrate from MeOH/EtOAc/H2O;
occasionallycrystalsre-formedandthesehadampofabout190◦C, lit.[7]
195–196◦C from MeOH/EtOAc), [α]D 38.80 (c 0.5 in MeOH) [lit.[7]
[α]2D5 40.40 (c 0.5 in MeOH), 34.20 (c 0.9 in EtOH)]. δH (CD3OD) 3.96
(1H, br s, H12), 3.84–3.60 (6H, m, H7, H24, C26H2O, and C27H2O),
3.46–3.26 (1H, m, H3), 2.39–2.10 (2H, m, H4 and H9), 2.04–1.09 (22H,
m), 1.02 (3H, d, J 6.0, C21H3), 0.91 (3H, s, C19H3), 0.71 (3H, s,
C18H3). δC (CD3OD) 74.1 (C12), 72.9 (C3), 72.4 (C24), 69.1 (C7),
62.2 (C27), 61.3 (C26), 49.3 (C17), 48.3 (C25), 47.5 (C13), 43.2 (C14),
43.0 (C5), 41.1 (C8), 40.5 (C4), 37.2 (C20), 36.6 (C1), 35.9 (C10), 35.9
(C6), 33.4 (C23), 32.4 (C22), 31.2 (C2), 29.6 (C11), 28.9 (C16), 27.9
(C9), 24.3•(C15), 23.3 (C19), 18.2 (C21), 13.1 (C18). m/z (FAB+) 491
([M + Na] +), 469 ([M + 1]•+), 415.
Diethyl 7α,12α-Diformyloxy-3α,24S-dihydroxy-5β-cholestane-
26,27-dioate 8
A suspension of the β-keto diester 5 (0.634 g, 1.0 mmol), ben-
zene ruthenium(ii) chloride dimer (2.5 mg, 5.0 µmol), and ethanol
(12 mL) in a glass Parr hydrogenation bottle under a positive pres-
sure of argon was degassed by sonication for 20 min. (S)-(+)-2,2ꢀ-
Bis(diphenylphosphino)-1,1ꢀ-binaphthyl (S-BINAP; 3.5 mg, 5.6 µmol)
was added and the vessel was sealed, evacuated, and purged with hydro-
gen gas three times such that the final pressure was 80 psi. The mixture
was then warmed to 43◦C and shaken for 20 h. The cooled solution
was poured into water (50 mL) and extracted with dichloromethane
(2 × 60 mL).Theorganicphasewaswashedwithbrine, dried, andevapo-
rated under reduced pressure to yield the title compound (0.60 g, 98%) as
a tan solid, [α]D 52.28 (c 0.5 in EtOH). δH 8.05 (1H, s, OC(O)H12), 7.99
(1H, s, OC(O)H7), 5.19 (1H, br s, H12), 4.98 (1H, br s, H7), 4.27–4.16
(4H, m, OCH2CH3), 4.05–3.91 (1H, m, H24), 3.35 (1H, m, H3), 3.23
(1H, d, J 6.0, H25), 2.50–0.50 (m), 1.20 (6H, m, 2 × OCH2CH3), 0.85
(3H, s, CH3), 0.75 (3H, d, J 6.0, C(21)H3), 0.68 (3H, s, CH3). δC 168.8
and 168.2 (ester C=O), 160.7 and 160.6 (formyl C=O), 75.3 (C12),
71.4 (C7), 71.2 [C24(S)], 70.8 (C3), 61.6 (OCH2CH3), 56.9 (C25), 47.2
(C17), 44.9 (C13), 42.9 (C14), 40.9 (C5), 37.7 (C8), 35.1 (C20), 34.8
(C1 and C4), 34.2 (C10), 31.5 (C6), 31.2 (C23), 30.3 (C22), 28.5 (C9),
27.1 (C11 and C16), 25.5 (C12), 22.7 (C15), 22.3 (C19), 17.8 (C21),
14.0 (OCH2CH3), 12.1 (C18).
Acknowledgments
The authors acknowledge the financial support of McFarlane
Marketing (Australia) Pty Ltd and the excellent technical

38
R. Adhikari et al.
assistance of Noel Hart, Ahmed Tawfik, Mike Falkiner, and
Stuart Littler.
[7] H. Ishida, S. Kinoshita, R. Natsuyama, H. Nukaya, K. Tsuji, M.
Nagasawa,T. Kosuge, Chem. Pharm. Bull. (Tokyo) 1992, 40, 864.
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46, 1347.
[9] R. J. Clay, T. A. Collom, G. L. Karrich, J. Wemple, Synthesis
1993, 290.
[10] R. Noyori, Asymmetric Catalysis in Organic Synthesis 1994,
Ch. 2, Homogeneous Asymmetric Hydrogenation (John Wiley &
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[11] D. J.Ager, S.A. Laneman,Tetrahedron:Asymmetry 1997, 8, 3327.
doi:10.1016/S0957-4166(97)00455-2
[12] R. Noyori, Angew. Chem., Int. Ed. 2002, 41, 2008. doi:
10.1002/1521-3773(20020617)41:12<2008::AID-ANIE2008>
3.0.CO;2-4
[13] Preparation of this reagent from commercially available benzene
ruthenium(ii) chloride dimer: R. A. Zelonka, M. C. Baird, Can.
J. Chem. 1972, 50, 3063.
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