A facile synthesis of C-24 and C-25 oxysterols by in situ generated ethyl(trifluoromethyl)dioxirane

Article abstract of DOI:10.1016/j.steroids.2008.09.015

Experiments were performed to compare the regioselective hydroxylation of the isopropyl C-H bond at C-25 in 5α-cholestan-3β-yl acetate by in situ generated dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corresponding ketones and potassium peroxymonosulfate in aq. NaHCO₃, pH 7.5-8.0. Of the four dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reactivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate (overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.

Full text of DOI:10.1016/j.steroids.2008.09.015

steroids 7 4 ( 2 0 0 9 ) 81–87
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
A facile synthesis of C-24 and C-25 oxysterols by
in situ generated ethyl(trifluoromethyl)dioxirane
Shoujiro Ogawaa, Genta Kakiyamaa, Akina Mutoa, Atsuko Hosodaa,
Kuniko Mitamurab, Shigeo Ikegawab, Alan F. Hofmannc, Takashi Iidaa,∗
a
Department of Chemistry, College of Humanities & Sciences, Nihon University, Sakurajousui, Setagaya, Tokyo 156-8550, Japan
Faculty of Pharmaceutical Sciences, Kinki University, Kowakae, Higashi-osaka 577-8502, Japan
Department of Medicine, University of California, San Diego, La Jolla, CA 92093-0063, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Experiments were performed to compare the regioselective hydroxylation of the
isopropyl C–H bond at C-25 in 5␣-cholestan-3␤-yl acetate by in situ generated
dimethyldioxirane, methyl(trifluoromethyl)dioxirane, hexafluoro(dimethyl)dioxirane or
ethyl(trifluoromethyl)dioxirane (ETDO). The dioxiranes were generated from the corre-
sponding ketones and potassium peroxymonosulfate in aq. NaHCO3, pH 7.5–8.0. Of the four
dioxiranes examined, partially fluorinated, sterically bulky ETDO displayed the highest reac-
tivity and regioselectivity. Using in situ generated ETDO, a facile, synthesis was developed
for two naturally occurring oxysterols, i.e., 25-hydroxycholesterol, as well as its 3-sulfate
(overall yield of the sulfate, 24%) and 24-oxocholesterol (16%), starting from cholesterol.
© 2008 Published by Elsevier Inc.
Received 15 April 2008
Accepted 19 September 2008
Published on line 19 October 2008
Keywords:
Ethyl(trifluoromethyl)dioxirane
Remote-oxyfunctionalization
Hydroxylation
25-Hydroxycholesterol 3-sulfate
24-Oxocholesterol
1.
Introduction
plaque and are suggested to play an active role in plaque
development. Although a number of structurally different
Oxysterols are oxygenated derivatives of cholesterol having
a second oxygen function in addition to that at C-3 in the
steroid nucleus and bearing an iso-octyl or modified iso-octyl
side chain at C-17. Oxysterols are intermediates in bile acid
biosynthesis in the hepatocyte [1] and also serve to trans-
port cholesterol from brain to liver for conversion to bile acids
[2]. These compounds have a variety of biological proper-
ties such as cytotoxicity, carcinogenicity, and mutagenicity.
Moreover, oxysterols are present in human atherosclerotic
oxysterols have been hitherto isolated from biological tis-
sues, all of them have the 3␤-hydroxy-ꢀ5 steroid nucleus of
cholesterol. For example, high levels of the oxysterol conju-
gates of (24S)-24-hydroxycholesterol 3-sulfate-24-glucuronide
[3] and 5-cholesten-3␤,25-diol 3-sulfate [4,5] have recently
been found to be present in significant amounts in human
tissues.
The remote-oxyfunctionalization of unactivated ter-
tiary methine and/or secondary methylene C H bonds in
Abbreviations: DMDO, dimethyldioxirane; EI, electron ionization; ETDO, ethyl(trifluoromethyl) dioxirane; EtOAc, ethyl acetate; FAB,
fast atom bombardment; m.p., melting point; IR, infrared; HDDO, hexafluoro(dimethyl)dioxirane; 1H NMR, proton nuclear magnetic
resonance; 13C NMR, carbon 13 nuclear magnetic resonance; LR-MS, low-resolution mass spectrum; HR-MS, high-resolution mass spec-
trum; PIM, positive ion mode; NIM, negative ion mode; TLC, thin layer chromatography; MTDO, methyl(trifluoromethyl)dioxirane; THF,
tetrahydrofuran.
∗
Corresponding author.
E-mail address: takaiida@chs.nihon-u.ac.jp (T. Iida).
0039-128X/$ – see front matter © 2008 Published by Elsevier Inc.
doi:10.1016/j.steroids.2008.09.015

82
steroids 7 4 ( 2 0 0 9 ) 81–87
Fig. 1 – Chemical structures of dioxiranes examined and their oxidation products (1b–1d) with 5␣-cholestan-3␤-yl acetate
(1a).
hydrocarbons by non-microbial or non-enzymatic method
2.
Experimental
is of particular interest from the viewpoint of biomimetic
chemistry. The hope is to develop facile syntheses of bioactive
compounds from abundant natural sources using such “arti-
ficial enzymes” [6]. To this end, a variety of versatile oxygen-
transfer reagents has been developed by many groups of
workers.
2.1.
General procedure
Melting points (m.p.) were determined on a micro hot-stage
apparatus and are uncorrected. IR spectra were obtained in
KBr discs on a JASCO FT-IR 460 plus spectrometer (Tokyo,
Japan). 1H and 13C NMR spectra were obtained on a JEOL
JNM-EX 270 FT instrument at 270 and 68.8 MHz, respectively.
Low-resolution mass (LR-MS) spectra were recorded with a
JEOL GCmate gas chromatograph/mass spectrometer at 70 eV
electron ionization (EI) or fast atom bombardment (FAB) probe
using the positive (PIM) or negative ion mode (NIM). High-
resolution mass (HR-MS) spectra were also measured using
a JEOL GCmate with an EI probe under the PIM. Capillary
gas chromatographic (CGC) analysis was carried out by Shi-
madzu GC-2010 gas chromatograph (Kyoto, Japan) equipped
with a flame ionization detector using temperature program-
ming (from 260 to 300 ◦C, 2 ◦C/min); a chemically bonded
fused-silica capillary column (25QC3/BPX5; 25 m × 0.32 mm
i.d.; film thickness, 0.25 ␮m; SGE, Yokohama, Japan) was
fitted with the instrument. Normal phase TLC for nonsul-
fated compounds was performed on pre-coated silica gel
plates (0.25 mm layer thickness; E.Merck, Darmstadt, Ger-
many) using hexane–ethyl acetate mixtures (95:5–60:40, v/v)
as the developing solvent. Reversed-phase TLC for the sulfated
compounds was carried out on pre-coated RP-18F254S plates
using methanol–water–acetic acid mixtures (90:10:1, v/v/v)
as the developing solvent. A Sep-Pak Vac tC18 cartridge (5 g;
Waters, Milford, MA, USA) was used for solid-phase extraction.
Oxone® was purchased from Sigma–Aldrich Com. (St.
Louis, MO, USA). Acetone, trifluoro-2-propanone, 1,1,1-
trifluro-2-butanone, and hexafluoroacetone were available
from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan).
Dioxiranes, the smallest three-membered cyclic perox-
ides that contain a carbon atom, are of particularly interest,
because of their selective reactivity [7,8]. In addition, synthetic
procedures based on dioxirane oxidations can be performed
under extremely mild, nonhydrolytic conditions. The con-
version of hydrophobic natural compounds to hydrophilic
oxygen-containing derivatives would appear to be a highly
useful achievement of dioxirane chemistry. For this pur-
pose a variety of dialkyldioxiranes [9,10], their halogenated
analogs [11–17] and chiral dioxiranes [8,18,19] have been
employed.
Our previous papers have reported the utility of dimethyl-
dioxirane (DMDO) as an effective oxygen-donor to the specific
tertiary and/or secondary C–H bonds in a variety of struc-
turally different bile acids and steroids [20–22]. In these
studies, a concentrated DMDO/CHCl3 solution (see below)
[23–27] was prepared from acetone and potassium peroxy-
monosulfate (Oxone®, 2KHSO5·KHSO4·K2SO4). A concentrated
solution of methyl(trifluoromethyl)dioxirane (MTDO), gener-
ated from 1,1,1-trifluoro-2-propanone (TFP) [11–16] has been
shown to be superior to DMDO in reactivity and selectivity
[7,15] but to prepare this reagent, a large amount of expensive
TFP is required.
As part of our ongoing program to develop an improved,
biomimetic oxidant system [28,29], we report here the
use of ethyl(trifluoromethyl)dioxirane (ETDO), which in situ
is generated efficiently from commercially available 1,1,1-
trifluoro-2-butanone and Oxone® in a reaction flask. We
compared the efficiency of hydroxylation at C-25 of 5␣-
cholestan-3␤-yl acetate (1a) by DMDO, MTDO, ETDO, and
hexafluoro(dimethyl)dioxirane (HDDO) (Fig. 1). We were able
to develop a facile synthesis of two naturally occurring oxys-
terols, 25-hydroxycholesterol as such and its 3-sulfate (2c) and
24-oxocholesterol (3b), from cholesterol (4) by using ETDO gen-
erated in situ. Previously reported chemical syntheses of these
oxysterols are either multistep procedures with an extremely
low-yield from 4 or alternatively, use an expensive natural
product as the starting material.
2.2.
General oxidation procedure of 5˛-cholestan-3ˇ-yl
acetate (1a) with dioxiranes generated in situ
To a solution of 1a (0.23 mmol) in CH2Cl2 (1 mL) in a three-
necked reaction flask fitted with a dry-ice/acetone condenser,
a pressure equalizing graduated separatory funnel and a sili-
cone rubber septum, Oxone® (2.3 mmol) and water (3 mL) were
added. To the resulting two-layer system, the different ketones
(23 mmol) were added by a syringe through the septum. The

steroids 7 4 ( 2 0 0 9 ) 81–87
83
(KBr) ꢁmax cm−1: 1731 (C O), 3313 (OH). 1H NMR (CDCl3), ı: 0.68
(3H, s, 18-CH3), 0.93 (3H, d, J 5.4, 21-CH3), 1.02 (3H, s, 19-CH3),
1.21 (6H, s, 26- and 27-CH3), 2.03 (3H, s, OCOCH3), 4.61 (1H,
br. m, 3␣-H), 5.38 (1H, d, J 2.7, 6-H). LR-MS (FAB+), m/z: 467
(M+Na, 19%), 384 (M−AcOH, 44%), 367 (M−AcOH–H2O, 100%),
289 (M−S.C.–part of ring D, 9%), 255 (M−S.C.–AcOH, 19%), 213
(M−S.C.–AcOH–part of ring D–CH3, 16%).
resulting suspension was vigorously stirred in an ice-bath
and adjusted to mildly alkaline conditions of pH 7.5–8.0 with
1 M aq. NaHCO3. As the reaction proceeded, the suspension
changed gradually to a clear solution. After stirring at room
temperature for 12 h, the reaction product was extracted with
CH2Cl2. The combined extract was washed with water, dried
with Drierite®, and evaporated in vacuo. The above procedure
was repeated several times until most of 1a was exhausted;
the reaction was monitored by TLC and CGC.
A solution of the 3␤-acetate 2a (200 mg, 0.45 mmol) in 5%
methanolic KOH (20 mL) was refluxed for 1 h. After evapora-
tion of the solvent, the residue was dissolved in CH2Cl2, and
the solution was washed with water, dried with Drierite® and
evaporated in vacuo. Recrystallization of the oily residue from
aq. methanol gave the 3␤,25-dihydroxy-5-ene 2b as colorless
needles: yield, 163 mg (90%). m.p. 178–180 ◦C. (lit. 178–180 ◦C
[32]). IR (KBr), ꢁmax cm−1: 3301 (OH). 1H NMR (CDCl3) ı: 0.68
(3H, s, 18-CH3), 0.93 (3H, d, J 5.4, 21-CH3), 1.01 (3H, s, 19-CH3),
1.21 (6H, s, 26- and 27-CH3), 3.50 (1H, br. m, 3␣-H), 5.34 (1H, br.
s, 6-H). 13C NMR (CDCl3) ı: 11.9 (C-18), 18.7 (C-21), 19.4 (C-19),
20.7 (C-23), 21.1 (C-11), 24.3 (C-15), 28.2 (C-16), 29.2 and 29.3 (C-
26 and C-27), 31.6 (C-2), 31.9 (C-7), 31.9 (C-8), 35.7 (C-20), 36.4
(C-22), 36.5 (C-10), 37.2 (C-1), 39.8 (C-12), 42.3 (C-4 and C-13),
44.4 (C-24), 50.1 (C-9), 56.0 (C-17), 56.7 (C-14), 71.1 (C-25), 71.8
(C-3), 121.7 (C-6), 140.7 (C-5). LR-MS (EI), m/z: 402 (M+, 65%), 384
(M−H2O, 100%), 369 (M−H2O–CH3, 53%), 366 (M−2H2O, 21%),
351 (M−2H2O–CH3, 34%), 317 (M−H2O–ring A–part of ring B,
13%), 299 (M−2H2O–ring A–part of ring B, 29%), 273 (M−S.C.,
75%), 255 (M−H2O–S.C., 31%), 245 (24%), 231 (M−S.C.–ring D,
22%), 213 (M−H2O-CH3–S.C.–part of ring D, 43%). HR-MS (EI),
calculated for C27H46O2 [M+], 402.3498; found m/z: 402.3498.
2.2.1. 5˛,6ˇ-Dibromocholestan-3ˇ-yl acetate (5)
This compound was prepared by the usual method. m.p.
109–111 ◦C. (lit. 112–113 ◦C [30]). IR (KBr), ꢁmax cm−1: 1741 (C O).
1H NMR (CDCl3), ı: 0.71 (3H, s, 18-CH3), 0.85 and 0.88 (each 3H,
s, 26- and 27-CH3), 0.91 (3H, d, J 5.4, 21-CH3), 1.46 (3H, s, 19-
CH3), 2.05 (3H, s, OCOCH3), 4.83 (1H, m, 6␣-H), 5.48 (1H, br.
m, 3␣-H). LR-MS (FAB+), m/z: 611 (M+Na, 4%), 529 (M−AcOH,
26%), 447 (M−Br, 53%), 415 (M−S.C.–AcOH, 6%), 385 (11%), 367
(M−Br2–AcOH, 100%).
2.2.2. 5˛,6ˇ-Dibromo-25-hydroxycholestan-3ˇ-yl acetate
(6) (large-scale preparation)
The reaction was carried out by an essentially identical
procedure mentioned above. Thus, a solution of the 5␣,6␤-
dibromo-3␤-acetate (5; 5 g, 8.5 mmol) in CH2Cl2 (25 mL) was
added in a three-necked reaction flask. A vigorously stirred
suspension of 1,1,1-trifluoro-2-butanone (83 mL, 850 mmol)
was added to 100 mL Oxone® (50 g, 85 mmol) in distilled water
(100 mL) and stirring was continued. The pH of the suspension
was kept at 7.5–8.0 with 1 M aq. NaHCO3 during the period
of reaction. After stirring at room temperature for 12 h, the
reaction product was extracted with CH2Cl2 and the com-
bined extract was washed with water, dried with Drierite®, and
evaporated in vacuo. The procedure was repeated three times
(total reaction time, 27 h). The reaction product was puri-
fied by passage through a column of silica gel (100 g). Elution
with hexane–EtOAc (1:9, v/v) afforded the starting compound
5; 1.1 g (20%). Continued elution with hexane–EtOAc (2:8,
v/v) gave the desired 3␤-acetoxy-5␣,6␤-dibromo-25-hydroxy
compound 6, which was recrystallized from EtOAc–methanol
as colorless prisms: yield, 2.3 g (45%). m.p. 120–122 ◦C (lit.
127–128 ◦C [12]). IR (KBr) ꢁmax cm−1: 1723 (C O), 3521 (OH).
1H NMR (CDCl3) ı: 0.71 (3H, s, 18-CH3), 0.93 (3H, d, J 5.4,
21-CH3), 1.21 (6H, s, 26- and 27-CH3), 2.05 (3H, s, OCOCH3),
4.83 (1H, m, 6␣-H), 5.48 (1H, br. m, 3␣-H). LR-MS (FAB+),
m/z: 627 (M+Na, 12%), 527 (M−AcOH, 100%), 505 (M−Br–H2O,
28%), 465 (M−AcOH–Br, 36%), 445 (M−AcOH–H2O–Br, 92%), 415
(M−S.C.–AcOH, 20%), 367 (M−AcOH–H2O–Br2, 68%).
2.2.4. 3ˇ-Sulfooxy-25-hydroxycholest-5-ene (2c, as
sodium salt)
To a solution of the 3␤,25-dihydroxy-5-ene (30 mg, 0.07 mmol)
in dry pyridine (2 mL), sulfur trioxide–trimethylamine com-
plex (30 mg, 0.22 mmol) was added, and the suspension was
stirred at room temperature for 1 h [33]. The reaction mix-
ture was poured onto ice-cooled petroleum ether (20 mL) and
the precipitated solid was collected by filtration. After being
washed with petroleum ether, the solid product was dissolved
in methanol (1 mL). The resulting solution was adjusted to pH
8 by adding 1N NaOH, diluted with water (10 mL), and then
loaded onto a preconditioned Sep-Pak Vac tC18 cartridge. The
cartridge was successively washed with water (20 mL) and
then with 20% methanol (20 mL), and the desired 3␤-sulfooxy-
25-hydroxy-5-ene (2c) was eluted with methanol (20 mL). After
evaporation of the solvent, recrystallization of the residue
from methanol–EtOAc gave the analytically pure 2c in the
form of colorless amorphous solids: yield, 25 mg (70%). m.p.
164–165 ◦C. IR (KBr) ꢁmax cm−1: 3451 (OH). 1H NMR (CD3OD) ı:
0.72 (3H, s, 18-CH3), 0.96 (3H, d, J 5.4, 21-CH3), 1.03 (3H, s, 19-
CH3), 1.17 (6H, s, 26- and 27-CH3), 4.13 (1H, br. m, 3␣-H), 5.38
(1H, br. s, 6-H). LR-MS (FAB−), m/z: 481 (M−, 71%), 306 (10%),
199 (12%), 168 (15%), 153 (100%), 122 (22%), 97 (HSO4−, 68%), 80
(SO3−, 38%). HR-MS (FAB−), calculated for C27H45O5S, 481.2987;
found m/z: 481.2992.
2.2.3. 3ˇ,25-Dihydroxycholest-5-ene (2b)
To a solution of the 5␣,6␤-dibromo-25-hydroxy-3␤-acetate 6
(300 mg, 0.5 mmol) dissolved in ether (9 mL) and acetic acid
(100 ␮L), zinc powder (300 mg) was added in one portion
and the suspension was vigorously stirred at room temper-
ature for 1 h. Excess zinc was removed by filtration, and the
mother liquor was washed with brine, dried with Drierite®
and evaporated to dryness. Recrystallization of the residue
from EtOAc–methanol afforded the unsaturated 3␤-acetoxy-
25-hydroxy-ꢀ5 compound 2a in the form of colorless prisms:
yield, 200 mg (90%). m.p. 139–140 ◦C. (lit. 139–140 ◦C [31]). IR
2.2.5. 5˛,6ˇ-Dibromocholest-24-en-3ˇ-yl acetate (8)
A mixture of the 5␣,6␤-dibromo-25-hydroxyl-3␤-acetate 6 (2 g,
3.3 mmol) in 1,4-dioxane (50 mL) and conc. H2SO4 (4 mL) was
stirred at 0 ◦C for 2 h. Ice-water was added to the mixture and

84
steroids 7 4 ( 2 0 0 9 ) 81–87
the reaction product was extracted with EtOAc. The combined
organic layer was washed successively with 5% aq. NaHCO3
and brine, dried with Drierite®, and evaporated to dryness. The
brown residue was chromatographed on a column of silica gel
(60 g), eluting with hexane–EtOAc (95:5, v/v). Recrystallization
of a homogeneous fraction from EtOAc–methanol gave 5␣,6␤-
dibromocholest-24-en-3␤-yl acetate (8) as colorless needles:
yield, 1.4 g (70%). m.p. 102–103.5 ◦C. IR (KBr) ꢁmax cm−1: 1735
(C O). 1H NMR (CDCl3) ı: 0.71 (3H, s, 18-CH3), 0.93 (3H, d, J 5.4,
21-CH3), 1.46 (3H, s, 19-CH3), 1.59 and 1.60 (each 3H, s, 26- and
27-CH3), 2.08 (3H, s, –OCOCH3), 4.83 (1H, m, 6␣-H), 5.09 (1H, t, J
5.4, 24-H), 5.48 (1H, br. m, 3␣-H). 13C NMR (CDCl3) ␦: 12.2 (C-18),
17.6 (C-27), 18.6 (C-21), 20.1 (C-19), 21.3 (C-11), 24.0 (C-15), 24.7
(C-23), 25.7 (C-26), 26.2 (C-7), 28.1 (C-16), 30.8 (C-6), 35.6 (C-20),
36.0 (C-22), 36.5 (C-2), 37.2 (C-1), 39.6 (C-4), 41.9 (C-10), 41.9 (C-
12), 42.7 (C-13), 47.2 (C-8), 55.1 (C-9), 56.0 (C-17), 56.1 (C-14), 74.0
(C-3), 88.1 (C-5), 125.1 (C-24), 131.0 (C-25), 170.4 (–OCOCH3). LR-
MS (FAB+) m/z: 609 (M+Na, 9%), 585 (M+, 6%), 527 (M−AcOH,
78%), 505 (19%), 473 (M−S.C., 19%), 447 (M−AcOH–Br, 69%),
365 (M−Br2–AcOH, 100%), 329 (25%), 307 (44%). HR-MS (FAB+),
calculated for C29H46O2NaBr2 [M+Na]+, 607.1762; found, m/z:
607.1764.
combined organic layer was washed with water, dried over
Drierite®, and evaporated to dryness. Recrystallization of the
oily residue from EtOAc gave analytically pure 24-oxocholest-
5-en-3␤-yl acetate (3a) as colorless needles: yield, 450 mg
(90%). m.p. 128–130 ◦C (lit. 127–128 ◦C [34]; 130–132 ◦C [35]). IR
(KBr) ꢁmax cm−1: 1703 (C O, ketone), 1730 (C O, ester). 1H NMR
(CDCl3) ı: 0.67 (3H, s, 18-CH3), 0.92 (3H, d, J 8.1, 21-CH3), 1.02
(3H, s, 19-CH3), 1.08 and 1.10 (each 3H, s, 26- and 27-CH3), 2.03
(3H, s, OCOCH3), 4.60 (1H, br. m, 3␣-H), 5.38 (1H, d, J 2.7, 6-H).
LR-MS (FAB+), m/z: 465 (M+Na, 13%), 383 (M−AcOH, 100%), 365
(10%), 255 (M−H2O–S.C., 12%), 213 (M−AcOH–CH3–S.C.–part of
ring D, 10%).
Alkaline hydrolysis of 3a (500 mg, 1.1 mmol) with 5%
methanolic KOH, as described for the preparation of 2b,
gave 24-oxochol-5-en-3␤-ol (3b), which recrystallized from
aq. methanol as colorless prisms: yield, 440 mg (99%). m.p.
131–133 ◦C (lit. 133–134 ◦C [34]). IR (KBr) ꢁmax cm−1: 1714 (C O),
3381 (O–H). 1H NMR (CDCl3) ı: 0.68 (3H, s, 18-CH3), 0.92 (3H, d,
J 8.1, 21-CH3), 1.01 (3H, s, 19-CH3), 1.09 (each 3H, s, 26- and 27-
CH3), 3.52 (1H, m, 3␣-H), 5.35 (1H, br. s, 6-H). 13C NMR (CDCl3)
ı: 11.9 (C-18), 18.3 (C-21), 18.4 and 18.5 (C-26 and C-27), 19.4
(C-19), 21.1 (C-11), 24.2 (C-15), 28.1 (C-16), 29.8 (C-2), 31.6 (C-
7), 31.9 (C-8 and C-22), 35.4 (C-20), 36.5 (C-10), 37.2 (C-1 and
C-23), 39.7 (C-12), 40.8 (C-25), 42.3 (C-4 and C-13), 50.1 (C-9),
55.9 (C-17), 56.7 (C-14), 71.8 (C-3), 121.7 (C-6), 140.7 (C-5), 215.5
(C-24). LR-MS (EI) m/z: 400 (M+,100%), 385 (M−CH3, 22%), 382
(M−H2O, 72%), 367 (M−CH3–H2O, 35%), 357 (M−part of S.C.
CH(CH3)2 , 9%), 339 (M−H2O–part of S.C., 10%), 314 (52%), 299
(29%), 289 (M−H2O–ring A–part of ring B, 34%), 271 (43%), 255
(M−H2O–S.C., 47%), 229 (M−H2O–S.C.–part of ring D, 29%), 213
(M−H2O–CH3–S.C.–part of ring D, 78%). HR-MS (EI), calculated
for C27H44O2 [M+]: 400.3341; found m/z: 400.3337.
2.2.6. 24ꢂ-Hydroxycholest-5-en-3ˇ-yl acetate (9b)
To a magnetically stirred solution of the 3␤-acetoxy-5␣,6␤-
dibromo-5-ene 8 (1.2 g, 2.0 mmol) in dry tetrahydrofuran (THF)
(20 mL), 1.0 M BH3/THF solution (20 mL) was added dropwise
with ice-bath cooling. The mixture was stirred at room tem-
perature for 1 h under a nitrogen stream. NaOH (3N, 10 mL) and
then 30% H2O2 (10 mL) were slowly added with ice-bath cool-
ing, and the resulting mixture was stirred at 0 ◦C for 30 min.
The product was extracted with CHCl3, and combined extract
was washed with water, dried with Drierite®, and evaporated
to dryness. The oily residue, which is consisted of an epimeric
mixture (9a) of 24R- and 24S-hydroxy-5␣,6␤-dibromides, was
dissolved in ether (35 mL) and acetic acid (0.4 mL). Zinc pow-
der (1.0 g) was added to the mixture with vigorous stirring and
the resulting suspension was stirred at room temperature for
1 h. Excess zinc was removed by filtration, and the mother
liquor was washed with brine, dried with Drierite®, and evap-
orated to dryness. Chromatography of the oily residue on a
column of silica gel (50 g) and elution with benzene-EtOAc
(96:4, v/v) afforded an epimeric mixture of the title compound
9b, which recrystallized from aqueous methanol as color-
less amorphous solids: yield, 540 mg (60%). m.p. 133–134 ◦C.
IR (KBr) ꢁmax cm−1: 1737 (C O), 3348 (OH). 1H NMR (CDCl3) ı:
0.68 (3H, s, 18-CH3), 0.89–0.94 (9H, m, 21-, 26- and 27-CH3),
1.02 (3H, s, 19-CH3), 2.03 (3H, s, –OCOCH3), 3.33 (1H, m, 24␰-H),
4.62 (1H, br. m, 3␣-H), 5.38 (1H, br. s, 6-H). LR-MS (FAB+), m/z:
467 (M+Na, 15%), 385 (M−AcOH, 100%), 367 (85%), 283 (17%),
255 (M−AcOH–S.C., 27%), 213 (M−AcOH–S.C.–part of ring D-
CH3, 19%). HR-MS (FAB+), calculated for C29H48O3Na [M+Na]+:
467.3501; found, m/z: 467.3500.
3.
Results and discussion
As mentioned above, dioxiranes are powerful and versatile
oxidants, particularly for the remote-oxyfunctionalization of
unactivated tertiary and/or secondary C–H bonds in sub-
strates, in analogy with cytochrome P450 enzymes in vivo.
Volatile and labile dioxiranes can be generated by a large
excess of an appropriate ketone and Oxone® under condi-
tions where pH is controlled using NaHCO3 as buffer. The
generated dioxirane was collected either as an isolated stock
solution in the parent ketone [9–15,23–27] or in its concen-
trated form by extraction into CHCl3 [20–22,26] (or CCl4 [27]).
The dioxirane solution can then be stored in a −20 ◦C freezer
to prevent decomposition. Otherwise, dioxiranes are gener-
ated in situ in a reaction flask [25,27]. For example, DMDO
could be generated to a concentration of ca. 0.10 M as an
acetone solution [23,24], which could still be further concen-
trated (to ca. 0.18 M) by extraction with CHCl3 [20–22,26] or CCl4
[27]. Using the isolated (or concentrated) solution of dioxirane
allows the oxidation to be performed under extremely mild,
strictly neutral, nonhydrolytic conditions. However, the isola-
tion method has a practical drawback, with the exception of
DMDO, since a large excess amount of expensive ketone pre-
cursor (e.g., TFP) is required. The in situ generation method of
dioxiranes using the expensive ketones has been shown to be
feasible particularly in preparative, large-scale reactions, but
2.2.7. 24-Oxocholest-5-en-3ˇ-ol (3b)
Jones reagent (1 mL) was added dropwise to a solution of the
24␰-hydroxy-ꢀ5-3␤-acetate 9b (500 mg, 1.1 mmol) in acetone
(25 mL) under 0 ◦C, and the mixture was stirred for 30 min
at room temperature. Isopropanol (2 mL) was added, and the
oxidation product was extracted with CH2Cl2 (50 mL). The

steroids 7 4 ( 2 0 0 9 ) 81–87
85
respectively) [20]. A comparison of the in situ generated MTDO
and ETDO reactions also revealed that after 27 h, yields of
1a, 1c, 1d, and unknowns by MTDO were 39%, 4%, 4%, and
1%, respectively, but those by ETDO were 13%, 6%, 8%, and
7%, respectively. The result evidently suggests that a more
bulky ETDO is superior to MTDO in reactivity and attacks
preferentially a less sterically hindered C–H bond at C-25 in
1a. Although a mechanism of the observed differences in the
reactivity among the four dioxiranes examined is unclear, the
coexistence of both an electron-withdrawing fluoro group and
an electron-donating alkyl group in dioxirane molecules may
be one of the essential factors.
Based on above the results, our next effort was to develop a
facile synthesis of naturally occurring 25-hydroxycholesterol
and its 3-sulfate (2c) as well as 24-oxocholesterol (3b) start-
ing from cholesterol (4). The synthetic routes are shown in
Fig. 3. A key step in the syntheses involves the hydroxyla-
tion at C-25 of 5␣,6␤-dibromocholestan-3␤-yl acetate (5) [12]
with in situ generated ETMO. In order to prevent simultane-
ous oxidation of the 3␤-hydroxyl group and the ꢀ5-bond in 4
by ETDO, it was converted to the 3␤-acetoxy-5␣,6␤-dibromide
5 in two steps [37]. Treatment of 5 with in situ generated
ETDO at room temperature for 27 h afforded the expected
25-hydroxy-5␣,6␤-dibromide 6 in 45% isolated yield after chro-
matographic purification. Procedures and product isolation
are quite straightforward, since ETDO is quite volatile and
easily removed. The 1H and 13C NMR spectral data of 6 were
in good agreement with those reported in the literature [12].
Debromination of 6 with zinc powder in ether/acetic acid,
followed by alkaline hydrolysis of the resulting product with
methanolic KOH gave 25-hydroxycholesterol (2b, 90% yield),
which is a key intermediate useful in the synthesis of various
oxysterols.
Fig. 2 – Time-course of the conversion (%) of
5␣-cholestan-3␤-yl acetate (1a) to
25-hydroxy-5␣-cholestan-3␤-yl acetate (1b) by dioxiranes
generated in situ.
a detailed study has not yet been reported except for DMDO
[8,25,27].
This reasoning led us to examine the reactivity and regios-
electivity of dioxiranes generated in situ under identical
experimental conditions. Our initial effort was directed to
define optimal conditions for the in situ generation of dioxi-
ranes. We tested the previously unreported HDDO and ETDO
as well as the known DMDO and MTDO. We used 5␣-cholestan-
3␤-yl acetate as the target molecule (1a).
As described in detail in Section 2, each of the four vari-
ants of dioxiranes was generated in situ at room temperature
from acetone (or 1,1,1-trifluoroacetone, hexafluoroacetone,
and 1,1,1-trifluro-2-butanone) and Oxone® in water. To the
suspension was added a solution of 1a in CH2Cl2. It was essen-
tial to maintain the pH of the two-layer system (CH2Cl2/water)
at 7.5–8.0 by adding aq. NaHCO3 during the period of reac-
tion with vigorous stirring. Fig. 2 shows the time-course of the
conversion (%) of 1a to 25-hydroxy-5␣-cholestan-3␤-yl acetate
(1b). According to a previous report [12], 1a was converted to
1b in 78% yield at 0 ◦C for 3 h with an isolated stock solu-
tion of MTDO/TFP (ca. 0.8 M) but in 30% yield at 20◦C for 24 h
with a DMDO/acetone solution (0.1 M), indicating that the for-
mer is more effective than the latter. Under the conditions
examined in our experiments, 1a was transformed into 1b
regioselectively except for one case (see below), but yields and
optimal reaction times were significantly influenced by the
structures of the oxidants. Thus, when 1a was subjected to
the DMDO oxidation, the formation of 1b gradually increased
until the maximum conversion of ca. 40% was obtained after
72 h (six times). Unexpectedly, the hydroxylation of 1a with
fully fluorinated HDDO did not proceed at all. On the con-
trary, partially fluorinated MTDO [12,36] and ETDO, both of
which have trifluoromethyl and alkyl groups, showed a simi-
lar reactivity and inserted an oxygen atom at C-25 effectively
to give 1b in the maximum conversion of ca. 52% and 66%,
respectively, with a considerable reduction in the reaction
time (27 h, three times). The prolonged reaction with MTDO
(or ETDO) decreased rapidly the conversion to 1b and caused
a second-order reaction forming increasing amounts of the
double-functionalized 17␣,25- and 14␣,25-diols (1c and 1d,
The compound 2b was sulfated cleanly at C-3 with sulfur
trioxide-trimethylamine complex to afford the desired 25-
hydroxycholesterol 3-sulfate (2c) [33]. The analytically pure 2c
was efficiently obtained by solid-phase extraction using a Sep-
Pak Vac tC18 cartridge; the total yield from cholesterol (4) was
24%. The sulfate 2c has recently been identified in primary
rat hepatocytes [4] and also detected in the nuclei of normal
human liver tissue [5].
When the 25-hydroxy-5␣,6␤-dibromide 6 was subjected to
the dehydration reaction with conc. H2SO4, it was converted
to the ꢀ24-unsaturated compound 8 exclusively; theꢀ25(26)
-
isomer was not formed at all. The trisubstituted ꢀ24-structure
in 8 was characterized by the olefinic carbon signals appear-
ing at 125.1 (C-24) and 131.0 (C-25) ppm, respectively, in the
13C NMR and the proton signal resonating at 5.09 ppm (H-24)
as triplet (J 5.4 Hz) in the 1H NMR. In addition, the 13C chemical
shifts of the C20–C27 in the side chain were in good agreement
with those reported for desmosterol (3␣-hydroxycholest-5,24-
diene) [38].
Hydroboration of 8 with BH3/THF complex, followed by
oxidative cleavage of the resulting alkylborane with alkaline
hydrogen peroxide gave an anti-Markovnikovs’ type regiose-
lective formation of the 24␰-hydroxy compound 9a. Without
isolating each of the 24R- and 24S-hydroxy epimers at this
stage, debromination of 9a with zinc powder in ether–acetic
acid afforded the 24␰-hydroxy-ꢀ5 compound 9b in isolated
yield of 60%, which showed a single spot on TLC and a sin-

86
steroids 7 4 ( 2 0 0 9 ) 81–87
Fig. 3 – Synthetic route to 3␤-sulfooxy-25-hydroxycholest-5-ene (2c) and 24-oxocholesterol (3b) from cholesterol (4).
[3] Meng LJ, Griffiths WJ, Nazer H, Yang Y, Sjövall J. High levels
of (24S)-24-hydroxycholesterol 3-sulfate, 24-glucuronide in
the serum and urine of children with severe cholestatic liver
disease. J Lipid Res 1997;38:926–34.
[4] Ren S, Hylemon P, Zhang Z-P, Rodriguez-Agudo D, Marques
D, Li X, et al. Identification of a novel sulfonated oxysterol,
5-cholesten-3␤,25-diol 3-sulfonate, in hepatocyte nuclei and
mitochondria. J Lipid Res 2006;47:1081–90.
[5] Ren S, Li X, Rodriguez-Agudo D, Gil G, Hylemon P, Pandak
WM. Sulfated oxysterol, 25HC3S, is a potent regulator of
lipid metabolism in human hepatocytes. Biochem Biophys
Res Commun 2007;360:802–8.
[6] Reese PB. Remote functionalization reactions in steroids.
Steroids 2001;66:481–97.
gle peak by CGC. Finally, oxidation of 9b with Jones reagent,
followed by the alkaline hydrolysis of the resulting oxidation
product 3a gave the desired 24-oxocholesterol (3b) in an excel-
lent yield of 99%. Thus, 3b, which has been isolated from
various biological fluids [34,39,40] was obtained from choles-
terol (4) in eight steps with overall yield of 16%. The present
method for syntheses of oxysterols starting from cholesterol
(4) is very simple and inexpensive.
In conclusion, ETDO generated in situ from 1,1,1-trifluoro-
2-butanone/Oxone® system is a new, attractive synthetic
reagent that can be used for regioselective oxyfunctionaliza-
tion of readily available natural compounds such as choles-
terol. Further applications of ETDO to the synthesis of bioac-
tive molecules are now being conducted in our laboratory.
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This work was supported by a Nihon University Multidisci-
plinary Research Grant for 2007–2008 (to T.I.), a Grant-in-Aid
for Scientific Research (C) (to T.I., Grant 19,510,223) (to S.I.,
Grant 19,590,165) for 2007–2008, a Grant-in Aid for Young Sci-
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Ministry of Education, Sciences, Sports, and Culture of Japan,
a Research for Promoting technological Seeds for 2007 (to T.I.),
a SUNBOR grant from the Suntory Institute for Bioorganic
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