Synthesis of an isomeric mixture (24RS,25RS) of sodium scymnol sulfate

Article abstract of DOI:10.1016/j.steroids.2007.12.006

This is the first reported multistep synthesis of the shark bile sterol sodium scymnol sulfate epimeric at the C-24 hydroxyl and C-27 sulfate positions. The starting cholic acid was protected as the tetrahydropyran ether (THP) derivative, reduced to the C-24 alcohol and oxidized to the protected aldehyde. This aldehyde was then coupled with methyl 3-hydroxypropionate using 2 equiv. of lithium diethylamide at -65 °C to produce methyl (24RS,25RS)-24,27-dihydroxy-3α,7α,12α,tris[(tetrahydropyran-2-yl)oxy]-5β-cholestan-26-oate. After protecting the 24 and 27 hydroxyls as the THP derivatives, this fully protected ester was then reduced to the monoalcohol. The monoalcohol was sulfated using the sulfur trioxide-triethylamine complex in dimethylformamide. The protective THP groups were removed with methanolic HCl and the sulfate was converted to the sodium salt with sodium ethoxide in methanol. This general synthetic scheme has application to produce a range of monosulfated sterols.

Full text of DOI:10.1016/j.steroids.2007.12.006

steroids 7 3 ( 2 0 0 8 ) 424–429
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Synthesis of an isomeric mixture (24RS,25RS)
of sodium scymnol sulfate
Donald W. Harney, Theodore A. Macrides∗
Natural Products Research Group, School of Medical Sciences, RMIT University, PO Box 71, Bundoora 3083, Victoria, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
This is the first reported multistep synthesis of the shark bile sterol sodium scymnol
sulfate epimeric at the C-24 hydroxyl and C-27 sulfate positions. The starting cholic
acid was protected as the tetrahydropyran ether (THP) derivative, reduced to the C-
24 alcohol and oxidized to the protected aldehyde. This aldehyde was then coupled
with methyl 3-hydroxypropionate using 2 equiv. of lithium diethylamide at −65 ◦C to
produce methyl (24RS,25RS)-24,27-dihydroxy-3␣,7␣,12␣,tris[(tetrahydropyran-2-yl)oxy]-5␤-
cholestan-26-oate. After protecting the 24 and 27 hydroxyls as the THP derivatives, this
fully protected ester was then reduced to the monoalcohol. The monoalcohol was sulfated
using the sulfur trioxide–triethylamine complex in dimethylformamide. The protective THP
groups were removed with methanolic HCl and the sulfate was converted to the sodium
salt with sodium ethoxide in methanol. This general synthetic scheme has application to
produce a range of monosulfated sterols.
Received 30 August 2007
Received in revised form
29 November 2007
Accepted 6 December 2007
Published on line 15 December 2007
Keywords:
Sodium scymnol sulfate
Cholic acid
Shark bile
Chemical synthesis
© 2007 Elsevier Inc. All rights reserved.
1.
Introduction
and/or (24R,25S) epimers. Our group had synthesized epimeric
unsulfated (24RS) scymnol [10] previously. (24R) scymnol was
The natural sodium scymnol sulfate has been purified by our
group from shark bile by preparative HPLC for use in com-
mercial skin care products [1]. We have investigated many
of the wide-ranging biological properties of scymnol and its
sulfate including its potent antioxidant [2,3] and hepatopro-
tective activities [4,5]. Ishida et al. have also investigated its
protective properties against vascular endothelial cell injury
[6–8].
The sodium scymnol sulfate isolated from shark bile con-
tains the 24R alcohol substituent and either or both of the 27R
and 27S sulfate esters [9] (see Fig. 1). Thus, as a further pro-
gression in the potential replacement of the natural product
with a synthetic product, we designed the synthetic scheme
shown in Fig. 2 to allow monosulfation at the C-27 position.
This scheme leads to the (24RS,25RS) epimeric mixture of
alcohols whereas the natural product comprises the (24R,24R)
synthesized subsequently by Adhikari et al. [11]. However a
sulfated form of scymnol had not been previously synthesized.
2.
Experimental
2.1.
General
1H NMR were carried out on samples purified from the
crude reaction products by silica gel column chromatogra-
phy using Bruker 300 and 400 MHz Avance Spectrometers
using deuterochloroform (or other indicated solvents) as the
reference or an internal deuterium lock. The multiplicity
of the signals is indicated as s = singlets, m = multiplet and
br = broad. 13C NMR spectra were recorded on an Avance
400 MHz instrument using an internal deuterium lock and
∗
Corresponding author. Tel.: +61 3 99257070; fax: +61 3 99257063.
E-mail address: macrides@rmit.edu.au (T.A. Macrides).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.12.006

steroids 7 3 ( 2 0 0 8 ) 424–429
425
use. All reagents were analytical grade and purchased from
regular commercial sources. Cholic acid (3␣,7␣,12␣-5␤-cholan-
24-oic acid) ≥99% was obtained from Fluka BioChemika
(Buchs, Switzerland). The sulfur trioxide–triethylamine com-
plex was prepared by the method of Tserng and Klein
[12].
Fig. 1 – (24R,25RS) sodium scymnol sulfate.
2.2.
Synthesis
2.2.1. Tetrahydropyran-2-yl-
proton decoupling. The chemical shifts are given as ı values
(ppm) and referenced to tetramethylsilane for deuterochloro-
form and 2,2,3,3-d(4)-3-(trimethylsilyl)propionic acid sodium
salt (TSP) for deuterium oxide (D2O) and deuteromethanol
(CD3OD) as solvent. High resolution mass spectroscopy was
carried out using a Bruker BioApex II FTICR electrospray
in positive ion mode using ammonium acetate in acetoni-
trile and water mixture. Microanalysis was performed by
the University of Otago, Dunedin New Zealand. Analyti-
cal TLC was carried out using precoated Merck Kieselgel
60 F254 silica gel aluminium plates from E. Merck (Darm-
stadt, Germany). Visualization was performed by spraying
with phosphomolybdic acid (5%, w/v) and sulfuric acid in
glacial acetic acid (7.5%, w/v) followed by heating for 5 min at
110 ◦C. Column chromatography was performed with Merck
Kieselgel 60 (230–400 mesh). Melting points are uncorrected.
Reagents were purified by standard techniques. Tetrahy-
drofuran (THF) and diethyl ether (ether) were dried with
sodium using benzophenone ketyl as the indicator. Petroleum
spirits refers to the fraction boiling in the range 40–60 ◦C.
The 3,4-dihydro-2H-pyran (DHP) was freshly distilled before
3˛,7˛,12˛,tris[(tetrahydropyran-2-yl)oxy]-5ˇ-cholan-24-
oate (compound 2,
Fig. 2)
A
mixture of cholic acid (16.42 g, 40 mmol) and p-
toluenesulfonic acid (40 mg) in DCM (100 ml) was magnetically
stirred for 10 min at 22 ◦C and then 3,4-dihydro-2H-pyran
(17.5 g, 208 mmol, 1.3 equiv.) added in stages over 30 min.
An exotherm raised the temperature to 39.5 ◦C and the
insoluble cholic acid dissolved during the reaction. The
reaction returned to 22 ◦C and after a further 3 h of stirring
the crude product 2 was concentrated in a rotary evapora-
tor under vacuum to yield a colourless oily residue (37.1 g)
that was used in the next reaction after 1.5 h; Rf 0.5 (ethyl
acetate/petroleum spirits = 1/3). The product on standing
yielded a second main spot at Rf 0.15––presumably due
to equilibration between the eight possible tetrahydropy-
ran (THP) acetals. 1H NMR (CDCl3) 0.63–0.75 (3H, m, H-18),
0.86–1.07 (6H, m, H-19, H-21), 3.32–4.22 (11H, m, H-3␤, H-7␤,
H-12␤, 4CH2O), 4.54–5.01 (4H, m, 4OCHO). Positive ion high
resolution MS: calculated for C44H72O9Na [M + Na+]: 767.5074.
Found: 767.5077.
Fig. 2 – (1) Cholic acid; (2) tetrahydropyran-2-yl-3␣,7␣,12␣,tris[(tetrahydropyran-2-yl)oxy]-5␤-cholan-24-oate; (3)
3␣,7␣,12␣,tris[(tetrahydropyran-2-yl)oxy]-5␤-cholan-24-ol; (4) 3␣,7␣,12␣,tris[(tetrahydropyran-2-yl)oxy]-5␤-cholan-24-al; (5)
methyl 3-hydroxypropionate; (6) methyl 24,26-dihydroxy-3␣,7␣,12␣,tris[(tetrahydropyran-2-yl)oxy]-5␤-cholestan-26-oate;
(7) methyl 3␣,7␣,12␣,24,26-penta[(tetrahydropyran-2-yl)oxy]-5␤-cholestan-26-oate; (8)
3␣,7␣,12␣,24,26-penta[(tetrahydropyran-2-yl)oxy]-5␤-cholestan-26-ol; (9)
(24RS,25RS)-3␣,7␣,12␣,24,26-penta[(tetrahydropyran-2-yl)oxy]-5␤-cholestan-27-yl triethylammonium sulfate; (10)
(24RS,25RS)-3␣,7␣,12␣,24,26-pentahydroxy-5␤-cholestan-27-yl sulfonic acid; (11)
(24RS,25RS)-3␣,7␣,12␣,24,26-pentahydroxy-5␤-cholestan-27-yl sodium sulfate.

426
steroids 7 3 ( 2 0 0 8 ) 424–429
ing to −65 ◦C. Toluene (5 ml) was added to the protected
aldehyde 4 (11.12 g, 17.2 mmol) and distilled in a rotary evap-
orator under vacuum in order to remove any residual water
or DCM, which would react with lithium diethylamide. The
protected aldehyde 4 in THF (12 ml) was added to the lithi-
ated methyl-3-hydroxypropionate solution over a period of
30 min at −65 ◦C. After 2 h the reaction mixture was removed
from the cooling bath and ether (55 ml) and 20% aqueous
ammonium chloride (35 ml) were added. The mixture was
warmed to room temperature and the organic layer was sep-
arated. The aqueous layer was washed with ether (60 and
30 ml) and the combined organic layer and ether extracts were
dried (Na2SO4). The solution was rotary evaporated under vac-
uum to give 15.9 g of the crude product. Purification of the
crude product by silica column chromatography using a step-
wise elution with DCM/petroleum spirits (3/1) and increasing
in stages to 100% DCM gave the title compound 6. A sec-
ond chromatography was required on some impure fractions.
Following rotary evaporation under vacuum, the title com-
pound 6 (4.515 g, 6.027 mmol) was isolated as a colourless oil.
This corresponded to a yield of 35% from purified aldehyde 4
and 16.3% from cholic acid. Rf 0.25 (ether/DCM = 3/2); 1H NMR
(CDCl3) 0.60–0.73 (3H, m, H-18), 0.82–1.04 (6H, m, H-19, H-21),
2.51–2.60 and 2.63–2.70 (1H, dm, HC–COO), 3.35–3.56 (H-3␤,
3OCHax), 3.56–3.67 (1H, br, H-7␤), 3.67–3.81 (4H, H-12␤, CH3)
3.81–4.20 (6H, m, 3OCHeq, CH2OH, CHOH), 4.57–4.83 (3H, m,
OCHO). MS: calculated for C43H76O10N [M + NH4+]: 766.5464.
Found: 766.5501.
2.2.2. 3˛,7˛,12˛,Tris[(tetrahydropyran-2-yl)oxy]-5ˇ-
cholan-24-ol (compound 3,
Fig. 2)
A dry flask was charged with dry ether (120 ml) and dry THF
(25 ml) containing crude compound 2 (37.1 g). The mixture at
22 ◦C was mechanically stirred under dry nitrogen and LiAlH4
(50 ml, 1 M in ether) added over 10 min. During this time the
spontaneous reflux was moderated with room temperature
water bath cooling. After further reflux heating of 1.5 h the
resulting clear solution was cooled and ethyl acetate (17.5 ml)
was added over 7 min. Reflux heating was recommenced for
a further 15 min, after which water (3.5 ml) was added drop-
wise. The reaction was cooled to room temperature and the
resulting precipitate was stirred for another 30 min. The reac-
tion mixture was filtered through a bed of Celite and washed
with ether (300 ml). The crude product was concentrated in
a rotary evaporator under vacuum, with toluene (6 ml) being
added towards the end to azeotrope out residual water. The
crude title compound 3 was isolated as a colourless oil (27.2 g).
Rf 0.4 (ethyl acetate/petroleum spirits = 4/6); 1H NMR (CDCl3)
0.62–0.75 (3H, m, H-18), 0.84–1.05 (6H, m, H-19, H-21), 3.33–4.23
(11H, m, H-3␤, H-7␤, H-12␤, 2H-24, 3CH2O), 4.55–4.97 (3H, m,
OCHO).
2.2.3. 3˛,7˛,12˛,Tris[(tetrahydropyran-2-yl)oxy]-5ˇ-
cholan-24-al (compound 4,
Fig. 2)
A mixture of of pyridinium chlorochromate (15.09 g, 70 mmol)
and anhydrous sodium acetate (1.15 g, 14 mmol) in DCM
(80 ml) was mechanically stirred at 22 ◦C while crude oil 3
(27.2 g) in DCM (10 ml) was added in one lot. After an ini-
tial exotherm, the reaction returned to 22 ◦C and after 3.5 h
dry ether (80 ml) was added, stirring continued for another
30 min and finally the solvent was decanted. The resulting
tarry residue was triturated with dry ether (2× 20 ml) and the
solvent washes decanted. The reaction mixture was concen-
trated in a rotary evaporator under vacuum to yield the crude
product (31.4 g). Purification of the crude product by column
chromatography with silica gel using stepwise elution with
ether in petroleum spirits increasing from 5 to 50%, yielded
a colourless oily residue of compound 4 (11.12 g, 17.24 mmol)
after rotary evaporation under vacuum. This product rep-
resented a yield of 46.6% from cholic acid. Rf 0.5 (ethyl
acetate/petroleum spirits = 1/3); 1H NMR (CDCl3) 0.61–0.72 (3H,
m, H-18), 0.81–1.05 (6H, m, H-19, H-21), 3.34–4.20 (9H, m, H-
3␤, H-7␤, H-12␤, 3CH2O), 4.59–4.81 (3H, m, OCHO), 9.75 (1H, s,
H-24).
2.2.5. Methyl-(24RS,25RS)-3˛,7˛,12˛,24,27-
penta[(tetrahydropyran-2-yl)oxy]-5ˇ-cholestan-26-oate
(compound 7, Fig. 2)
Compound 6 (7.46 g, 8.13 mmol) and p-toluenesulfonic acid
(45 mg) in DCM (70 ml) was magnetically stirred while DHP
(2.04 g, 24.2 mmol) was added over 5 min resulting in a 5 ◦C
exotherm. The reaction was allowed to proceed for a further
3 h after which the reaction mixture was concentrated in a
rotary evaporator under vacuum to give the title compound 7
(9.69 g) as a crude colourless oil. Rf 0.75 (ether/DCM = 1/4); 1H
NMR (CDCl3) 0.60–0.73 (3H, m, H-18), 0.82–1.04 (6H, m, H-19, H-
21), 2.20–2.40 (1H, m, H-25), 3.36–3.58 (6H, m, H-3␤, 5OCHax),
3.58–3.66 (1H, m H-7␤), 3.66–3.80 (4H, m, H-12␤, CH3), 3.80–4.18
(8H, CHOTHP, CH2OTHP, 5OCHeq), 4.54–4.84 (5H, m, OCHO).
MS: calculated for C53H92O12N [M + NH4+]: 934.6614. Found:
934.6590.
2.2.6. (24RS,25RS)-3˛,7˛,12˛,24,27-
Penta[(tetrahydropyran-2-yl)oxy]-5ˇ-cholestan-26-ol
2.2.4. Methyl-(24RS,25RS)-24,27-dihydroxy-
(compound 8, Fig. 2)
3˛,7˛,12˛,tris[(tetrahydropyran-2-yl)oxy]-5ˇ-cholestan-26-
oate (compound 6,
Fig. 2)
Crude fully protected ester 7 (9.69 g, 8.13 mmol) in dry ether
(150 ml) was mechanically stirred under dry nitrogen and
LiAlH4 (12.9 ml of 1 M in ether) was added over 3 min and the
reaction mixture exotherm resulted in mild reflux. After a
further 2 h reflux the solution was cooled to room tempera-
ture and ethyl acetate (5 ml) was added over a 2-min period.
The reaction mixture was refluxed for a further 10 min, and
cooled again to room temperature after which water (1.3 ml)
was added drop-wise and a precipitate formed. The reaction
mixture was stirred for a further 30 min and filtered through
a bed of Celite, washing with ether (200 ml). The combined
A solution of diisopropylamine (3.83 g, 37.8 mmol) in dry
THF (100 ml) was mechanically stirred under dry nitrogen.
After cooling to −65 ◦C in dry ice/ethanol, 1.6 M butyl lithium
(23.7 ml, 37.8 mmol) in hexane was added over 3 min. The
reaction was warmed to −20 ◦C and held for 20 min before
recooling to −65 ◦C. Methyl-3-hydroxypropionate 5 (1.975 g,
1.82 mmol) in THF (3 ml) was added over 12 min. The reaction
was warmed to −20 ◦C and held for 10 min before recool-

steroids 7 3 ( 2 0 0 8 ) 424–429
427
ether solution was concentrated in a rotary evaporator under
vacuum to give the crude title compound 8 (9.4 g). Purifi-
cation by silica column chromatography using a stepwise
elution with DCM/petroleum spirits (1/1–0/1) immediately fol-
lowed by ether/petroleum spirits (15/75 and 1/3) gave pure
8 (6.33 g, 6.90 mmol) as a colourless oil. This represented a
yield of 77.6% based on compound 6. Rf 0.3 (ether/DCM = 3/7);
1H NMR (CDCl3) 0.60–0.73 (3H, m, H-18), 0.81–1.08 (6H, m,
H-19, H-21), 3.34–3.58 (H-3␤, 5OCHax), 3.58–3.69 (1H br, H-
7␤), 3.69–3.81 (3H H-12␤, CH2OH), 3.81–4.18 (8H, m, CHOTHP,
CH2OTHP, 5OCHeq), 4.44–4.85 (5H, m, OCHO). MS: calculated
for C52H92O11N [M + NH4+]: 906.6665. Found: 906.6625.
2.2.7. (24RS,25RS)-3˛,7˛,12˛,24,26-
Penta[(tetrahydropyran-2-yl)oxy]-5ˇ-cholestan-27-yl
triethylammonium sulfate (compound 9, Fig. 2)
Compound 8 (4.40 g, 4.80 mmol) in dry dimethylformamide
(DMF) (15 ml) was warmed to obtain solution. The solution was
cooled to room temperature and sulfur trioxide–triethylamine
complex (1.28 g, 7.06 mmol, 1.47 equiv.) was added and mag-
netically stirred for 3.5 h after which time methanol (1.5 ml)
was added and the reaction allowed to proceed with stirring
for a further 30 min to react excess reagent. The solution was
concentrated in a rotary evaporator under vacuum to remove
DMF and the resulting crude oil of title compound 9 (6.35 g)
was used immediately in the next step. An attempt to purify
this compound in a previous preparation led to partial depro-
tection as indicated by a reduction of the acetal protons by
NMR analysis. It is proposed that the THP-protected triethy-
lammonium alkyl sulfate 9 acted as a mild acidic catalyst
during chromatography with methanol and DCM causing par-
tial deprotection, whereas an NMR of the crude compound 9
showed the required five acetal protons indicating no depro-
tection. MS on the crude product: found mass of 986.6160
which corresponded to [M − Et3N + NH4+] calc. 986.6239; Rf
0.75 (BuOH/CH3COOH/H2O = 8/1/1).
Fig. 3 – The four stereoisomers for the synthetic compound
11 of Fig. 2.
2.2.8. (24RS,25RS)-3˛,7˛,12˛,24,26-Pentahydroxy-5ˇ-
cholestan-27-yl sodium sulfate (compound 11,
Fig. 2)
2.2.8.1. Deprotection
to
(24RS,25RS)-3˛,7˛,12˛,24,26-
pentahydroxy-5ˇ-cholestan-27-yl sulfonic acid (compound
10, Fig. 1). The crude compound 9 (6.35 g) was dissolved in
methanol (100 ml) and stirred magnetically at room temper-
ature. One molar HCl was freshly prepared from 8.3 ml of 38%
aqueous HCl and made up to 100 ml with methanol. Addition
of the 1 M HCl solution (17 ml, 17 mmol) was completed over
a 5-min period. The reaction was allowed to proceed at room
temperature and the deprotection was carefully monitored by
TLC. The deprotection was complete after 3 h and showed one
main spot on TLC at Rf 0.4 (BuOH/CH3COOH/H2O = 8/1/1). The
resulting deprotected sulfonic acid compound 10 as shown in
Fig. 2 was then converted at room temperature to the sodium
salt by the addition of sodium ethoxide (1.80 g, 26.4 mmol) in
methanol (15 ml) that was added over a 2-min reaction period
with constant stirring. A small drop of the reaction mixture
added to a drop of water indicated a pH 10 had been reached.
Gaseous CO2 was bubbled through the reaction mixture for
15 min and this resulted in a drop to pH 8. The reaction mix-
ture was concentrated in a rotary evaporator under vacuum
to a low volume and silica gel (25 ml) was added. Further
evaporation of methanol gave a dry powder, which was added
to the top of a large chromatographic column of silica gel in
methanol (5%, v/v) in DCM. Fractions were collected while
increasing the methanol in a stepwise manner from 5 to 10, 15
to 20, 25 and finally 30 and 50% in DCM. The title compound 11
(2.13 g, 3.74 mmol) was collected and the yield was 78% based
on the purified protected monoalcohol compound 8. Rf 0.3
(BuOH/CH3COOH/H2O = 8/1/1); calculated for C27H47O9SNa:
C, 56.8; H, 8.3; S, 5.6; Na, 4.0. Found: C, 55.6; H, 8.3; S, 5.6; Na,
3.9; 1H NMR (CD3OD) 0.71 (3H, s, H-18), 0.92 (3H, m, H-19),
1.0–1.06 (3H, m, H-21), 2.18–2.36 (2H, m, H-4 and 9), 3.27–3.43
(m, H-3␤ + CH3OH), 3.43–3.66 (3H, m, H-24, H-26), 3.79 (1H, m,
H-7␤,) 3.97 (1H, m, H-12␤), 4.07–4.17 (1.5H, m, H-27), 4.17–4.22
(0.5H, m, H-27); 13C NMR (CD3OD) 15.28 (C18), 20.07, 20.19
(C21), 25.36 (C19), 26.10 (C15), 29.24 (C9), 30.46 (C16), 30.86
(C11), 32.15, 32.77, 32.96 (C23), 33.21 (C2), 34.46, 34.68 (C22),
37.00 (C6), 37.41 (C10), 38.13 (C1), 38.75 (C20), 41.24 (C4), 42.53
(C8), 44.23 (C5), 47.99, 48.36 (C25), 48.66 (C13), 49.04 (C14),

428
steroids 7 3 ( 2 0 0 8 ) 424–429
49.31 (C17), 61.75, 62.44, 62.52 (C26), 68.94, 69.80, 69.96 (C27),
70.88 (C7), 73.06, 73.29, 73.54 (C24), 74.34 (C3), 75.75 (C12);
MS: calculated for C27H52O9NS [M− + NH4+ + H+]: 566.3339.
Found: 566.3357; mp: 173 ◦C and after rechromatography
185–186 ◦C.
Table 1 – 13C NMR data of the two natural scymnol
sulfate and four synthetic stereoisomers
Position
Isheda et al. [9]
Present work
24R,25R
24R,25S
24R,25R, 24R,25S
(24S,25R; 24S,25R)
20
21
22
23
24
25
26
27
38.6
19.9
34.5
32.9
73.1
48.3
61.7
69.7
38.6
19.9
34.6
33.1
72.9
48.5
62.3
68.9
38.74
20.17, 20.19
34.46, 34.67
32.15, 32.77, 32.96
73.05, 73.27
47.98, 48.35
61.74, 62.42, 62.51
68.93, 69.78, 69.95
3.
Results and discussion
The protective group chosen for this synthesis was the THP
group rather than the silyl ether for the following reasons. It is
more amenable to commercial synthesis, and can be removed
under mild conditions without causing desulfation. However,
the NMR of the protected intermediates is more difficult to
interpret due to the chirality of each THP group.
This first reported synthesis of sodium scymnol sulfate
commences with cholic acid. This reacted with DHP and the
resulting THP protected THP cholate ester 2 was reduced with
lithium aluminium hydride and worked up by addition of a
small amount of water to produce a precipitate that was fil-
tered off. The ester had been stripped of the DCM solvent
before reduction. Investigation of an alternative reduction
with Red Al® in DCM resulted in problematic emulsions and
was not pursued further. The alcohol 3 was oxidized to the
aldehyde 4 with pyridinium chlorochromate in the presence
of anhydrous sodium acetate [13] and purified by column chro-
matography. Methyl-3-hydroxypropionate (5) was prepared
from 3-hydroxypropionitrile and methanolic HCl containing
a little water [14]. The pure aldehyde 4 was coupled using
2 equiv. of lithium diethylamide in THF at −65 ◦C according
to the method of Sunazuka et al. [15]. The reaction produced
compound 6 which was chromatographed and then protected
as the THP derivative 7. The methyl ester group at C-26 was
reduced to the monohydroxyl compound 8. The triethylamine
salt of the C-27 monosulfate 9 was prepared by reaction of
the monohydroxyl compound 8 and Et3N·SO3 in DMF accord-
ing to the method of Tserng and Klein [12]. After stripping
the DMF under high vacuum, the THP protective groups were
removed in methanolic HCl to produce compound 10. Care
was taken to prevent desulfation by excessive methanolic HCl
treatment [16]. Sodium ethoxide was dissolved in methanol
and gradually added to the compound 10 reaction mixture,
changing the pH to the range of 9–10. The pH was then
adjusted to between pH 7 and 8 and the crude mixture was
stripped of solvent, absorbed onto a portion of silica gel and
chromatographed to yield pure (24RS,25RS) sodium scymnol
sulfate 11. This was characterised using 13C and 1H NMR and
compared with the two [24R,25R] and [24R,25S] sterioisomers
of natural sodium scymnol sulfate using the results of Ishida
et al. [9]. Our 13C NMR results comparison is summarised in
Table 1. Extra chemical shifts were observed for the unnat-
ural isomers (24S,25R) and (24S,25S) produced in our product
(24RS,25RS) sodium scymnol sulfate 11 at positions 21, 23, 26
and 27 as shown in Fig. 4. Fig. 3 shows the steric orientations
at the 24 and 25 positions for the four sterioisomers of our
synthetic compound 11.
Fig. 4 – Positions in the sodium scymnol sulfate side-chain.
sodium scymnol sulfate for use in mouse pharmacokinetic
studies [17] using the synthetic method in this paper.
The synthetic sodium scymnol sulfate was also confirmed
by high resolution MS and microanalysis.
In conclusion, we have successfully completed the syn-
thesis of (24RS,25RS) sodium scymnol sulfate and the general
synthetic scheme presented in this paper may be useful in
the synthesis of a range of sterol monosulfated products com-
mencing from various bile acids.
Acknowledgements
The authors wish to thank Ms Nicolette Kalafatis for tech-
nical laboratory support and Dr Paul Wright for assistance
in manuscript preparation. Mass spectral analyses were per-
formed by Mr Stuart Thomson and Ms Sally Duck of Monash
University. We wish to thank Dr Greg Simpson, Deputy Chief
of the CSIRO Molecular and Health Science Technologies Clay-
ton, Victoria for technical support, and in particular CSIRO
staff Carl Braybrook for MS interpretation and Dr Roger J.
Mulder for 13C NMR spectroscopy on the synthetic sodium
scymnol sulfate. This work was supported by the Australian
Research Council (ARC) Collaborative Research Grant Program
(Grant No. LP0219239) and the Industry Partner, McFarlane
Marketing (Aust.) Pty Ltd, Melbourne, Vic., Australia.
r e f e r e n c e s
We believe that the observed protective effect of scymnol is
due to its free radical inhibitory effect in biological systems [2].
If this is so, the 24R and 24S sterioisomers may be equivalent
in activity. We have also produced 14C-radiolabelled 24RS,25RS
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