Synthesis of 7alpha-hydroxy derivatives of regulatory oxysterols.

Article abstract of DOI:10.1016/S0039-128X(00)00131-8

7alpha-Hydroxy derivatives of oxysterols are of considerable interest because of their possible involvement in regulation of cholesterol metabolism. This paper describes stereoselective syntheses and complete characterization of the 7alpha-hydroxy derivatives of four key oxysterols: 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S), 25-epoxycholesterol.

Full text of DOI:10.1016/S0039-128X(00)00131-8

Steroids 65 (2000) 529–535
Synthesis of 7␣-hydroxy derivatives of regulatory oxysterols૾
Dansu Li, Thomas A. Spencer*
Department of Chemistry, Dartmouth College, Hanover, NH 03755, USA
Received 22 March 2000; received in revised form 15 May 2000; accepted 26 May 2000
Abstract
7␣-Hydroxy derivatives of oxysterols are of considerable interest because of their possible involvement in regulation of cholesterol
metabolism. This paper describes stereoselective syntheses and complete characterization of the 7␣-hydroxy derivatives of four key
oxysterols: 25-hydroxycholesterol, 27-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. © 2000 Elsevier Sci-
ence Inc. All rights reserved.
Keywords: Oxysterols; 7␣-Hydroxyoxysterols; Synthesis
1. Introduction
on cholesterol biosynthesis [6]. However, it has also been
reported that in human diploid fibroblasts the 7␣-hydroxy
derivatives of both 1 and 2 do repress HMG-CoA reductase
activity [7].
Recent evidence indicates that oxysterols are involved in
regulation of cholesterol 7␣-hydroxylase, the enzyme that cat-
alyzes the rate-limiting step in the conversion of cholesterol to
bile acids. Mangelsdorf’s group [1] reported that specific ox-
ysterols activate transcription of an LXRE-luciferase construct
through the orphan nuclear transcription factor LXR␣, and
suggested that this might be part of a feedback regulation of
crucial metabolic pathways, such as steroid hormone or bile
acid biosynthesis. Shortly thereafter, Lehmann et al. [2], re-
ported that the receptors LXR␣ and LXR␤ are activated by
specific oxysterols, and that they induce transcription of a
cholesterol 7␣-hydroxylase promoter, implicating oxysterols
as regulators of this key enzyme. Mangelsdorf et al. have also
described an extended investigation of structural requirements
for oxysterol ligands for the LXR␣ and LXR␤ receptors [3]. It
has been observed that some of these same oxysterols are also
subject to enzymatic 7␣-hydroxylation [4]. It has, in fact, been
suggested that such 7␣-hydroxylation of 25-hydroxycholes-
terol (1) (Scheme 1) may be important to inactivate 1 as a
regulator of HMG-CoA reductase and the LDL receptor [5], in
another putative regulatory role for oxysterols. In support of
this possibility, it has been reported that the 7␣-hydroxy de-
rivative of 27-hydroxycholesterol (2) has no inhibitory effect
Further studies of the effects of 7␣-hydroxylation of oxys-
terols on different aspects of cholesterol regulation are clearly
needed. As part of such studies, it is obviously important to
have available samples of the pertinent 7␣-hydroxylated ox-
ysterols. This paper describes the synthesis and complete char-
acterization of the 7␣-hydroxy derivatives of four key oxys-
terols: 25-hydroxycholesterol (1), 27-hydroxycholesterol (2),
24(S)-hydroxycholesterol (3), and 24(S),25-epoxycholesterol
(4). These were selected on the basis of their previous impli-
cation as possible regulators of cholesterol metabolism. 25-
Hydroxycholesterol (1) is one of the most potent oxysterols in
HMG-CoA reductase repression assays [8,9] and because it is
commercially available, it has been by far the most widely
used “representative” oxysterol. Observations consistent with a
regulatory role for 1 have recently been reported [10,11].
27-Hydroxycholesterol (2) has been identified in mouse liver
[12], and has been proposed as a regulator of cholesterol
metabolism [13–15], although not without dispute [16]. 24(S)-
Hydroxycholesterol (3) and 24(S),25-epoxycholesterol (4) ap-
peared to be particularly promising candidate oxysterols on the
basis of our earlier investigations [17,18] and the recent finding
of their high affinities as ligands for the LXR receptors [1,2].
The 7␣-hydroxy derivative 5 (Scheme 2) of 25-hydroxy-
cholesterol (1) has been previously prepared by a different
route from that described herein, requiring isolation from a
mixture with its 7␤-isomer [19]. The 7␣-hydroxy derivative
૾ This research was supported by NIH grant HL52069.
* Corresponding author. Tel.: ϩ1-603-646-2805; fax: ϩ1-603-646-
3946.
E-mail address: taspen@dartmouth.edu (T.A. Spencer).
0039-128X/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(00)00131-8

530
D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
Scheme 1.
6 of 27-hydroxycholesterol (2) has also been reported, but
without complete separation from its 7␤-isomer and com-
plete characterization [20]. During the course of this work,
Corey and Grogan [21] reported preparation of 7␣-hydroxy-
24(S),25-epoxycholesterol (8) (Scheme 3), by a synthesis
slightly different from that reported herein. This paper pre-
sents full details of stereoselective syntheses of the 7␣-
hydroxy derivatives 5–8 of all four key oxysterols 1–4.
shifts are reported in units of ␦. Melting points (m.p.) were
determined using a Thomas-Hoover apparatus in capillary
tubes and are uncorrected. Thin-layer chromatography
(TLC) was carried out on EM plastic sheets pre-coated with
silica gel 60 F-254 (Whatman, Tewksbury, MA, USA).
Visualization was obtained by exposure to 5% phosphomo-
lybdic acid in 2-propanol. Flash chromatography was car-
ried out on EM Reagent silica gel 60 (230–400 mesh),
unless otherwise noted. MgSO4 was used to dry all organic
layers.
2. Experimental
CH2Cl2 was distilled from calcium hydride. Tetrahydro-
furan and ether were distilled from sodium/benzophenone.
Pyridine was distilled from calcium hydride onto 3Å mo-
lecular sieves. Acetonitrile was dried over 3Å molecular
sieves for 24 h and then distilled onto 3Å molecular sieves.
CrO3 was dried in vacuo for 12 h over KOH before use. All
2.1. General
NMR spectra, unless otherwise noted, were taken in
CDCl3 on a 300-MHz Varian spectrometer. The chemical
Scheme 2.

D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
531
Scheme 3.
reagents, unless otherwise noted, were obtained from Al-
drich Chemical Co. (Milwaukee, WI, USA).
0.93 (d, J ϭ 6.6 Hz, 3H), 0.70 (s, 3H); 13C NMR 202.2,
166.1, 166.0, 164.1, 133.3, 132.7, 132.3, 130.5, 129.9,
129.6, 128.6, 128.5, 127.1, 83.6, 73.1, 55.1, 50.2, 50.1,
45.7; 43.4, 41.8, 38.9, 38.7, 38.1, 36.5, 36.3, 35.9, 28.8,
27.8, 26.6, 26.4, 26.4, 21.4, 20.7, 19.1, 17.6, 12.3. Analysis
Calculated for C41H52O5: C, 78.80; H, 8.39. Found: C,
78.58; H, 8.55.
2.2. Cholest-5-ene-3␤,25-diol 3␤,25-Dibenzoate (9)
According to a procedure by Kim et al. [22], to a solution
of 43.6 mg (0.108 mmol) of 1 (Steraloids Inc.) in 2.0 ml of
dry pyridine was added 0.5 ml (4 mmol) of benzoyl chlo-
ride. The mixture was stirred at room temperature for 3 h
under N2, diluted with 10 ml of MeOH and 20 ml of hexane,
washed with 10 ml each of 10% HCl, H2O, saturated
NaHCO3 solution and H2O, dried, filtered, evaporated to
give 65.4 mg (98%) of 9: m.p. 100–103°C (lit. [23] m.p.
100–102°C); 1H NMR 8.03 (m, 4H), 7.54 (m, 2H), 7.45 (m,
4H), 5.43 (m, 1H), 4.87 (m, 1H), 1.26 (s, 6H), 1.07 (s, 3H),
0.93 (d, J ϭ 6.6 Hz, 3H), 0.69 (s, 3H); 13C NMR 166.1,
165.8, 139.8, 132.9, 132.6, 132.2, 131.0, 129.7, 129.6,
128.4, 128.3, 122.9, 83.5, 74.7, 56.8, 56.3, 50.2, 42.5, 41.7,
39.9, 38.4, 37.2, 36.8, 36.4, 35.8, 32.1, 32.0, 28.4, 28.1,
26.3, 26.3, 24.5, 21.2, 20.6, 19.6, 18.8, 12.1.
2.4. Cholest-5-ene-3␤,7␣,25-triol 3␤,25-Dibenzoate (15)
According to a modification of a procedure by Kumar et
al. [25], to a solution of 18 mg (0.035 mmol) of 12 in 1 ml
of dry THF at Ϫ78°C was added 0.1 ml of 1 M L-Selectride
in THF. The resulting solution was stirred at Ϫ78°C for 5 h
under N2 and 0.1 ml each of water, 6 N NaOH and 30%
H2O2 were added. Most of the THF was evaporated, 10 ml
of water was added, and the mixture was extracted with 3 ϫ
10 ml of CH2Cl2. The organic layers were dried, filtered,
and evaporated to give 17.0 mg of residue, which was
chromatographed (3:17 EtOAc:hexane) to afford 12.8 mg
(71%) of 15. Recrystallization from ether-hexane gave col-
orless 15: m.p. 136.5–137.2°C; 1H NMR 8.03 (m, 4H), 7.55
(m, 2H), 7.44 (m, 4H), 5.69 (d, J ϭ 5.1 Hz, 1H), 4.93 (m,
1H), 3.87 (m, 1H), 1.26 (s, 6H), 1.07 (s, 3H), 0.94 (d, J ϭ
6.6 Hz, 3H), 0.70 (s, 3H); 13C NMR 166.1, 165.9, 145.4,
133.0, 132.6, 132.3, 130.9, 129.8, 129.6, 128.5, 128.4,
125.1, 83.5, 74.2, 65.5, 56.1, 49.6, 42.4, 42.4, 41.7, 39.4,
38.2, 37.8, 37.7, 37.0, 36.4, 35.9, 28.5, 27.8, 26.4, 24.5,
20.9, 20.6, 18.9, 18.5, 11.9. Analysis Calculated for
C41H54O5: C, 78.54; H, 8.69. Found: C, 78.46; H, 8.62.
2.3. 3␤,25-Dihydroxycholest-5-en-7-one 3␤,25-Dibenzoate
(12)
According to a procedure by Parish and Wei [24], to a
solution of 26.3 mg (0.0431 mmol) of 9 in 5 ml of dry
benzene were added 35.0 mg of 3A molecular sieves and
232 mg (1.08 mmol) of PCC. After the mixture was refluxed
for 24 h under N2, 10 ml of brine was added carefully and
the resulting mixture was extracted with 3 ϫ 10 ml of
CH2Cl2. The combined organic layers were evaporated to
give a brown residue, which was taken up in 50 ml of ether
and filtered. The filtrate was dried, filtered, and evaporated
to give 28.2 mg of residue that was chromatographed (1:9
EtOAc:hexane) to give 23.4 mg (87%) of 12. Recrystalli-
zation from CH2Cl2-hexane gave colorless 12: m.p. 148.9–
2.5. Cholest-5-ene-3␤,7␣,25-triol (5)
A solution of 12.8 mg (0.0204 mmol) of 15 in 10 ml of
MeOH containing 0.5 g of KOH was heated at reflux for
1 h. Most of MeOH was evaporated, 10 ml of H2O was
added, and the resulting mixture was extracted with 3 ϫ 10
ml of CH2Cl2. The combined organic layers were washed
1
149.4°C; H NMR 8.02 (m, 4H), 7.56 (m, 2H), 7.44 (m,
4H), 5.75 (m, 1H), 4.98 (m, 1H), 1.27 (s, 6H), 1.07 (s, 3H),

532
D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
with 10 ml of brine, dried, filtered, and evaporated to give
a yellow residue, which was chromatographed (4:1 EtOAc:
hexane) twice to give 10 mg of white solid. Further purifi-
cation by HPLC on a 5-␮m Ultrasphere ODS semi-prepar-
ative HPLC column (10 ϫ 300 mm, Beckman) with 1:9
H2O:MeOH as efluant at a flow rate of 3 ml/min and
recrystallization from CH2Cl2-acetone afforded 4.3 mg
(51%) of 5: m.p. 234 to 235°C (lit. [19] m.p.
1.03 (d, J ϭ 6.9 Hz, 3H), 0.94 (d, J ϭ 6.3 Hz, 3H), 0.70 (s,
3H); 13C NMR 202.1, 166.9, 166.0, 164.0, 133.2, 133.0,
130.7, 130.5, 129.8, 129.7, 128.6, 128.5, 127.0, 73.0 70.1,
54.9, 50.1, 50.0, 45.6, 43.3, 38.9, 38.6, 38.0, 36.2, 35.9,
34.1, 32.9, 29.9, 28.8, 27.7, 26.5, 23.5, 21.4, 19.0, 17.5,
17.5, 12.2. FAB-HRMS (M-Hϩ) Analysis Calculated for
C41H51O5: 623.3737. Found: 623.3735. Analysis Calculated
for C41H52O5: C, 78.80; H, 8.39. Found: C, 78.83; H, 8.38.
1
234.5–235.5°C); H NMR (CD3OD) 5.53 (d, J ϭ 5.1 Hz,
1H), 3.75 (m, 1H), 3.45 (m, 1H), 1.16 (s, 6H), 0.99 (s, 3H),
0.96 (d, J ϭ 6.3 Hz, 3H), 0.71 (s, 3H). (lit. [19] H NMR
2.8. (25R)-Cholest-5-ene-3␤,7␣,26-triol 3␤,26-Dibenzoate
(16)
1
(pyridine-d5) 5.57 (m, 1H), 5.3–4.5 (m, 1H), 3.92 (m, 1H),
1.28 (s, 6H), 0.93 (s, 3H), 0.61 (s, 3H)).
As in the preparation of 15 from 12, 38.6 mg (0.0619
mmol) of 13 was converted to 45.0 mg of crude 16, which
was recrystallized from acetone-CH2Cl2 three times to give
2.6. (25R)-Cholest-5-ene-3␤,26-diol 3␤,26-Dibenzoate
(10)
1
35.0 mg (93%) of 16: m.p. 143.4–144.4°C; H NMR 8.06
(m, 4H), 7.55 (m, 2H), 7.46 (m, 4H), 5.69 (d, J ϭ 5.1 Hz,
1H), 4.92 (m, 1H), 4.17 (m, 2H), 3.87 (m, 1H), 1.07 (s, 3H),
1.03 (d, J ϭ 6.9 Hz, 3H), 0.95 (d, J ϭ 6.3 Hz, 3H), 0.70 (s,
3H); 13C NMR 166.9, 166.1, 145.4, 133.0, 130.8, 130.7,
129.8, 129.7, 128.5, 128.5, 125.1, 74.2, 70.2, 65.5, 56.0,
49.6, 42.4, 42.3, 39.3, 38.2, 37.7, 37.7, 37.0, 36.2, 35.9,
34.1, 32.9, 28.5, 27.8, 24.5, 23.4, 20.9, 19.0, 18.5, 17.2,
11.9. Analysis Calculated for C41H54O5: C, 78.54; H, 8.68.
Found: C, 78.03; H, 8.78.
As in the preparation of 9 from 1, 76.4 mg (0.190 mmol)
of 2, prepared from diosgenin by the method of Kim et al.
[22], was converted to crude 10, which was chromato-
graphed (1:19 EtOAc:hexane) to give 102 mg (87%) of 10.
Recrystallization from methanol-CH2Cl2 gave colorless 10:
1
m.p. 136–137°C (lit. [22] m.p. 136.5–137.5°C); H NMR
8.07 (m, 4H), 7.56 (m, 2H), 7.46 (m, 4H), 5.43 (m, 1H),
4.87 (m, 1H), 4.13 (m, 2H), 1.08 (s, 3H), 1.04 (d, J ϭ 6.6
Hz, 3H), 0.95 (d, J ϭ 6.3 Hz, 3H), 0.70 (s, 3H) (lit. [22] 1H
NMR 8.05 (m, 4H), 7.54 (m, 2H), 7.44 (m, 4H), 5.42 (m,
1H), 4.86 (m, 1H), 4.21 (m, 1H), 4.12 (m, 1H), 1.07 (s, 3H),
1.02 (s, 3H), 0.93 (d, J ϭ 6.5 Hz, 3H), 0.69 (s, 3H)); 13C
NMR 166.8, 166.1, 139.8, 132.9, 132.9, 131.0, 130.7,
129.7, 128.5, 128.4, 122.9, 74.7, 70.1, 56.8, 56.2, 50.1, 42.5,
39.9, 38.4, 37.2, 36.8, 36.2, 35.9, 34.1, 32.9, 32.1, 32.0,
28.4, 28.1, 24.4, 23.5, 21.2, 19.5, 18.9, 17.2, 12.0; (lit. [22]
13C NMR 166.6, 166.0, 139.6, 132.8, 132.7, 130.7, 130.5,
129.6, 129.5, 128.3, 128.2, 122.7, 74.5, 70.0, 56.6, 56.0,
50.0, 42.2, 39.7, 38.2, 37.0, 36.6, 36.0, 35.7, 33.9, 32.7,
31.9, 28.2, 27.8, 24.2, 23.3, 21.0, 19.4, 18.7, 17.0, 11.8.)
2.9. (25R)-Cholest-5-ene-3␤,7␣,26-triol (6)
As in the preparation of 5 from 15, 39.1 mg (0.0625
mmol) of 16 was converted to crude 6, which was chro-
matographed (7:3 EtOAc:hexane) to give 20.9 mg (80%) of
6. Recrystallization from CH2Cl2-acetone gave colorless 6:
1
m.p. 214–216°C; H NMR (CD3OD) 5.52 (d, J ϭ 5.4 Hz,
1H), 3.74 (m, 1H), 3.47 (m, 1H), 3.34 (m, 2H), 0.99 (s, 3H),
0.94 (d, J ϭ 6.6 Hz, 3H), 0.88 (d, J ϭ 6.6 Hz, 3H), 0.70 (s,
3H); 13C NMR (CD3OD) 146.8, 125.1, 72.2, 68.7, 66.0,
57.6, 50.8, 43.5, 43.4, 43.0, 40.8, 39.1, 38.6, 37.6, 37.3,
37.0, 34.5, 32.3, 30.9, 29.6, 25.3, 24.7, 22.0, 19.4, 18.8,
17.2, 12.3. Analysis Calculated for C27H46O3: C, 77.45; H,
11.08. Found: C, 77.20; H, 11.06.
2.7. (25R)-3␤,26-Dihydroxycholest-5-en-7-one 3␤,26-
Dibenzoate (13)
According to a procedure by Blair et al. [26], to a solu-
tion of 101.5 mg (0.163 mmol) of 10 in 0.6 ml of dry
pyridine and 20 ml of dry CH2Cl2 was added 0.6 g of CrO3.
The mixture was heated at reflux overnight under N2,
cooled, and decanted. After addition of 3 ϫ 10 ml of
CH2Cl2 and 10 ml of ether, the resulting mixture was
washed with 4 ϫ 7 ml of saturated NaHCO3 solution and
the combined aqueous layers were extracted with 15 ml of
ether. The combined organic layers were washed with 2 ϫ
10 ml of 5% HCl, 10 ml of saturated NaHCO3 solution and
10 ml of brine, dried, filtered, and evaporated to give 215.6
mg of brown residue, which was chromatographed (1:9
EtOAc:hexane) to afford 56.0 mg (54%) of 13: m.p. 167.5–
2.10. Cholest-5-ene-3␤,24(S)-diol 3␤,24-Dibenzoate (11)
To a solution of 21.6 mg (0.0537 mmol) of 3, prepared
by the procedures of Zhang Z, Li D, Blanchard DE, Lear
SE, Erickson SK, Spencer TA (being prepared for publica-
tion), in 1.0 ml of dry pyridine was added 0.5 ml (4 mmol)
of benzoyl chloride. After the mixture was stirred at room
temperature overnight, 5 ml of MeOH was added carefully,
and the resulting mixture was extracted with 2 ϫ 20 ml of
EtOAc. The combined organic layers were washed with 10
ml each of 10% HCl, H2O, saturated NaHCO3 solution, and
H2O, dried, filtered, and evaporated to give 145 mg of
yellow solid, which was chromatographed (1:19 EtOAc:
hexane) to give 28.3 mg (86%) of 11: m.p. 183.5–184.1°C
(lit. [27] m.p. 182–184°C); 1H NMR 8.06 (m, 4H), 7.56 (m,
1
168.3°C; H NMR 8.06 (m, 4H), 7.57 (m, 2H), 7.46 (m,
4H), 5.76 (m, 1H), 4.98 (m, 1H), 4.17 (m, 2H), 1.27 (s, 3H),

D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
533
2H), 7.46 (m, 4H), 5.42 (m, 1H), 4.96 (m, 1H), 4.87 (m,
2.13. Cholest-5-ene-3␤,7␣,24(S)-triol (7)
1H), 1.07 (s, 3H), 1.00 (d, J ϭ 6.9 Hz, 3H), 0.98 (d, J ϭ 6.6
Hz, 3H), 0.95 (d, J ϭ 6.6 Hz, 3H), 0.66 (s, 3H); 13C NMR
166.7, 166.3, 139.9, 133.0, 133.0, 131.1, 130.9, 129.8,
128.6, 128.5, 123.0, 80.0, 74.8, 56.9, 56.0, 50.3, 42.6, 40.0,
38.5, 37.3, 36.9, 36.0, 32.1, 31.9, 31.5, 28.4, 28.1, 27.8,
24.5, 21.3, 19.7, 19.2, 19.0, 17.6, 12.1 (lit. [28] 13C NMR
(25.2 MHz) 165.5, 139.4, 132.7, 130.8, 129.6, 128.3, 122.5,
79.6, 74.5, 56.6, 55.6, 50.0, 42.2, 39.7, 38.2, 37.0, 36.6,
35.6, 31.8, 31.8, 31.2, 31.1, 28.1, 27.9, 27.3, 24.3, 21.1,
19.3, 18.9, 18.7, 17.3, 11.8.
As in the preparation of 5 from 15, 17.5 mg (0.0280
mmol) of 17 was converted to crude 7, which was chro-
matographed (4:1 EtOAc:hexane) to give 11.4 mg (97%) of
colorless 7 that was homogeneous by TLC. Recrystalliza-
tion twice from acetone-hexane gave 7: m.p. 203–204°C;
1H NMR (CD3OD) ␦ 5.53 (d, J ϭ 5.4 Hz, 1H), 3.76 (m,
1H), 3.47 (m, 1H), 3.21 (m, 1H), 1.00 (s, 3H), 0.97 (d, J ϭ
6.6 Hz, 3H), 0.91 (d, J ϭ 6.0 Hz, 3H), 0.89 (d, J ϭ 6.0 Hz,
3H), 0.72 (s, 3H). 13C NMR (CD3OD) 146.8, 125.1, 78.3,
72.2, 66.0, 57.5, 50.8, 43.5, 43.4, 43.0, 40.8, 39.1, 38.6,
38.2, 37.6, 34.7, 33.8, 32.3, 31.8, 29.5, 25.2, 22.0, 19.7,
19.6, 18.8, 17.7, 12.3; EI-HRMS (Mϩ-H2O) Analysis Cal-
culated for C27H42O: 400.3341. Found: 400.3349.
2.11. 3␤,24(S)-Dihydroxycholest-5-en-7-one 3␤,24-
Dibenzoate (14)
According to a procedure by Ratcliffe and Rodehorst
[29], to a solution of 16.0 mg (0.0256 mmol) of 11 in 0.3
ml of pyridine and 10 ml of dry CH2Cl2 was added 0.3 g
of CrO3. The mixture was heated at reflux overnight
under N2, decanted and evaporated to give a black resi-
due, which was taken up in 50 ml of ether and filtered.
The filtrate was washed with 10 mL of 5% NaHCO3
solution and 10 ml of brine, dried, filtered, and evapo-
rated to give 38.0 mg of yellow oil which was chromato-
graphed (1:9 EtOAc:hexane) to afford 12.7 mg (78%) of
14: m.p. 143–147°C. Recrystallization from CH2Cl2-hex-
ane three times gave 14: m.p. 151.6 to 152.4°C; 1H NMR
8.05 (m, 4H), 7.56 (m, 2H), 7.45 (m, 4H), 5.75 (s, 1H),
4.96 (m, 2H), 1.27 (s, 3H), 1.00 (d, J ϭ 6.9 Hz, 3H), 0.98
(d, J ϭ 6.9 Hz, 3H), 0.95 (d, J ϭ 6.3 Hz, 3H), 0.67 (s,
3H); 13C NMR 202.2, 166.7, 166.0, 164.1, 133.3, 133.0,
131.1, 130.5, 129.9, 129.8, 128.6, 128.6, 127.1, 80.0,
73.1, 54.6, 50.2, 50.0, 45.7, 43.4, 38.9, 38.7, 38.1, 36.3,
35.9, 31.8, 31.4, 28.7, 27.8, 27.7, 26.5, 21.4, 19.2, 19.2,
17.6, 17.6, 12.2. Analysis Calculated for C41H52O5: C,
78.80; H, 8.39. Found: C, 78.56, H, 8.37.
2.14. 24(S),25-Epoxycholesteryl-3␤-acetate (18)
To a solution of 40.4 mg (0.101 mmol) of 4, prepared as
previously described [30], in 1.5 ml of dry pyridine was
added 0.7 ml of acetic anhydride. The resulting mixture was
stirred under N2 for 12 h, 10 ml of saturated NaHCO3
solution was added, and the mixture was extracted with 3 ϫ
10 ml of CH2Cl2. The combined organic layers were dried,
filtered, and evaporated to give 109.7 mg of residue, which
was chromatographed on aluminum oxide (active neutral,
gamma, Ϫ60 mesh, AlfaAesar; 1:19 EtOAc:hexane) to give
36.7 mg (82%) of 18. Recrystallization from CH2Cl2-hex-
ane afforded colorless 18: m.p. 113 to 115°C (lit. [31] m.p.
114.5–116.5°C); 1H NMR 5.38 (m, 1H), 4.60 (m, 1H), 2.68
(m, 1H), 2.03 (s, 3H), 1.30 (s, 3H), 1.27 (s, 3H), 1.02 (s,
3H), 0.94 (d, J ϭ 6.3 Hz, 3H), 0.70 (s, 3H); 13C NMR 170.7,
139.8, 122.8, 74.2, 65.1, 58.3, 56.9, 56.2, 50.2, 42.6, 39.9,
38.3, 37.2, 36.8, 35.9, 32.8, 32.0, 29.9, 28.4, 28.0, 25.9,
25.2, 24.5, 21.7, 21.2, 19.5, 18.9, 18.8, 12.1.
2.15. 24(S),25-Epoxycholest-5-en-3␤-ol-7-one, 3␤-Acetate
(19)
2.12. Cholest-5-ene-3␤,7␣,24(S)-triol 3␤,24-Dibenzoate
(17)
According to a procedure by Salvador et al. [32], to a
solution of the crude product (40.6 mg) containing 18,
prepared as described above from 28.5 mg (0.0713 mmol)
of 4, and 8.1 mg of CuI in 2 ml of dry CH3CN was added
0.2 ml (5.0–6.0 M solution in decane, 1.0–1.2 mmol) of
t-BuOOH. The resulting mixture was stirred at 50°C for
25 h under N2, 10 ml of 10% Na2SO3 was added, and the
mixture was extracted with 3 ϫ 10 ml of CH2Cl2. The
combined organic layers were washed with 10 ml of satu-
rated NaHCO3 solution, 10 ml of H2O, and 10 ml of brine,
dried, filtered, and evaporated to give 60.4 mg of yellow
residue that was crystallized twice from CH2Cl2-hexane to
give 22.3 mg (69% from 4) of 19: m.p. 175.5–177.5°C; 1H
NMR 5.70 (s, 1H), 4.72 (m, 1H), 2.69 (m, 1H), 2.05 (s, 3H),
1.31 (s, 3H), 1.27 (s, 3H), 1.21 (s, 3H), 0.95 (d, J ϭ 6.6 Hz,
3H), 0.69 (s, 3H); 13C NMR 202.2, 170.6, 164.2, 127.0,
72.5, 65.1, 54.8, 50.2, 50.0, 45.7, 43.4, 38.9, 38.6, 38.0,
As in the preparation of 15 from 12, except for a reaction
time of three rather than 5 h, 34.9 mg (0.0558 mmol) of 14
was converted to 34.6 mg of crude 17, which was chro-
matographed (3:17 EtOAc:hexane) to give 24.2 mg (69%)
1
of 17: m.p. 171–173°C; H NMR 8.05 (m, 4H), 7.56 (m,
2H), 7.45 (m, 4H), 5.67 (d, J ϭ 5.1 Hz, 1H), 4.95 (m, 2H),
3.86 (m, 1H), 1.06 (s, 3H), 1.00 (d, J ϭ 6.9 Hz, 3H), 0.97
(d, J ϭ 6.6 Hz, 3H), 0.95 (d, J ϭ 5.7 Hz, 3H), 0.67 (s, 3H);
13C NMR 166.6, 166.1, 145.4, 133.0, 132.9, 131.1, 130.8,
129.8, 129.7, 128.5, 125.1, 80.0, 74.2, 65.4, 55.6, 49.6, 42.4,
42.3, 39.3, 38.2, 37.7, 37.7, 37.0, 35.8, 31.8, 31.4, 28.4,
27.8, 27.6, 24.4, 20.9, 19.1, 19.0, 18.5, 17.5, 11.8. FAB-
HRMS (M-Hϩ) Analysis Calculated for C41H53O5:
625.3893. Found: 625.3894.

534
D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
36.3, 35.8, 32.8, 28.8, 27.6, 26.6, 25.9, 25.2, 21.5, 21.4,
19.0, 18.9, 17.5, 12.2. Analysis Calculated for C29H44O4: C,
76.27; H, 9.71. Found: C, 76.40; H, 9.63.
conversion to the known dibenzoates 9 to 11 [22,25,27]
Allylic oxidation at C7 can be effected to afford 7-hydroxy
derivatives directly, but produces a mixture of 7␣ and 7␤
isomers [20]. Alternatively, oxidation can be effected to
form a 7-keto derivative which can be stereoselectively
reduced to the axial 7␣-hydroxy compound by use of a
hindered complex metal hydride, such as L-selectride [25].
Accordingly, each of 9 to 11 was subjected to allylic oxi-
dation (CrO3/pyridine for 9 [24] and 11 [29]; PCC for 10)
[27] to afford enones 12 to 14 in good yield, and these were
in turn reduced using L-selectride to yield the 7␣-hydroxy
2.16. 24(S),25-Epoxycholest-5-ene-3␤,7␣-diol 3␤-Acetate
(20)
As in the preparation of 15 from 12, 24.3 mg (0.0533
mmol) of 19 was converted to 20.4 mg of crude 20, which
was crystallized from ether-hexane to afford 17.3 mg (71%)
1
of 20: m.p. 185 to 186°C; H NMR 5.63 (d, J ϭ 4.8 Hz,
1
1H), 4.65 (m, 1H), 3.85 (bs, 1H), 2.69 (m, 1H), 2.04 (s, 3H),
1.31 (s, 3H), 1.27 (s, 3H), 1.01 (s, 3H), 0.95 (d, J ϭ 6.6 Hz,
3H), 0.69 (s, 3H); 13C NMR 170.7, 145.4, 124.9, 73.6, 65.4,
65.2, 58.4, 55.9, 49.6, 42.4, 39.3, 38.1, 37.7, 37.7, 37.0,
35.9, 32.8, 28.5, 27.7, 25.8, 25.2, 24.5, 21.6, 20.9, 18.9,
18.8, 18.4, 11.9. Analysis Calculated for C29H46O4: C,
75.94; H, 10.11. Found: C, 75.75; H, 10.08.
steroids 15 to 17 with complete stereoselectivity. The H
NMR spectra of each of the crude hydride reduction prod-
ucts showed only the relatively deshielded doublet for a
6␤-H and none of the relatively shielded singlet expected
for a 6␣-H [25]. Removal of the dibenzoate protecting
groups from 15 to 17 by treatment with KOH in MeOH then
produced the desired triols 5 to 7 in excellent yield.
Synthesis of 7␣-hydroxy-24(S),25-epoxycholesterol (8)
(Scheme 3) required a modified reaction sequence because
of the acid liability of the epoxide function. The known [31]
acetate 18 of 24(S),25-epoxycholesterol (4) was in this case
used as the protected substrate for allylic oxidation. Several
methods, including those used for the conversion of 9 to 11
to 12 to 14, caused destruction of the epoxide group as well
as allylic oxidation. Attempted direct allylic oxidation of 4
was also unsuccessful. The conversion of 18 to 19 was
achieved, however, by the procedure of Salvador et al. [32],
with t-BuOOH and CuI and CH3CN, as had been employed
by Corey and Grogan [21] for the analogous reaction on
24(S),25-epoxycholesteryl benzoate. Reduction with L-se-
lectride yielded 20 and cleavage of the acetate protecting
group then afforded 8. Thus, all four 7␣-hydroxylated oxy-
sterols 5 to 8 are now available as reference standards and
procedures have been developed for their synthesis in larger
amounts as required for further experimentation.
2.17. 24(S),25-Epoxycholest-5-ene-3␤,7␣-diol (8)
According to a modification of a procedure by Corey and
Grogan [21], a solution of 17.3 mg of 20 in 4 ml of 1:4
THF-MeOH containing 0.04 g of NaOH was stirred at rt for
2.5 h, 10 ml of water was added, and the resulting mixture
was extracted with 3 ϫ 10 ml of CH2Cl2. The combined
organic layers were washed with 10 ml of brine, dried,
filtered and evaporated to give 12.6 g of yellow residue that
was chromatographed on aluminum oxide (active neutral,
gamma, 60 mesh, AlfaAesar; 6:4 EtOAc:hexane) to give 8.3
mg (54%) of colorless 8 that was homogeneous by TLC:
1
m.p. 149.4–150.5°C; H NMR 5.61 (d, J ϭ 4.8 Hz, 1H),
3.86 (bs, 1H), 3.59 (m, 1H), 2.69 (m, 1H), 1.31 (s, 3H), 1.27
(s, 3H), 1.00 (s, 3H), 0.95 (d, J ϭ 6.6 Hz, 3H), 0.70 (s, 3H);
13C NMR 146.5, 124.1, 71.6, 65.6, 65.2, 58.4, 56.0, 49.7,
42.5, 42.4, 42.3, 39.4, 37.8, 37.7, 37.3, 36.0, 32.8, 31.7,
28.6, 25.9, 25.2, 24.6, 21.0, 19.0, 18.9, 18.5, 11.9; EI-
HRMS (Mϩ-H2O) Analysis Calculated for C27H40O:
398.3184. Found: 398.3184.
References
[1] Janowski BA, Willy PJ, Devi TR, Falck JR, Mangelsdorf DJ. An
oxysterol signalling pathway mediated by the nuclear receptor LXR␣.
Nature 1996;383:728–31.
3. Results and discussion
[2] Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB,
Su J-L, Sundseth SS, Winegar DA, Blanchard DE, Spencer TA,
Willson TM. Activation of the nuclear receptor LXR by oxysterols
defines a new hormone response pathway. J Biol Chem 1997;272:
3137–40.
[3] Janowski BA, Grogan MJ, Jones SA, Wisely GB, Kliewer SA, Corey
EJ, Mangelsdorf DJ. Structural requirements of ligands for the oxys-
terol liver X receptors LXR␣ and LXR␤. Proc Natl Acad Sci USA
1999;96:266–71.
[4] Axelson M, Shoda J, Sjo¨vall J, Toll A, Wikvall K. Cholesterol is
converted to 7␣-hydroxy-3-oxo-4-cholestenoic acid in liver mito-
chondria. J Biol Chem 1992;267:1701–4.
[5] Dueland S, Nenseter MS, MacPhee AA, Davis RA, Trawick JD.
Expression of 7␣-hydroxylase in non-hepatic cells results in liver
phenotypic resistance of the low density lipoprotein receptor to cho-
lesterol repression. J Biol Chem 1992;267:22695–8.
25-Hydroxycholesterol (1) is commercially available,
but adequate amounts of the other three oxysterols had to be
synthesized for conversion to their 7␣-hydroxy derivatives.
27-Hydroxycholesterol (2) was prepared from diosgenin by
the method reported by Schroepfer and co-workers [22].
24(S)-Hydroxycholesterol (3) was prepared from stigmas-
terol by our recently developed route (by the procedures of
Zhang, Li, Blanchard, Lear, Erickson, Spencer, unpublished
results) and 24(S),25-epoxycholesterol (4) was synthesized
using procedures we have recently reported [30].
The strategy for the preparation of the 7␣-hydroxy de-
rivatives is illustrated for diols 1 to 3 in Scheme 2. Initial
protection of the free hydroxyl groups was accomplished by

D. Li, T.A. Spencer / Steroids 65 (2000) 529–535
535
[6] Martin KO, Reiss AB, Lathe R, Javitt NB. 7␣-Hydroxylation of
27-hydroxycholesterol: biological role in the regulation of cholesterol
synthesis. J Lipid Res 1997;38:1053–8.
[7] Zhang J, Dricu A, Sjo¨vall J. Studies on the relationships between
7␣-hydroxylation and the ability of 25- and 27-hydroxycholesterol to
suppress the activity of HMG-CoA reductase. Biochim Biophys Acta
1997;1344:241–9.
[19] Nagano H, Poyser JP, Cheng K-P, Luu B, Ourisson G, Beck JPL.
Chimie et biochimie de drogues chinoises II-ste´rols hydroxyle´s cy-
totoxiques sur des cellules cance´reuses. Synthe`se et activite´ bi-
ologique. J Chem Res(m) 1977;9:2522–71.
[20] Shoda J, Axelson M, Sjo¨vall J. Synthesis of potential C27-intermedi-
ates in bile acid biosynthesis and their deuterium-labeled analogs.
Steroids 1993;58:119–25.
[8] Kandutsch AA, Chen HW. Inhibition of sterol synthesis in cultured
mouse cells by cholesterol derivatives oxygenated in the side chain.
J Biol Chem 1974;249:6057–61.
[21] Corey EJ, Grogan MJ. Stereocontrolled syntheses of 24(S),25-epoxy-
cholesterol and related oxysterols for studies on the activation of LXR
receptors. Tetrahedron Lett 1998;39:9351–4.
[9] Taylor FR, Saucier SE, Shown EP, Parish EJ, Kandutsch AA. Cor-
relation between oxysterol binding to a cytosolic binding protein and
potency in the repression of hydroxymethylglutaryl coenzyme A
reductase. J Biol Chem 1984;259:12382–7.
[10] Johnson KA, Morrow CJ, Knight GD, Scallen TJ. In vivo forma-
tion of 25-hydroxycholesterol from endogenous cholesterol after a
single meal, dietary cholesterol challenge. J Lipid Res 1994;35:
2241–53.
[11] Lala DS, Syka PM, Lazarchik SB, Mangelsdorf DJ, Parker KL,
Heyman RA. Activation of the orphan nuclear receptor steroidogenic
factor 1 by oxysterols. Proc Natl Acad Sci USA 1997;94:4895–4900.
[12] Saucier SE, Kandutsch AA, Gayen AK, Swahn DK, Spencer TA.
Oxysterol regulators of 3-hydroxy-3-methylglutaryl-coA reductase in
liver. Effect of dietary cholesterol. J Biol Chem 1989;264:6863–9.
[13] Esterman AL, Baum H, Javitt NB, Darlington GJ. 26-Hydroxycho-
lesterol: regulation of hydroxymethylglutaryl-coA reductase activity
in Chinese hamster ovary cell culture. J Lipid Res 1983;24:1304–9.
[14] Andersson S, Davis DL, Dahlba¨ck H, Jo¨rnvall H, Russell DW. Clon-
ing, structure, and expression of the mitochondrial cytochrome P-450
sterol 26-hydroxylase, a bile acid biosynthesis enzyme. J Biol Chem
1989;264:8222–9.
[22] Kim H-S, Wilson WK, Needleman DH, Pinkerton FD, Wilson DK,
Quiocho FA, Schroepfer GJ Jr. Inhibitors of sterol synthesis. Chem-
ical synthesis, structure, and biological activities of (25R)-3␤,26-
dihydroxy-5␣-cholest-8(14)-en-15-one, a metabolite of 3␤-hydroxy-
5␣-cholest-8(14)-en-15-one. J Lipid Res 1989;30:247–61.
[23] Beckwith ALJ. Hydroxylation of 5␣,6␤-dibromocholestan-3␤-yl ac-
etate by chromic acid. J Chem Soc 1961:3162–4.
[24] Parish EJ, Wei TY. Allylic oxidation of ⌬5-steroids with pyridinium
chlorochromate (PCC) and pyridinium dichromate (PDC). Synth
Commun 1987;17:1227–33.
[25] Kumar V, Amann A, Ourisson G, Luu B. Stereospecific synthesis of
7␤- and 7␣-hydroxycholesterols. Synth Commun 1987;17:1279–86.
[26] Blair IA, Phillipou G, Seaborn C. Synthesis of C-7–2H2 steroids for
human metabolism studies. J Labelled Compd Radiopharm 1978;15:
645–55.
[27] Ikekawa N, Morisaki M, Koizumi N, Sawamura M, Tanaka Y, De-
Luca HF. Synthesis and biological activity of 24␰1- and 24␰2-hy-
droxyvitamin D3. Biochem Biophys Res Commun 1975;62:485–91.
[28] Koizumi N, Fujimoto Y, Takeshita T, Ikekawa N. Carbon-13 nuclear
magnetic resonance of 24-substituted steroids. Chem Pharm Bull
1979;27:38–42.
[15] Rennert H, Fischer RT, Alvarez JG, Trzaskos JM, Strauss JF III.
Generation of regulatory oxysterols: 26-hydroxylation of cholesterol
by ovarian mitochondria. Endocrinology 1990;127:738–46.
[16] Lund E, Breuer O, Bjorkhem I. Evidence that 24- and 27-hydroxy-
lation are not involved in the cholesterol-induced down-regulation of
hydroxymethylglutaryl-CoA reductase in mouse liver. J Biol Chem
1992;267:25092–7.
[17] Erickson SK, Lear SR, Gayen AK, Spencer TA. Oxysterol regulation
of cholesterol metabolism. Circulation 1994;90:420.
[18] Spencer TA. The squalene dioxide pathway of steroid biosynthesis.
Acc Chem Res 1994;27:83–90.
[29] Ratcliffe R, Rodehorst R. Improved procedure for oxidation with the
chromium-trioxide-pyridine complex. J Org Chem 1970;35:4000–2.
[30] Tomkinson NCO, Willson TM, Russel JS, Spencer TA. Efficient,
stereoselective synthesis of 24(S),25-epoxycholesterol. J Org Chem
1998;63:9919–23.
[31] Emmons GT, Wilson WK, Schroepfer GJ Jr. 24,25-Epoxysterols.
Differentiation of 24R- and 24S-epimers by 13C nuclear magnetic
resonance spectroscopy. J Lipid Res 1989;30:133–8.
[32] Salvador JAR, Melo MLS, Campos Neves AS. Copper-catalysed
allylic oxidation of ⌬5-steroids by t-butyl hydroperoxide. Tetrahedron
Lett 1997;38:119–22.